.SUBTILISIN DY VARIANTS HAVING DECREASED ADSORPTION AND INCREASED HYDROLYSIS

TECHNICAL FIELD

The present invention relates to novel enzyme variants useful in a variety of cleaning compositions, and DNA sequences encoding such enzyme variants.

BACKGROUND

Enzymes make up the largest class of naturally occurring proteins. Each class of enzyme generally catalyzes (accelerates a reaction without being consumed) a different kind of chemical reaction. One class of enzymes known as proteases, are known for their ability to hydrolyze (break down a compound into two or more simpler compounds with the uptake of the H and OH parts of a water molecule on either side of the chemical bond cleaved) other proteins. This ability to hydrolyze proteins has been taken advantage of by incorporating naturally occurring and protein engineered proteases as an additive to laundry detergent preparations. Many stains on clothes are proteinaceous and wide-specificity proteases can substantially improve removal of such stains.

Unfortunately, the efficacy level of these proteins in their natural, bacterial environment, frequently does not translate into the relatively unnatural wash environment. Specifically, protease characteristics such as thermal stability, pH stability, oxidative stability and substrate specificity are not necessarily optimized for utilization outside the natural environment of the enzyme.

The amino acid sequence of the protease determines the characteristics of the protease. A change of the amino acid sequence of the protease may alter the properties of the enzyme to varying degrees, or may even inactivate the enzyme, depending upon the location, nature and/or magnitude of the change in the amino acid sequence. Several approaches have been taken to alter the wild-type amino acid sequence of proteases in an attempt to improve their properties, with the goal of increasing the efficacy of the protease in the wash environment. These approaches include altering the amino acid sequence to enhance thermal stability and to

improve oxidation stability under quite diverse conditions

Despite the variety of approaches described in the art, there is a continuing need for new effective variants of proteases useful for cleaning a variety of surfaces.

Objects of the Present Invention

It is an object of the present invention to provide Subtilisin DY enzyme variants having improved hydrolysis versus the wild-type of the enzyme

It is also an object of the present invention to provide cleaning compositions comprising these subtilisin enzyme variants

SUMMARY

The present invention relates to Subtilisin DY variants having a modified ammo acid sequence of wild-type Subtilisin DY ammo acid sequence, the wild-type amino acid sequence comprising a first loop region _, , a second loop region, a third loop region, a fourth loop region and a fifth loop region, wherein the modified ammo acid sequence comprises different ammo acids than that occurring in wild-type Subtilisin DY (i.e., substitution) at specifically identified positions in one or more of the loop regions whereby the Subtilisin DY variant has decreased adsorption to, and increased hydrolysis of. an insoluble substrate as compared to the wild-type Subtilisin DY The present invention also relates to DNA sequences encoding such Subtilisin DY variants The present invention also relates to compositions comprising such Subtilisin DY variants for cleaning a variety of surfaces.

DESCRIPTION I. Subtilisin DY Variants

This invention pertains to subtilisin enzymes, in particular Subtilisin DY, that have been modified by mutating the various nucleotide sequences that code for the enzyme, thereby modifying the amino acid sequence of the enzyme The modified subtilisin enzymes (hereinafter, "Subtilisin DY variants") of the present invention have decreased adsorption to and increased hydrolysis of an insoluble substrate as compared to the wild-type subtilisin. The present invention also pertains to DNA sequences encoding for such Subtilisin DY variants.

The subtilisin enzymes of this invention belong to a class of enzymes known as proteases. A protease is a catalyst for the cleavage of peptide bonds. One type of protease is a serine protease. A serine protease is

distinguished by the fact that there is an essential serine residue at the active site

The observation that an enzyme's rate of hydrolysis of soluble substrates increases with enzyme concentration is well documented It would therefore seem plausible that for surface bound substrates, such as is encountered in many cleaning applications, the rate of hydrolysis would increase with increasing surface concentration This has been shown to be the case (Brode, P F III and D S Rauch, LANGMUIR, "Subtilisin BPN' Activity on an Immobilized Substrate", Vol 8, pp 1325-1329 (1992)) In fact, a linear dependence of rate upon surface concentration was found for insoluble substrates when the surface concentration of the enzyme was varied (Rubingh, D N and M D Bauer, "Catalysis of Hydrolysis by Proteases at the Protein-Solution Interface " in POLYMER SOLUTIONS, BLENDS AND INTERFACES, Ed by I Noda and D N Rubingh, Elsevier, p 464 (1992)) Surprisingly, when seeking to apply this principle in the search for variant proteases which give better cleaning performance, we did not find that enzymes which adsorb more give better performance In fact, we surprisingly determined the opposite to be the case decreased adsorption by an enzyme to a substrate resulted in increased hydrolysis of the substrate (i e better cleaning performance)

While not wishing to be bound by theory, it is believed that improved performance, when comparing one variant to another, is a result of the fact that enzymes which adsorb less are also less tightly bound and therefore more highly mobile on the surface from which the insoluble protein substrate is to be removed At comparable enzyme solution concentrations, this increased mobility is sufficient to outweigh any advantage that is conferred by delivering a higher concentration of enzyme to the surface

The mutations described herein are designed to change (i e , decrease) the adsorption of the enzyme to surface-bound soils In Subtilisin DY, certain am o acids form exterior loops on the enzyme molecule For purposes of discussion, these loops shall be referred to as first, second, third, fourth and fifth loop regions Specifically, positions 58-65 form the first loop region, positions 94-106 form the second loop region, positions 125-132 form the third loop region, positions 153-166 form the fourth loop region, positions 186-190 form the fifth loop region, and positions 199-219 form the sixth loop region (position numbering analagous to positions in the ammo acid sequence for wild-type subtilisin Subtilisin DY (SEQ ID NO 1 ))

It is believed that these loop regions play a significant role in the adsorption of the enzyme molecule to a surface-bound peptide, and specific mutations in one or more of these loop regions will have a significant effect on this adsorption While not wishing to be bound by theory, it is believed that the loop regions are important to the adsorption of the Subtilisin DY molecule for at least two reasons First, the ammo acids which comprise the loop regions can make close contacts with any surfaces to which the molecule is exposed Second, the proximity of the loop regions to the active-site and binding pocket of the Subtilisin DY molecule gives them a roie in the catalytically productive adsorption of the enzyme to surface- bound substrates (peptides/protem soils)

As used herein, "variant" means an enzyme having an ammo acid sequence which differs from that of wild-type

As used herein, "mutant Subtilisin DY DNA" means a DNA sequence coding for a Subtilisin DY variant.

As used herein, "wild-type Subtilisin DY" refers to an enzyme represented by SEQ ID NO 1 The ammo acid sequence for Subtilisin DY is further described by Nedkov, P , Oberthur, W and Braunitzer, G , BIOL CHEM , Vol 366, pp 421-430 (1985), incorporated herein by reference

As used herein, the term "Subtilisin DY wild-type ammo acid sequence" encompasses SEQ ID NO 1 as well as SEQ ID NO 1 having modifications to the ammo acid sequence other than at any of positions 58, 59, 60, 61 , 62, 64, 65, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 125, 126, 127, 128, 129, 130, 131 , 132, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 186, 187, 188, 189, 190, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218 or 219

As used herein, "more hydrophilic am o acid" refers to any other ammo acid having greater hydrophilicity than a subject ammo acid with reference to the hydrophilicity table below The following hydrophilicity table (Table 1 ) lists ammo acids in descending order of increasing hydrophilicity (see Hopp, T P , and Woods, K R., "Prediction of Protein Antigenic Determinants from Ammo Acid Sequences", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, Vol 78, pp. 3824-3828, 1981 , incorporated herein by reference).

TABLE 1

Am o Acid Hydrophilicity Value

Table 1 also indicates which am o acids carry a charge (this characteristic being based on a pH of from about 8-9) The positively charged ammo acids are Arg and Lys, the negatively charged ammo acids are Glu and Asp, and the remaining ammo acids are neutral In a preferred embodiment of the present invention, the substituting ammo acid is either neutral or negatively charged, more preferably negatively charged (i e , Glu or Asp)

Therefore, for example, the statement "substitute Gin with an equally or more hydrophilic ammo acid which is neutral or has a negative charge" means Gin would be substituted with Asn (which is equally hydrophilic to Gin), or Ser, Glu or Asp (which are more hydrophilic than Gin), each of which are neutral or have a negative charge, and have a greater hydrophilicity value as compared to Gin Likewise, the statement "substitute Pro with a more hydrophilic ammo acid which is neutral or has a negative charge" means Pro would be substituted with Gin, Asn, Ser, Glu or Asp

In one embodiment of the present invention, the Subtilisin DY variant has a modified ammo acid sequence of Subtilisin DY wild-type ammo acid sequence, wherein the wild-type ammo acid sequence comprises a substitution at one or more positions in one or more of the first loop region, the second loop region, the third loop region, the fourth loop region, the fifth loop region or the sixth loop region, whereby the Subtilisin DY variant has

decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Subtilisin DY

In a preferred embodiment of the present invention, the substituting ammo acid for one or more of the positions in one or more of the loop regions is with reference to Table 1 , neutral or negatively charged and equally or more hydrophylic, preferably more hydrophylic, than the am o acid at the subject position in the wild-type ammo acid sequence A Substitutions in the First Loop Region

When a substitution occurs in the first loop region, the substitution occurs at one or more of positions 58, 59, 60, 61 , 62, 64 or 65

When a substitution occurs at position 58, the substituting am o acid is Asn Asp, Gin, Glu, Gly, Pro or Ser

When a substitution occurs at position 59, the substituting ammo acid

When a substitution occurs at position 60, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 61 , the substituting am o acid

When a substitution occurs at position 62, the substituting ammo acid is Asn Asp, Gin, Glu Pro or Ser

When a substitution occurs at position 64, the substituting ammo acid is Asn, Asp Gin, Glu, Pro or Ser

When a substitution occurs at position 65, the substituting am o acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser B Substitutions in the Second Loop Region

When a substitution occurs in the second loop region, the substitution occurs at one or more of positions 94, 95, 96, 97, 98, 99, 100 101 , 102, 103, 104, 105 or 106

When a substitution occurs at position 94, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr

When a substitution occurs at position 95, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Met, Pro, Ser , Thr or Val

When a substitution occurs at position 96, the substituting ammo acid

When a substitution occurs at position 97, the substituting ammo acid

When a substitution occurs at position 98, the substituting ammo acid

When a substitution occurs at position 99, the substituting am o acid is Asn Asp Gin, Glu Pro or Ser

When a substitution occurs at position 100, the substituting am o

When a substitution occurs at position 101 , the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 102, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser

When a substitution occurs at position 103, the substituting ammo acid is Ala Asn Asp Cys, Gin Glu Gly His, He, Leu, Met, Pro, Ser, Thr or Val

When a substitution occurs at position 104, the substituting ammo

When a substitution occurs at position 105, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr

When a substitution occurs at position 106, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val C Substitutions in the Third Loop Region

When a substitution occurs in the third loop region, the substitution occurs at one or more of positions 125, 126, 127, 128, 129, 130, 131 or 132

When a substitution occurs at position 125, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Met, Pro, Ser, Thr or Val

When a substitution occurs at position 126, the substituting ammo acid is Asn Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 127, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 128, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly or Ser

When a substitution occurs at position 129, the substituting ammo

When a substitution occurs at position 130, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 131 , the substituting ammo

When a substitution occurs at position 132, the substituting ammo

acid is Asn Asp, Gin, Glu, Gly, Pro or Ser D - Substitutions in the Fourth Loop Region

When a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 153, 154 155 156, 157, 158 159, 160, 161 162, 163, 164, 165 or 166

When a substitution occurs at position 153, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 154, the substituting ammo acid is Asp, Gin, Glu or Ser

When a substitution occurs at position 155, the substituting ammo

When a substitution occurs at position 156, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 157, the substituting ammo

When a substitution occurs at position 158, the substituting ammo

When a substitution occurs at position 159, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a subst'tution occurs at position 160, the substituting ammo

When a substitution occurs at position 161 , the substituting am o acid is Asn, Asp, Glu or Ser

When a substitution occurs at position 162, the substituting am o acid is Asp, Gin, Glu or Ser

When a substitution occurs at position 163, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser

When a substitution occurs at position 164, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly His, Leu, Met, Pro, Ser, Thr or Val

When a substitution occurs at position 165, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 166, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr or Val E Substitutions in the Fifth Loop Region

When a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 186, 187, 188, 189 or 190

When a substitution occurs at position 186, the substituting ammo acid, is Asn Asp Gin, Glu Gly, His, Pro, Ser or Thr

When a substitution occurs at position 187, the substituting ammo

When a substitution occurs at position 188, the substituting ammo acid is Ala Asn, Asp, Cys Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr, Tyr or Val

When a substitution occurs at position 189, the substituting am o

When a substitution occurs at position 190, the substituting ammo F Substitutions in the Sixth Loop Region

When a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 199, 200, 201 , 202, 203, 204, 205, 206 207 208, 209, 210, 21 1 , 212, 213, 214 215, 216, 217, 218 or 219

When a substitution occurs at position 199, the substituting ammo acid is Asn Asp, Gin, Glu, Gly, His, Pro, Ser or Thr

When a substitution occurs at position 200, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly or Ser

When a substitution occurs at position 201 , the substituting ammo acid is Asn Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 202, the substituting ammo acid is Ala Asn, Asp, Cys, gin, Glu, Gly His, Met, Pro, Ser or Thr

When a substitution occurs at position 203, the substituting ammo

When a substitution occurs at position 204, the substituting am o acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Met, Pro, Ser, or Thr

When a substitution occurs at position 205, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He. Leu, Met, Pro, Ser, Thr or Val

When a substitution occurs at position 206, the substituting ammo

When a substitution occurs at position 207, the substituting am o acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser

When a substitution occurs at position 208, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr or Val

When a substitution occurs at position 209, the substituting am o acid is Asn. Asp, gin. Glu, Gly or Ser

When a substitution occurs at position 210, the substituting ammo

When a substitution occurs at position 211 , the substituting ammo acid is Asp. Gin, Glu or Ser

When a substitution occurs at position 212, the substituting amino acid is Asn, Asp, gin, Glu, Gly, Pro or Ser.

When a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr or Val.

When a substitution occurs at position 214, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser

When a substitution occurs at position 215, the substituting amino

When a substitution occurs at position 216, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Met, Pro, Ser, Thr or Val.

When a substitution occurs at position 217, the substituting amino acid is Asp, gin, Glu or Ser.

When a substitution occurs at position 218, the substituting amino acid is Asn, Asp, Gin. Glu, Pro or Ser.

When a substitution occurs at position 219, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser. G Preparation of enzyme variants

Example 1 Mutant Subtilisin DY DNA

A phagemid ("DYP") containing the wild type Subtilisin DY gene is constructed. The 2.8 Kbp Pvu II restriction enzyme fragment of plasmid pUC119, (Vieira, J. and Messing, J., "Production of Single-Stranded Plasmid DNA", 153 METHODS IN ENZYMOLOGY 3-11 (1989)) is cloned into the Pvu II site of plasmid pUB110 (Bacillus Genetic Stock Center, Columbus, OH 1 E9) The pUC119-pUB110 hybrid plasmid is named pJMA601. Into the SamHI restriction site of pJMA601 is cloned the polymerase chain reaction - amplified subtilisin DY gene from chromosomal DNA giving phagemid DYP. Phagemid DYP is transformed into Escherichia coli Ung- stram CJ236 and a single stranded uracil-containing DNA template is produced using the VCSM13 helper phage (Kunkel, T.A., J.D. Roberts and

R.A. Zakour, "Rapid and efficient site-specific mutagenesis without phenotypic selection", METHODS IN ENZYMOLOGY, Vol. 154, pp. 367-382, (1987): as modified by Yuckenberg, P.D.. F. Witney, J. Geisselsoder and J. McClary. "Site-directed in vitro mutagenesis using uracil-containing DNA and phagemid vectors". DIRECTED MUTAGENESIS - A PRACTICAL APPROACH, ed. M.J. McPherson, pp. 27-48, (1991 ); both of which are incorporated herein by reference). A single primer site-directed mutagenesis modification of the method of Zoller and Smith (Zoller, M.J., and M. Smith, "Oligonucleotide-directed mutagenesis using M13-dehved vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA", NUCLEIC ACIDS RESEARCH, Vol. 10, pp. 6487-6500, (1982), incorporated herein by reference) is used to produce all mutants (basically as presented by Yuckenberg, et al., 1991 , above). Oligonucleotides are made using an Applied Biosystem Inc. 380B DNA synthesizer. Mutagenesis reaction products are transformed into Escherichia coli strain MM294 (American Type Culture Collection E. coli. 33625). All mutants are confirmed by DNA sequencing and the isolated DNA is transformed into the Bacillus subtilis expression strain BG2036 (Yang, M. Y., E. Ferrari and D. J. Henner, (1984), "Cloning of the Neutral Protease Gene of Bacillus subtilis and the Use of the Cloned Gene to Create an In Vitro-derived Deletion Mutation", JOURNAL OF BACTERIOLOGY, Vol. 160, pp. 15-21 ). For some of the loop mutants a modified DYP with a frameshift-stop codon mutation in the corresponding loop is used to produce the uracil template. Oligonucleotides are designed to restore the proper reading frame and to encode for random substitutions at positions 58, 59, 60, 61 , 62, 64, 65, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 125, 126, 127, 128, 129, 130, 131 , 132, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 186, 187, 188, 189, 190, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218 or 219 (equimolar and/or variable mixtures of all four nucleotides for all three bases at these codons). Mutations that correct for the frameshift-stop and produce a functional enzyme are identified by their ability to digest casein. The random substitutions are determined by DNA sequencing.

Example 2 Fermentation The Bacillus subtilis cells (BG2036) containing a subtilisin mutant of

interest are grown to mid-log phase in a one liter culture of LB-glucose broth and- inoculated into a Biostat ED fermenter (B Braun Biotech, Iπc , Allentown Pennsylvania) in a total volume of 10 liters The fermentation media contains Yeast Extract starch, antifoam, buffers and trace minerals (see FERMENTATION A PRACTICAL APPROACH, Ed B McNeil and L M Harvey, 1990) The broth is kept at a constant pH of 7 0 during the fermentation run Chloramphenical is added for antibiotic selection of mutagenized plasmid The cells are grown overnight at 37°C to an A 00 of about 60 and harvested

Example 3 Purification

The fermentation broth is taken through the following steps to obtain pure enzyme The broth is cleared of Bacillus subtilis cells by centπfugation, and clarified by removing fine particulates with a 100K cutoff membrane This is followed by concentration on a 10K cutoff membrane, and flow dialysis to reduce the ionic strength and adjust the pH to 5 5 using 0 025M MES buffer (2-(Λ/-morpholιno)ethanesulfonιc acid) The enzyme is further purified by loading it onto either a cation exchange chromatography column or an affinity adsorption chromatography column and eluting it from the column with a NaCI or a propylene glycol gradient (see Scopes, R K , PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, Spπnger-Verlag, New York (1984), incorporated herein by reference)

The pNA assay (DelMar, E G , C Largman, J W Brodπck and M C Geokas ANAL BIOCHEM , Vol 99, pp 316-320, (1979), incorporated herein by reference) is used to determine the active enzyme concentration for fractions collected during gradient elution This assay measures the rate at which p-nitroani ne is released as the enzyme hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-prolme-phenylalanine-p- nitroanilide (sAAPF-pNA) The rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a spectrophotometer and is proportional to the active enzyme concentration In addition, absorbance measurements at 280 nm are used to determine the total protein concentration The active enzyme/total-protein ratio gives the enzyme purity, and is used to identify fractions to be pooled for the stock solution

To avoid autolysis of the enzyme during storage, an equal weight of propylene glycol is added to the pooled fractions obtained from the chromatography column Upon completion of the purification procedure the

purity of the stock enzyme solution is checked with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the absolute enzyme concentration is determined via an active site titration method using trypsin inhibitor type ll-T: turkey egg white purchased from Sigma Chemical Company (St. Louis, Missouri). The measured conversion factors will show which changes made in the enzyme molecule at the various positions result in the enzyme variant having increased activity over the wild-type, against the soluble substrate pNA.

In preparation for use, the enzyme stock solution is eluted through a Sephadex-G25 (Pharmacia, Piscataway, New Jersey) size exclusion column to remove the propylene glycol and exchange the buffer. The MES buffer in the enzyme stock solution is exchanged for 0.1 M Tris buffer (Tris(hydroxymethyl-aminomethane) containing 0.01 M CaCl2 and pH adjusted to 8.6 with HCl. All experiments are carried out at pH 8.6 in Tris buffer thermostated at 25°C. H. Characterization of enzyme variants

Example 4 Model Surface Preparation

Aminopropyl controlled pore glass (CPG) purchased from CPG Inc. (Fairfield, New Jersey) is used as a support for covalently attaching the sAAPF-pNA substrate purchased from Bachem, Inc. (Torrence, California). The reaction is carried out in dimethyl sulfoxide and (1-ethyl-3-[3- (dimethylamino)propyl] carbodiimide hydrochloride) (EDC) is used as a coupling agent. Upon completion (monitored by pNA assay), the excess solvent is removed, and the CPG:sAAPF-pNA is rinsed with dimethyl sulfoxide (DMSO) and doubly-distilled water. This is followed by oven drying with a N2 purge at about 70°C. The reaction scheme and preparation of the immobilized substrate are conducted as described by Brode, P.F. Ill, and D.S. Rauch, "Subtilisin BPN': Activity on an Immobilized Substrate," LANGMUIR, Vol. 8, p. 1325-1329, (1992), incorporated herein by reference.

The CPG surface will have 62,000 ± 7,000 pNA molecules/μm ² . The surface area will remain unchanged from the value of 50.0m2/g reported by CPG Inc. for the CPG as received. This suggests that the procedure used to add sAAPF-pNA to CPG does not damage the porous structure (mean diameter is 486 A).

Example 5 Surface Hydrolysis Assay

Using CPG sAAPF-pNA, adsorption of an enzyme variant and hydrolysis of a CPG-bound peptide can be measured in a single experiment A small volume of enzyme variant stock solution is added to a flask containing Tris buffer and CPG sAAPF-pNA which has been degassed The flask is shaken on a wrist-action shaker for a period of 90 minutes during which the shaker is stopped at various time intervals (for example, every 2 minutes during the early stages of adsorption hydrolysis - e g , the first 20 minutes - and every 10 minutes towards the end of the experiment) The CPG sAAPF-pNA is allowed to settle and the solution is sampled Both the experimental procedure and the calculation of the adsorption and hydrolysis are conducted as described by Brode er al , 1992, above

All enzymes are monitored for stability against autolysis and should show no appreciable autolytic loss over the time course of this experiment Therefore, enzyme adsorption can be determined by measuring solution depletion The difference between the initial enzyme variant concentration and the concentration measured at each individual time point gives the amount of enzyme variant adsorbed The amount of pNA hydrolyzed from the surface is measured by taking an absorbance reading on an aliquot of the sample at 410 nm The total amount of pNA hydrolyzed is calculated by adding the amount sampled and the amount remaining in the flask This value is corrected by subtracting the amount of pNA that is hydrolyzed by Tris buffer at pH 8 6 when no enzyme is present This base-hydrolysis ranges from 7-29% of the total hydrolysis depending on the efficiency of the enzyme

Example 6 Soluble Substrate Kinetic Analysis

The rates of hydrolysis of the soluble substrate sAAPF-pNA are monitored by measuring the adsorbance increase as a function of time at 410 nm on a DU-70 spectrophotometer The enzyme concentration is held constant and is prepared to be in the range of 6-10 nanomolar while the substrate concentration is varied from 90-700 μM sAAPF-pNA for each kinetic determination An adsorbance data point is taken each second over a period of 900 seconds and the data are transferred to a LOTUS™ spreadsheet (Lotus Development Corporation, Cambridge, Massachusetts)

Analysis for kinetic parameters is conducted by the standard Lineweaver Burk analysis in which the data in the initial part of the run (generally the first minute) are fit to a linear regression curve to give v ₀ The v ₀ and s ₀ data are plotted in the standard inverse fashion to give KM and k _ca t I Example Subtilisin DY variants

Subtilisin DY variants of the present invention which have decreased adsorption to and increased hydrolysis of surface bound substrates are exemplified in Tables 2-36, below In describing the specific mutations, the original am o acid occurring in wild-type is given first, the position number second, and the substituted ammo acid third TABLE 2 Loop 1 - Single Mutation Variants

Thr58Asn Thr58Asp Thr58Gln Thr58Glu Thr58Gly Thr58Pro Thr58Ser Asp59Glu GlyβOAsn GlyβOAsp GlyβOGln GlyβOGlu GlyβOPro GlyβOSer AsnβlAsp AsnβlGln AsnβlGlu AsnβlSer Gly62Asn Glyβ2Asp Gly62Gln Gly62Glu Gly62Pro

Thr65Asp Thr65Gln Thr65Glu Thr65Gly

Thr65Pro

TABLE3

Loop 1 - Double Mutation Variants

TABLE 4

Loop 1 - Triple Mutation Variants

Thr58Asn + GlyβOGln + Gly64Pro Thr58Gly + AsnβlSer + ThrβδSer Asp59Glu + GlyβOPro + Thr65Ser

A.snβlGlu + Gly62Pro + Gly64Asn Thr58Asp + GlyβOAsn + Thr65Gln T r58Ser + Gly62Ser + Thr65Gly Thr58Pro + Asp59Glu + Gly62Pro GlyβOGlu + AsnβlGln + ThrβδGln GlyβOGlu + Gly64Asn + Thrβ5Pro GlyβOAsn + Gly62Asp + Thr65Ser Thr58Asn + AsnβlGlu + Thr65Gln Thr58Asn + Asp59Glu + Glyβ2Pro GlyβOGln + Glyβ2Gln + Gly64Glu AsnβlGln + Gly62Pro + Thr65Ser AsnβlGln + Gly62Asn + Thr65Gln AsnβlSer + Gly62Gln + Thr65Gly GlyβOSer + Gly64Gln + Thr65Gly Thr58Pro + GlyβOPro + Gly64Gln Thr58Asn + GlyβOAsn + Thr65Pro Asp59Glu + Glyβ2Gln + Thr65Asn Thr58Ser + GlyβOGln + Gly62Asp Thr58Asn + Gly64Ser + Thr65Asn GlyβOPro + Glyβ2Asp + Thr65Gln AsnβlGlu + Gly64Ser + ThrβδGly Thr58Pro + AsnβlAsp + Gly64Gln Asp59Glu + AsnβlSer + Gly62Ser Thr58Asn + GlyβOAsp + Thrβ5Asn Asp59Glu + AsnβlGln + Gly62Asn Thr58Asn + GlyβOAsp + AsnβlAsp Thr58Asn + GlyβOGlu + AsnβlGlu

TABLE 5

Loop 1 - Quadruple Mutation Variants

GlyβOGlu -r AsnolGlu + Gly62Glu + Thr65Gly

GlyβOAsp + Asn61A.sp + Gly62Asp + Thr65Gln

Thr58Asp + Asp59Glu + GlyβOGlu + ThrβδSer

Asp59Gl + AsnβlAsp + Gly62A-sp + Thr65Gln

Asp59Glu + AsnβlAsp + Gly62Glu + ThrβδGly

Asp59Glu + GlyβOAsp + Gly62Asp + ThrβδGln

Thr58Ser + Asp59Glu + GlyβOAsp + Gly62Asp

TABLE6

Loop 2 - Single Mutation Variants

Val 94Ala Val 94Asn Val 94Asp Val 94Cys Val 94Gln Val 94Glu Val 94Gly Val 94His Val 94Met Val 94Pro Val 94Ser Val 94Thr Leu 95Ala Leu 95Asn Leu 95Asp Leu 95Cys Leu 95Gln Leu 95Glu Leu 95Gly Leu 95Hιs Leu 95Ile Leu 95Met Leu 95Pro Leu 95Ser Leu 95Thr Leu 95Val Asn 96Asp Asn 96Gln Asn 96Glu Asn 96Ser Ser 97Asp Ser 97Glu Ser 98Asp Ser 98Glu Gly 99Asn Gly 99Asp Gly 99Gln Gly 99Glu Gly 99Pro Gly 99Ser SerlOOAsp

SerlOOGlu GlylOlAsn GlylOlAsp GlylOlGln GlylOlGlu GlylOlPro GlylOlSer Thrl02Asn Thrl02Asp Thrl02Gln Thrl02Glu Thrl02Gly Thrl02Pro Thrl02Ser Tyrl03Ala Tyrl03Asn Tyrl03Asp Tyrl03Cys Tyrl03Gln Tyrl03Glu Tyrl03Gly Tyrl03His Tyrl03Ile Tyrl03Leu Tyrl03Met Tyrl03Pro Tyrl03Ser Tyrl03Thr Tyrl03Val Serl04Asp Serl04Glu Alal05Asn Alal05Asp Alal05Gln Alal05Glu Alal05Gly Alal05His Alal05Pro Alal05Ser Alal05Thr IlelOβAla IlelOβAsn IlelOβAsp IlelOβCys IlelOβGln IlelOβGlu IlelOβGly IlelOβHis IlelOβLeu IlelOβMet IlelOβPro IlelOβSer IlelOβThr

IlelOβVal

TABLE 7

Loop 2 - Double Mutation Variants

Val 94Thr 96Asp

Ser 97Glu O ^β Leu

Thrl02Asp 03Met

Ser 97Asp 05Kis

Asn 96Gln OOAsp

GlylOlAsn OβGln

Asn 96Glu 03Ser

Tyrl03Thr OβPro

SerlOOAsp 03Ile

GlylOlGlu 03Leu

SerlOOAsp 02Asn

Val 94His 95Thr

Serl04Asp 05Asn

TABLE 8

Loop 2 - Triple Mutation Variants

Val 94Gln SerlOOGlu

Ser 98Asp IlelOβAsn Val 94Gly IlelOβVal Val 94Ser Serl04Glu Val 94His IlelOβGly Val 94Pro IlelOβCys Val 94Thr IlelOβCys Val 94Met GlylOlSer Val 94Ala Gly 99Gln Leu 95Gly Alal05Gly Val 94Cys Tyrl03Ala GlylOlAsn IlelOβAla Gly 99Ser IlelOβVal Asn 96Gln Tyrl03Thr Leu 95Met IlelOβLeu Asn 96Glu Alal05His SerlOOGlu Tyrl03Val Ser 98Glu IlelOβMet Asn 96Asp Alal05Thr GlylOlAsp Tyrl03Thr Val 94Ala Tyrl03His Thrl02Gly Tyrl03Glu + Alal05Gly Ser 97Glu GlylOlSer Thrl02Gln Val 94Pro Gly 99Ser GlylOlAsp Ser 97Asp Gly 99Ser IlelOβGly Val 94Cys Leu 95Val GlylOlGln SerlOOAsp GlylOlAsn Tyrl03Gln Thrl02Asp Tyrl03Leu IlelOβVal Val 94Cys Serl04Glu AlalOδAsn

Val 94Ser Thrl02Asn Alal05Thr Ser 98Glu Gly 99Asn Tyrl03Val Leu 95Val GlylOlGln Alal05Asp Ser 97Glu Gly 99Pro Thrl02Ser Val 94Ser GlylOlGlu + IlelOβAla Gly 99Asn GlylOlAsp + Thrl02Glu

TABLE 9

Loop 2 - Quadruple Mutation Variants

Thrl02Glu + Tyrl03Val Tyrl03Ile + Alal05Thr SerlOOGlu + Thrl02Ser GlylOlPro + IlelOβAla Tyrl03Ile + Alal05Ser Gly 99Pro + SerlOOGlu Tyrl03Ile + AlalOδSer Thrl02Ser + Alal05Pro Serl04Asp + Alal05Gln SerlOOGlu + IlelOβSer Tyrl03Gly + Alal05Pro Tyrl03Leu + AlalOSPro Gly 99Asn + Tyrl03Thr Ser 97Asp + Thrl02Asn Thrl02Ser + IlelOβLeu Alal05Pro + IlelOβThr Tyrl03Gln + IlelOβHis GlylOlGln + Alal05Thr Asn 96Gln + Serl04Glu SerlOOGlu + Thrl02Pro GlylOlAsp + Tyrl03Ser Serl04Asp + Alal05Asp Tyrl03Met + Alal05Pro

95Ser Ser 97Asp Ser 98Asp 02Ser ber 97Glu Ser 98Glu Alal05Pro OβAsn Ser 97Glu Ser 98Glu GlylOlSer OβCys Ser 97Asp Ser 98Asp GlylOlPro 02Pro

Leu 95Glu SerlOOGlu GlylOlAsp 02Ser Val 94Me Leu 95Glu Asn 96Glu 01Asp

J-JΘU 95Hιs Gly 99Glu SerlOOGlu OlGlu

As 96Glu Gly 99Glu GlylOlSer OβAsn Leu 95Val Asn 96Asp Ser 98Glu 03Gln Asn 96Asp Ser 98Glu Gly 99Ser 02Asn Asn 96Glu Ser 98Glu GlylOlSer 02Pro Leu 95Val Asn 96Glu Ser 98Asp OβAla Val 94Gly Asn 96Glu Ser 98Asp 03Thr Ser 98Glu Gly 99Gln SerlOOAsp OlPro Ser 98Glu Gly 99Gln SerlOOAsp 02Gln Asn 96Glu SerlOOGlu Tyrl03Gln OβPro Asn 96Asp Gly 99Asn SerlOOGlu 03Ile Leu 95Glu Asn 96Gln Ser 98Glu 99Glu 97Asp Ser 98Glu SerlOOAsp OβVal 97Asp Ser 98Asp SerlOOGlu 02Ser 97Asp Ser 98Asp + SerlOOGlu OlGln

Leu 95Thr Ser 97Asp + Ser 98Glu OOAsp Val 94Met Asn 96Glu + Gly 99Asp OlAsp Ser 98Asp SerlOOGlu + GlylOlGlu 05Ser Val 94Cys Ser 98Asp + SerlOOAsp OlAsp Val 94Glu SerlOOGlu + GlylOlGlu 03Ile Leu 95Glu Ser 97Asp + Ser 98Asp 05Ser Leu 95Ala Asn 9βAsp + GlylOlAsp 02Asp Val 94Cys Gly 99Asp + GlylOlAsp βPro Lea 95Ser SerlOOGlu + GlylOlGln 2Glu Leu 95Met SerlOOAsp + Thrl02Glu βGln Asn 96Ser GlylOlAsp + Tyrl03Asn βAsp Leu 95Cys GlylOlGlu + Alal05Pro βGlu Val 94Asp Gly 99Glu + SerlOOAsp 5Ser Ser 97Glu Ser 98Glu + GlylOlAsp 3Ser Ser 97Asp SerlOOGlu + Thrl02Gln 3Thr Ser 97Glu SerlOOAsp + Tyrl03Ser 5Gly

TABLE 10

Loop 3 - Single Mutation Variants

Leul25Ala Leul25Asn Leul25Asp Leul25Cys Leul25Gln Leul25Glu Leul25Gly Leul25His Leul25Ile Leul25Met Leul25Pro

Leul25Ser Leul25Thr Leul25Val Glyl26Asn Glyl26Asp Glyl26Gln Giyl26Glu Glyl26Pro Glyl26Ser Glyl27Asn Glyl27Asp Glyl27Gln Giyl27Glu Glyl27Pro Glyl27Ser Prol28Asn Prol28Asp Prol28Gln Prol28Glu Prol28Gly Prol28Ser Serl29Asp Serl29Glu Glyl30Asn Glyl30Asp Glyl30Gln Glyl30Glu Glyl30Pro Glyl30Ser Serl31Asp Serl31Glu Thrl32Asn Thrl32Asp Thrl32Gln Thrl32Glu Thrl32Gly Thrl32Pro Thrl32Ser

TABLE 11

Loop 3 - Double Mutation Variants

Leul25Gln + Serl29Glu

TABLE 12

Loop 3 - Triple Mutation Variants

Glyl27Pro Serl31Glu Thrl32Asp Leul25Asn Serl31Asp Thrl32Asp Prcl28Gln Serl31Asp Thrl32Glu Prcl28Gly Serl31Asp Thrl32Asp Glyl26Gln Serl29Asp Glyl30Asp Leul25Ile Serl29Asp Glyl30Glu Glyl27Glu Prol28Asp Serl29Glu Serl29Asp Glyl30Asp Serl31Asp Serl29Glu Glyl30Asp Serl31Glu Serl29Glu Glyl30Glu Serl31Asp Gl l26Glu Glyl27Glu Prol28Glu Glvl30Glu Serl31Asp Thrl32Asp

TABLE 13

Loop 3 - Quadruple Mutation Variants

Glyl2βSer + Prol28Glu + Glyl30Ser + Thrl32Ser Leul25Cys - Glyl2 ^"7 Gln + Serl29Glu + Thrl32Pro Leul25Glr. + Glyl26Pro + Prol28Ser + Serl29Glu Leul25Thr ^■ > ^■ Serl29Glu + Glyl30Ser + Thrl32Pro GIyl27Gln + Prol28Gln + Serl29Glu + Glyl30Pro Glyl27Asn + Prol28Asn + Serl31Glu + Thrl32Gly Leul25Met + Glyl27Glu + Glyl30Asn + Thrl32Gly Leul25Met + Glyl27Asn + Prol28Asn + Serl29Glu Leul25Met + Glyl26Ser + Glyl27Gln + Serl31Asp Leul25Asn + Glyl26Asp + Glyl27Gln + Thrl32Ser Leul25Gln + Glyl27Gln + Glyl30Glu + Thrl32Pro Leul25Ala + Glyl30Asn + Serl31Asp + Thrl32Pro Leul25Met + Glyl26Pro + Glyl27Ser + Serl31Asp Leul25Cys + Glyl26Pro + Serl29Glu + Thrl32Asn LeuI25Gly -r Prol28Gly + Serl31Asp + Thrl32Gln Leul25Met + Glyl30Asn + Serl31Asp + Thrl32Gln Leul25Val + Glyl26Pro + Glyl30Gln + Thrl32Asp Glyl2βAsn + Glyl27Pro + Prol28Gln + Serl29Glu Leul25Gln + Glyl26Glu + Glyl27Glu + Glyl30Asn Leul25Val + Glyl26Asp + Glyl27Glu + Glyl30Pro Leul25Asp + Glyl26Glu + Glyl27Pro + Thrl32Pro Leul25Glu + Glyl26Glu + Glyl27Asn + Thrl32Ser Leul25Gln + Glyl27Pro + Glyl30Glu + Serl31Asp Leul25Cys + Glyl30Glu + Serl31Asp + Thrl32Gln Glyl26Gln + Glyl27Glu + Prol28Glu + Glyl30Asn Glyl26Ser + Glyl27Asp + Prol28Asp + Glyl30Ser Leul25Thr + Glyl27Ser + Serl31Glu + Thrl32Asp Prol28Asn + Glyl30Asn + Serl31Asp + Thrl32Glu Leul25Met + Glyl26Ser + Serl29Asp + Glyl30Asp Glyl2βGln + Prol28Gln + Serl29Glu + Glyl30Glu Glyl27Asn + Prol28Asn + Serl29Glu + Glyl30Glu Glyl27Ser + Serl29Glu + Glyl30Asp + Thrl32Gln Glyl27Asp + Prol28Glu + Serl29Asp + Glyl30Asn Glyl27Asp + Prol28Glu + Serl29Asp + Thrl32Ser Leul25Gln + Serl29Glu + Glyl30Asp + Serl31Asp Leul25Cys -r Serl29Glu + Glyl30Asp + Serl31Glu

Glyl2 Prol28Glu Serl29Glu Glyl30Asp Leul2 Glyl26Asp Glyl27Glu Prol28Glu Glyl2 Prol28Gln Serl29Glu Thrl32Ser Leul2 Glyl27Asp Prol28Ser Serl29Asp Glyl2 Glyl27Glu Prol28Gly Serl29Asp Leul2 Glyl27Asp Serl29Glu Glyl30Glu Glyl2 Prol28Ser Serl29Glu Glyl30Asp Glyl2 Serl29Glu Glyl30Glu Thrl32Pro Leul2 Glyl27Glu Serl29Asp Glyl30Asp Glyl2 Glyl27Asp Serl29Glu Glyl30Pro Glyl2 Glyl27Glu Serl29Asp Thrl32Pro Leul2 Glyl26Asp Glyl27Asp Serl29Asp Glyl2 Serl29Asp Serl31Glu Thrl32Gln Glyl2 Prol28Asn Serl29Asp Serl31Asp Prol2 Serl29Glu Serl31Asp Thrl32Ser Glyl2 Serl29Asp Serl31Glu Thrl32Ser Leul2 Serl29Glu Glyl30Pro Serl31Asp GIyl2 Serl29Glu Glyl30Ser Serl31Asp Serl29Glu Serl31Glu Thrl32Gln

Glyl2 Prol28Gln Serl29Glu Serl31Asp Leul2 Glyl26Ser Serl29Asp Serl31Glu Glyl2 Prol28Gln Serl29Asp Serl31Asp Glyl2 Prol28Ser Serl29Asp Serl31Glu Glyl2 Prol28Asp Serl2 Asp Thrl32Asn

TABLE 14

Loop 4 - Single Mutation Variants

Glyl53Asn Glyl53Asp Glyl53Gln Glyl53Glu Glyl53Pro Glyl53Ser Asnl54Asp Asnl54Gln Asnl54Glu Asnl54Ser Serl55Asp Serl55Glu Glyl56Asn Glyl56Asp GlylδβGln Glyl56Glu Glyl56Pro Glyl56Ser Serl57Asp Serl57Glu Serl58Asp Serl58Glu Glyl59Asn Glyl59Asp

Glyl59Gin Glyl59Glu Glyl59Pro Glyl59Ser SerlβOAsp SerlβOGlu GlnlβlAsn GlnlβlAsp GlnlβlGlu GlnlβlSer Asnl62Asp Asnl62Gln Asnl62Glu Asnl62Ser Thri63Asn Thrl63Asp Thrl63Gln Thrl63Glu Thrl63Gly Thrl63Pro Thrl63Ser Ilel64Ala Ilel64Asn Ilelβ4Asp Ilel64Cys Ilel64Gln Ilel64Glu Ilel64Gly Ilel64His Ilel64Leu Ilel64Met Ilel64Pro Ilelβ4Ser Ilel64Thr Ilel64Val Glyl65Asn Glyl65Asp GlylβδGln Glyl65Glu GlylβδPro Glyl65Ser TyrlββAla TyrlββAsn TyrlββAsp TyrlββCys TyrlββGIn TyrlββGlu TyrlββGly TyrlββHis Tyrlββlle TyrlββLeu TyrlββMet TyrlββPro

TyrlββSer T rlββThr TvrlββVal

TABLE 15

Loop 4 - Double Mutation Variants

Glyl59Glu Glyl65Gln SerlβOGlu Ilel64His Glyl53Ser Serl60Asp Glyl59Gln Glyl65Glu Glyl53Glu Glyl59Gln Glyl56Glu Thrl63Gly Glyl56Ser TyrlββAsn Glyl53Asp Thrl63Asn Asnl54Ser Serl57Glu Thrl63Ser Ilel64Pro Serl55Glu Asnl62Ser Glyl56Pro Ilel64Gly Thrl63Glu Glyl65Asn Glyl53Gln Serl55Glu Ilel64Ser TyrlββCys

TABLE 16

Loop 4 - Triple Mutation Variants

Serl55Asp + Glyl56Asp Asnl62Ser Serl57Asp + Serl58Glu Glyl59Pro SerlβOAsp + Glnl ^β lAsp Ilel64Ala Glyl59Gln + Serl ^β OAsp Glnl61Asp Glyl56Asp + Serl57Asp Serl58Asp Giyl59Asp + GlnlβlGlu Glyl65Gln Glyl53Pro + Glyl59Glu GlnlβlGlu Serl57Glu + Serl58Asp GlnlβlGlu Serl57Glu + Glyl59Glu Glyl65Ser Serl57Glu + Glyl59Glu SerlβOGlu Serl58Asp + SerlβOAsp Ilel64Thr Serl58Asp + SerlβOGlu TyrlββVal Asnl54Gln + Serl58Asp SerlβOAsp Serl58Glu + SerlβOAsp TyrlββHis Glyl53Asp + Asnl54Glu Glyl65Glu Serl55Asp + Serl57Asp Tyrlββlle Serl55Glu + Serl57Asp Glyl59Pro Glyl53Asn + Serl57Glu Thrl63Glu Serl55Asp + Serl57Asp Serl58Glu Serl57Asp + Glyl59Asp GlnlβlGlu Serl55Asp + GlylδβGlu Serl58Asp Asnl54Gln + SerlβOGlu Asnl62Asp Serl58Glu + SerlβOGlu Asnl62Glu Serl57Glu + GlnlβlAsp Thrl63Asp Asnl54Glu + Serl55Glu Serl57Glu Serl57Asp + SerlβOGlu TyrlββGly

Glyl53Asn + Serl57Glu + SerlβOAso

TABLE 17

Loop 4 - Quadruple Mutation Variants

Serl58Asp + Glyl59Glu + GlnlβlSer + Ilel64Thr

Glyl53Gln + Asnl54Glu + Serl55Glu + TyrlββGly

Glyl53Ser + Serl55Asp + Glyl56Asp + Ilel64Gln

Asήl54Ser + Serl55Glu + Glyl56Asp + TyrlββVal

Serl55Glu + Glyl56Glu + Thrl63Asn + Ilel64Leu

Serl57Glu + Serl58Asp + GlnlβlAsn + Ilel64Cys

SerlβOGlu + GlnlβlAsp + Thrl63Gln + Glyl65Pro

Serl57Asp + Serl58Glu + Glyl59Asp + Ilel64Cys

Asnl54Gln + SerlβOGlu + GlnlβlGlu + Asnl62Asp

Glyl53Glu + Glyl59Gln + GlnlβlAsn + Glyl65Glu

Asnl54Ser + Serl58Asp + Glyl59Asn + GlnlβlGlu

Asnl54Glu + Glyl56Asp + Thrlβ3Ser + TyrlββAsn

Serl58Glu + SerlβOAsp + GlnlβlAsn + Thrl63Pro

Asnl54Glu + Serl55Asp + Glyl56Gln + Thrl63Asp

Serl58Glu + GlnlβlGlu + Thrl63Asp + Ilel64His

Serl55Glu ÷ Serl57Asp + Asnl62Ser + Glyl65Pro

Serl55Asp -r SerI57Asp + Glyl59Pro + Ilel64Cys

Glyl59Glu + SerlβOAsp + Asnl62Glu + Ilel64Asn

Serl57Glu + GlnlβlAsp + Thrl63Asp + Glyl65Gln

Asnl54Glu + Serl55Glu + Serl57Asp + Glyl59Pro

Asnl54Gln + SerlβOAsp + GlnlβlAsp + Thrl63Asp

SerlβOAsp + GlnlβlAsp + Asnl62Gln + Thrl63Asp

Serl55Asp + Thrlβ3Asp + Ilel64Gly + GlylβδAsp

Glyl56Ser + Serl58Asp + Thrl63Asp + Ilelβ4Leu

Serl55Glu + Serl58Glu + Thrl63Glu + Ilel64Met

Glyl53Glu + GlylδβPro + Thrlβ3Asp + TyrlββHis

Glyl56Ser + Serl57Asp + SerlβOGlu + Asnl62Asp

Glyl53Asn + Serl57Glu + Asnl62Asp + Ilel64Leu

Glyl53Glu + Serl55Glu + Serl57Asp + Ilel64Met

GlylδβPro + Serl57Asp + Glyl59Gln + SerlβOGlu

Glyl53Pro + Serl55Glu + GlylδβGlu + GlylβδAsp

Serl55Asp + Serl58Glu + GlnlβlAsn + GlylβδPro

Serl55Asp + Serl58Glu + Glyl59Gln + Ilel64Gly

Glyl53Asp + Serl57Glu + Glyl59Pro + Thrl63Glu

Serl55Asp + GlylδβPro + GlnlβlSer + GlylβδAsp

Glyl56Glu + Serl57Glu + SerlβOAsp + Ilel64Ser

Asnl54Ser + Glyl56Asp + Serl58Asp + SerlβOAsp

Glyl53Gln + Serl55Asp + Serl58Asp + Glyl59Glu

Asnl54Asp + GlylδβGlu + Serl58Glu + Ilel64Ala

TABLE 18

Loop 5 - Single Mutation Variants

AlalδβAsn Alal86Asp Alal86Gln AlalδβGlu AlalδβGly Alal86His AlalδβPro Alal86Ser Alal86Thr

Serl87Asp Serl87Glu Phel88Ala Phel88Asn Phel88Asp Phel88Cys Phel88Gln PhelδδGlu PhelδβGly Phel88His Phelδδlle PhelδδLeu PhelδδMet Phel88Pro Phel88Ser Phel88Thr PhelδδTyr PhelδδVal Serl89Asp Serl89Glu Serl90Asp Serl90Glu

TABLE 19

Loop 5 - Double Mutation Variants

Serl87Glu + Phel88Gly Alal86Glu + Phel88Asn Serl87Glu + Phel88His AlalδβSer + Phel88Asp Alal86Gly + Serl87Asp Phel88Ile + Serl90Glu Alal86Thr + Serl90Glu Alal86Gly + Serl90Glu Phel88His + Serl90Asp Phel88Leu + Serl90Asp Alal86Ser + Serl90Asp Serl67Glu + Phel88Ile AlalδβThr + Phel88His PhelδδGln + Serl90Glu AlalδβHis + Serl87Asp Phel88Tyr + Serl90Glu Alal86Pro + PhelδδThr AlalδβGln + Serl87Asp Serl87Glu + PhelδδAla AlalδβAsp + Phel88Ile Alal86Ser + Phel88Pro Alal86Asn + Serl87Glu Phelδ8His + Serl90Glu Alal86Glu + Phel8δSer AlalδβSer + Phel88Leu PhelδδPro + Serl90Glu Serl87Asp + PhelδδThr AlalδβHis + PhelδδAla AlalδβAsn + Phel88Asp Phelδδlle + Serl90Asp AlalδβThr + PhelδδMet AlalδβAsp + PhelδδHis Serlδ7Asp + PhelδδAla AlalδβPro + Serl90Glu

TABLE20

Loop 5 - Triple Mutation Variants

AlalδβHis + Phel88Ala + Serl90Asp AlalδβAsn + Serl87Asp + PhelδδAla AlalδβGly + PhelδδAsn + Serl90Asp AlalδβPro + PhelδδMet + Serl90Asp AlalδβGly + Phel68Leu + Serl90Glu Alal86Gln + Serl87Asp + Phelδδlle AlalδβThr + Serlδ7Glu + PhelδδCys AlalδβSer + PhelδδAla + Serl90Glu AlalδβGln + Serl87Asp + PhelδδSer AlalδβGln + Serl87Glu + Phelδδlle AlalδβSer + PhelδδGly + Serl90Asp AlalδβSer + Serlδ7Asp + PhelδδMet AlalδβGly + Serlδ7Glu + PhelδδGly AlalδβGln + PhelδδPro + Serl90Asp

Alal8βAsn + PhelδδAla Serl90Glu Alal86Ser + Phelδ8Pro Serl90Glu Alal8βAsn Serl87Glu PhelδδPro AlalδβGln Phel88Gly Serl90Glu Alal8βAsn Serl87Glu Phelδδlle Alal8βHis Serlδ7Glu + PhelδδGly AlalSβHis PhelδδCys + Serl90Asp Alal8βThr Serlδ7Glu + PhelδδVal Alal86Gln Serlδ7Glu + PhelδδMet Alal86His PhelδδAla + Serl90Glu Alal8βGly PhelδδMet + Serl90Glu Alal8βSer + Serlδ7Glu + PhelδδHis Alal8βAsn + PhelδδGly + Serl90Glu Alal8βAsn + PhelδδPro Serl90Asp Alal8βAsn + PhelδδTyr Serl90Asp Alal8βGly + Phelδ8Thr Serl90Glu Alal86His + Serlδ7Asp PhelδδLeu Alal8βSer + Phe188Pro + Serl90Asp AlaloβThr + Serlδ 7Glu + PhelδδPro AlalδβPro + PhelδδPro + Serl90Glu Alal86Thr + PhelδβAsn + Serl90Asp Alal86Pro + Serlδ7Glu + Phel88His Alal86Asp + Serlδ7Glu Phel86Met AlalδβAsp Serlδ7Glu PhelδδGln AlalδβAsp Serlδ7Glu PhelδδCys AlalδβGlu Serlδ7Asp PhelδδPro AlalδβAsp Serlδ7G1U PhelδβTyr Alal86Glu Serlδ7Asp PhelδδThr Alal8βAsp Serlδ7Asp PhelδδVal Alal8βGlu Serlδ7Glu PhelδδAsn AlalSβAsp Serlδ7Asp PhelδδHis Alal8βAsp Serlδ7Glu Phel88Ala Alal86His Serlδ7Asp Phe18δAsp AlalδβGly Serlδ7Asp PhelδδAsp AlalδβGln Serlδ7Glu PhelδδGlu AlalδβAsn Serlδ7Asp PhelδδGlu Alal86Pro Serlδ7Glu PhelδδAsp Alal86Asp Serlδ7Glu PhelδδAsp Alal8βGlu Serlδ7Glu PhelδδGlu AlalSβAsp Serlδ7Asp PhelδδGlu Alal86Thr PhelδδAsp Serl90Glu AlalδβSer + Phel88Glu Serl90Glu AlalδβThr + Phe188Glu Serl90Asp AlalδβPro + Phel8δGlu Serl90Glu Serlδ7Glu + PhelδβGlu Serl90Glu Serlδ7Asp + PhelδδAsp Serl90Glu

TABLE21

Loop 5 - Quadruple Mutation Variants

AlalδβGly Serlδ7Glu PhelδδAsp Serl90Glu AlalδβGly Serlδ7Asp PhelδδAsp Serl90Asp

AlalδβGln + Serlδ7Asp + PhelδδAsp + Serl90Asp AlalδβGln + Serl67Glu + PhelδδAsp + Serl90Glu AlalδβSer + Serl87Asp + Phel88Glu + Serl90Glu AlalδβPro + Serl67Glu + PhelδδAsp + Serl90Asp Alal86Thr + Serl87Glu + Phel88Asp + Serl90Asp Alal86Thr + Serl87Glu + Phel88Asp + Serl90Glu Alal86Hιs + Serl87Asp + Phel88Glu + Serl90Asp AlalδβAsn + Serl87Glu + PhelδδAsp + Serl90Asp AlalδβGly + Serl87Glu + Phel88Glu + Serl90Asp AlalδβGln + Serlδ7Asp + Phelδ8Glu + Serl90Glu AlalδβGln + Serlδ7Asp + PhelδδGlu + Serl90Asp AlalδβGly + Serl87Glu + PhelδδAsp + Serl90Asp AlalδβAsp + Serl87Glu + PhelδδGly + Serl90Asp AlalδβAsp + Serlδ7Asp + PhelδδGln + Serl90Asp AlalδβGlu + Serlδ7Glu + PhelδδVal + Serl90Asp Alal86Asp + Serl87Glu + PhelδδPro + Serl90Glu Alalδ ^β Glu + Serl87Asp + Phel88Met + Serl90Glu AlalδβGlu + Serl87Asp + PhelδδAla + Serl90Glu AlalδβAsp + Serl87Asp + PhelδδHis + Serl90Asp Alal86Glu + Serl87Glu + Phel88Met + Serl90Glu Alal86Asp + Serlδ7Asp + PhelδδAla + Serl90Glu AlalδβGlu + Serlδ7Glu + PhelδδCys + Serl90Asp AlalδβGlu + Serl87Glu + PhelδδThr + Serl90Glu Alalδ ^β Asp + Serlδ7Asp + PhelδδHis + Serl90Glu AlalδβAsp + Serlδ7Asp + PhelδδGln + Serl90Glu AlalδβAsp + Serl87Asp + PhelδδTyr + Serl90Asp AlalδβGlu + Serlδ7Asp + PhelδδSer + Serl90Asp AlalδβGlu + Serl87Asp + PhelδδGly + Serl90Asp AlalδβAsp + Serl87Asp + PhelδδAsn + Serl90Asp AlalδβGlu + Serlδ7Glu + PhelδδCys + Serl90Glu AlalδβGlu + Serlδ7Asp + Phelδδlle + Serl90Glu Alal86Glu + Serlδ7Glu + PhelδδVal + Serl90Glu AlalθβAsp + Serl87Asp + PhelδδCys + Serl90Asp AlalδβAsp + Serlδ7Glu + PhelδδVal + Serl90Glu AlalδβAsp + Serl67Glu + PhelδδSer + Serl90Asp Alal86Asp + Serl87Asp + PhelδδAla + Serl90Asp AlalδβAsp + Serl87Asp + Phel88Gly + Serl90Glu AlalδβAsp + Serl67Asp + Phel88Thr + Serl90Glu AlalδβGlu + Serlδ7Glu + PhelδδHis + Serl90Glu AlalδβGlu + Serlδ7Asp + PhelδδPro + Serl90Glu AlalδβGlu + Serlδ7Glu + Phelδδlle + ^• Serl90Glu AlalδβGlu + Serl87Glu + PhelδδPro + Serl90Glu AlalδβGlu + Serl87Glu + PhelδδGly + Serl90Asp AlalδβGlu + Serl67Glu + PhelδδPro + Serl90Asp AlalδβGlu + Serlδ7Asp + PhelδδSer + Serl90Glu AlalδβAsp + Serl87Asp + PhelδδTyr + Serl90Glu AlalδβAsp + Serlδ7Glu + PhelδδHis + Serl90Glu AlalδβSer + Serl87Glu + PhelδδSer + Serl90Glu AlalδβHis + Serlδ7Glu + PhelδδSer + Serl90Glu AlalδβSer + Serl87Asp + PhelδδCys + Serl90Glu AlalδβSer + Serlδ7Glu + PhelδδAsn + Serl90Glu AlalδβGln + Serlδ7Glu + PhelδδAla + Serl90Asp AlalδβGln + Serlδ7Asp + PhelδδMet + Serl90Glu

AlalδβAsn Serl87Glu PhelδδHis Serl90Asp AlalδβThr Serl67Glu Phelδ8Ala Serl90Glu AlalδβPro Serl87Asp PhelδδCys Serl90Glu Alal86Pro Serl87Asp PhelδδLeu Serl90Asp Alal86Asn Serl87Asp PhelδδGln Serl90Glu

TABLE 22

Loop 6 - Single Mutation Variants

Alal99Asn Alal99Asp Alal99Gln Alal99Glu Alal99Gly Alal99His Alal99Pro Alal99Ser Alal99Thr Pro200Asn Pro2OOAsp Pro200Gln Pro200Glu Pro200Gly Pro200Ser Gly201Asn Gly201Asp Gly201Gln Gly201Glu Gly201Pro Gly201Ser Val202Ala Val202Asn Val202Asp Val202Cys Val202Gln Val202Glu Val202Gly Val202His Val202Met Val202Pro Val202Ser Val202Thr Ser203Asp Ser203Glu Val204Ala Val204Asn Val204Asp Val204Cys Val204Gln Val204Glu Val204Gly Val204His

Val204Met Val204Pro Val204Ser Val204Thr Tyr205Ala Tyr205Asn Tyr205Asp Tyr205Cys Tyr205Gln Tyr205Glu Tyr205Gly Tyr205His Tyr205Ile Tyr205Leu Tyr205Met Tyr205Pro Tyr205Ser Tyr205Thr Tyr205Val Ser206Asp Ser206Glu Thr207Asn Thr207Asp Thr207Gln Thr207Glu Thr207Gly Thr207Pro Thr207Ser Tyr208Ala Tyr20δAsn Tyr208Asp Tyr208Cys Tyr208Gln Tyr20δGlu Tyr20δGly Tyr208His Tyr208Ile Tyr208Leu Tyr208Met Tyr208Pro Tyr208Ser Tyr208Thr Tyr20δVal Pro209Asn Pro209Asp Pro209Gln Pro209Glu Pro209Gly Pro209Ser Ser210Asp Ser210Glu Asn211Asp Asn211Gln

Asn211Glu

Asn211Ser

Thr212Asn

Thr212Asp

Thr212Gln

Thr212Glu

Thr212Gly

Thr212Pro

Thr212Ser

Tyr213Ala

Tyr213Asn

Tyr213Asp

Tyr213Cys

Tyr213Gln

Tyr213Glu

Tyr213Gly

Tyr213His

Tyr213Ile

Tyr213Leu

Tyr213Met

Tyr213Pro

Tyr213Ser

Tyr213Thr

Tyr213Val

Thr214Asn

Thr214Asp

Thr214Gln

Thr214Glu

Thr214Gly

Thr214Pro

Thr214Ser

Ser215Asp

Ser215Glu

Leu216Ala

Leu216Asn

Leu216Asp

Leu216Cys

Leu216Gln

Leu216Glu

Leu216Ile

Leu216Met

Leu216Pro

Leu216Ser

Leu216Thr

Leu216Val

Asn217Asp

Asn217Gln

Asn217Glu

Asn217Ser

Gly21δAsn

Gly216Asp

Gly21δGln Gly21δGlu Gly21δPro Gly2l8Ser Thr219Asn Thr219Asp Thr219Gln Thr219Glu Thr219Gly Thr219Pro Thr219Ser

TABLE23

Loop 6 - Double Mutation Variants

TABLE 24

Loop 6 - Triple Mutation Variants

AIal99Thr + Asn211Asp + Gly218Ser Thr207Gln + Tyr213Gln + Leu216Met Aial99Gly + Val202Cys + Ser210Asp Val204Gln + Tyr213Ala + Thr214Pro Tyr213Leu + Asn217Ser + Thr219Glu Pro200Gly + Thr214Pro + Asn217Asp Pro200Gly + Leu216Val + Thr219Gln Pro200Gly + Gly201Ser + Ser210Asp Gly201Gln + Val202His + Thr212Glu Tyr206His + Asn211Ser + Asn217Asp Thr207Ser + Ser210Asp + Gly21δSer Thr212Asn + Leu216Asn + Thr219Glu Val202Pro + Val204Met + Pro209Asp Val202Asn + Thr212Pro + Thr214Glu Tyr20δlle + Thr212Asp + Tyr213His Ser210Asp + Thr212Ser + Leu216Ala Pro200Gln + Thr214Pro + Leu216His Tyr20δMet + Pro209Asp + Leu216Ile Val202Met + Leu216Gly + Thr219Gly Pro200Ser + Tyr205Val + Leu216Cys Val204Thr + Tyr205Glu + Asn217Gln Thr212Glu + Leu216Ile + Asn217Ser Val204Thr + Tyr206Pro + Asn211Glu Thr207Gly + Leu216Glu + Thr219Ser Ser203Glu + Tyr206Ala + Thr212Pro Alal99Ser + Val204His + Thr212Ser Alal99Asn + Val204Gln + Gly218Ser Pro200Gln + Thr214Pro + Gly218Asp Alal99Asn + Val202Met + Ser203Asp Pro200Gln + Ser210Asp + Asn211Gln Tyr205Ile + Thr214Asn + Leu216Met Pro200Gln + Pro209Glu + Thr219Ser Alal99Gly + Pro200Asn + Ser210Glu Val204Met + Asn217Gln + Thr219Ser Val202Thr + Thr214Asp + Gly21δSer Gly201Pro + Tyr205Met + Gly21δPro Val202Ser + Tyr205Ser + Asn217Asp Pro200Gly + Pro209Gln + Gly21δAsn Alal99Thr + Thr214Asn + Asn217Glu Thr207Asn + Thr212Pro + Gly21δAsp Gly201Pro + Asn211Ser + Thr212Asp Gly201Gln + Ser215Asp + Leu216Gly Val204Gln + Leu216Cys + Thr219Ser Thr207Ser + Thr214Gln + Ser215Glu Pro200Asn + Thr207Gly + Asn217Glu Thr212Asp + Tyr213Ala + Thr219Pro Thr207Asn + Tyr208Ile + Pro209Glu Alal99His + Asn211Glu + Thr212Pro Val204Thr + Asn211Asp + Leu216Ser Ser203Glu + Val204Ala + Thr219Gly Thr207Gly + Thr212Gly + Leu216His Gly201Asn + Thr207Gly + Tyr208Gly Gly201Asn + Val202Glu + Thr207Ser

Thr207Gln + Leu2lβGlu Gly21δSer Tyr205His + Ser2lOAsp Thr219Pro Pro200Gly + Tyr2 OδVal Ser210Asp Alal99Ser + Ser2 03Asp Tyr208Gln Gly201Ser + Asn2 UGlu Tyr213Pro Alal99Gln + Tyr2 05His Thr219Glu Pro200Ser + Thr2 07Pro Thr214Ser Pro200Gln + Tyr2 05Met Thr212Pro Alal99Gly + Val2 04His Asn211Glu Thr207Asn + Thr2 12Ser Thr219Asn Tyr205Pro + Thr2 07Gln Ser215Glu Val204His + Gly2lδGlu Thr219Gly Tyr205Pro + Tyr2 Oδlle + Ser215Glu Alal99Asn + Pro2 OOSer + Ser203Glu Tyr20δGln + Tyr2 13Met + Thr219Glu Val202Glu + Val2 04Thr + Asn217Ser Val204Gln + Pro2 09Gln + Thr214Gly Thr214Gln + Ser2 15Asp + Leu216Gly Val204Thr + Tyr2 13Ile + Ser215Glu Val204Gly + Asn2 UGln + Ser215Glu Pro200Ser + Val2 04Gly + Tyr213Ile Pro200Gly + Thr2 12Asp + Leu216His Asn211Glu + Tyr2 13Thr Leu216Pro Leu216Asn + Asn2 17Asp Gly218Ser Gly201Gln + Pro2 09Asn Ser215Glu Gly201Asn + Ser2 03Glu Pro209Asn Gly201Asn + Val2 02Ser Leu216Gly Asn211Asp + Tyr2 13His Thr219Gln Thr207Gln + Asn2 17Asp Gly218Ser Tyr20δGly + Ser2lOGlu Thr214Gly Thr207Gln + Tyr2 OδGln Tyr213Asp Alal99Asn + Tyr2 05Ile Gly21δPro Pro200Asn + Val2 02Gln Val204Ser Val204Asn + Ser2 15Glu Leu216Pro Pro200Ser + Gly2 OlSer Tyr20δAla Tyr20δThr + Tyr2 13Val Leu216Asp Thr207Gly + Thr2 12Ser Tyr213Ala Gly201Asn + Gly2 16Asp Thr219Asn Alal99Gly + Tyr2 05Cys Tyr208Thr Gly201Gln + Tyr2 13Cys Thr214Gly Alal99Gln + Pro2 OOSer + Thr214Asp Alal99Ser + Gly2 OlAsn Thr219Pro Val204Gly + Leu2 16Val Thr219Gln Gly201Asn + Ser2 03Glu Thr212Gly Gly201Ser + Ser2 03Glu Asn217Gln Pro200Gly + Thr2 07Gln Asn217Asp Alal99Thr + Tyr2 OδGln Thr219Pro Thr207Ser + Thr2 12Ser Gly218Asp Pro200Ser + Tyr2 Oδlle Asn217Ser Val204Pro + Tyr2 05Asp Thr214Pro Gly201Ser + Tyr2 05Glu Thr207Asn Pro200Ser + Thr2 07Gln Asn217Gln Gly201Asn + Val2 02Glu Leu216Cys

Alal99Ser + Pro209Ser + Gly2lδAsp Gly201Pro + Tyr205Met + Asn2HAsp Gly201Ser + Thr214Pro + Ser215Asp Val204Pro + Thr212Gly + Tyr213Val Gly201Pro + Tyr208Ser + Thr212Glu Alal99Gln + Ser203Asp + Leu2lβSer Val202Gly + Asn2UGlu + Tyr213Leu Ser203Asp + Thr212Gly + Thr214Gln Gly201Pro + Tyr205Gln + Thr207Gly Gly201Asn + Tyr208Asn + Tyr213Pro Pro200Ser + Tyr213Asp + Gly2lδPro Alal99Asn + Pro2OOGly + Thr214Ser Ser210Glu + Tyr213Gln + Gly218Asn Alal99Pro + Tyr213Thr + Ser215Asp Val202Pro + Tyr2OδCys + Asn2UGln Alal99Gln + Ser203Glu + Asn2USer Thr207Gln + Pro209Ser + Ser2lOGlu Pro200Ser + Val202Asn + Gly218Ser Tyr205Val + Tyr213Asn + Gly218Glu Val202Glu + Val204His + Tyr205Gln Alal99Gly + Thr207Pro + Asn2UGlu Val202Glu + Tyr2OδMet + Pro209Gly Val204Ser + Tyr208Ile + Leu2lβMet Tyr205Thr + Tyr208Ser + Asn217Ser Thr207Ser + Pro209Ser + Leu2lβAsn Gly201Asn + Pro209Asp + Tyr213Met Gly201Asn + Pro209Gln + Thr214Asn Pro200Asn + Val202Gln + Thr207Gln Alal99Asn + Ser203Glu + Val204Gln Ser203Asp + Pro209Gly + Leu2lβAla Alal99Thr + Pro200Asn + Ser2lOGlu Alal99Thr + Tyr205Leu + Asn217Glu Gly201Asn + Val202Met + Thr214Asp Thr212Gln + Thr214Glu + Thr219Ser Tyr205Glu + Tyr208Gly + Tyr213Leu Pro200Ser + Asn217Glu + Gly218Gln Ser210Glu + Thr214Pro + Leu2lβAsn Tyr208Asn + Pro209Asn + Tyr213Ser Alal99Pro + Gly2OlPro + Ser2lOAsp Gly201Gln + Thr207Gly + Gly216Glu Pro200Gln + Tyr208Gly + Ser215Asp Pro200Ser + Thr214Pro + Leu2lβGlu Alal99Gln + Asn217Asp + Gly2lδPro Pro200Asn + Val202Asp + Pro209Ser Thr212Gly + Tyr213Asn + Ser215Asp Gly201Gln + Tyr205Met + Thr219Glu Pro200Gly + Tyr213Gly + Thr219Glu Pro200Gly + Ser203Glu + Thr214Gln Tyr206Thr + Ser215Glu + Gly2lδSer Ser210Asp + Asn2USer + Leu2lβAsn Asn211Gln + Thr214Glu + Ser215Asp Pro209Glu + Ser2lOAsp + Leu2lβCys Tyr20δPro + Asn217Asp + Gly2lδAsp

Gly201Asn Thr207Gly + Asn211Gln Tvr205His Leu216Gly + Gly218Ser AΪal99Gln Tyr213Pro + Thr219Glu Alal99Gly Asn211Glu + Thr2l2Pro Gly201Pro Thr212Gln + Thr219Asn Pro200Gln Asn217Glu + Thr219Pro Gly201Ser Thr214Asn + Leu216Thr Gly201Gln Asn211Asp + Gly218Pro Ser203Asp Thr207Ser + Leu216Cys Val202His Ser215Asp + Leu216Gly Pro200Asn Val202Pro + Ser203Glu Pro200Ser Val202His + Leu21βThr Pro200Asn Leu216Ala + Gly21δPro Alal99Pro Val204His + Thr214Glu Gly201Gln Tyr213IIe + Gly21δAsn Alal99His Val204Gly + Asn211Gln Pro200Gln Val204Pro + Thr207Pro Gly201Asn Tyr213Ala + Gly2lδAsp Gly201Gln Thr214Gly + Gly216Glu Alal99Gln Gly201Asn + Thr214Asp Alal99Ser Leu216Val + Thr219Pro Pro200Gly Thr207Gln + Asn217Asp Alal99Thr Thr212Ser + Thr219Pro Pro200Ser Tyr206Ile + Gly218Asp Val204Pro Thr214Pro + Asn217Ser Gly201Pro Asn211Asp + Thr214Pro Gly201Ser Thr214Pro + Ser215Asp Gly201Pro Thr212Gly + Tyr213Ala Val202Asn Tyr213Leu + Thr219Pro Gly201Pro Thr207Gly + Thr212Gly Pro200Gln Ser215Glu + Thr219Gly Gly201Asn Tyr213Pro + Ser215Glu Pro200Ser Tyr213Asp + Gly218Pro Alal99Pro Thr212Asp + Thr219Ser Pro200Asn Thr207Gln + Thr212Gly Val204Cys Ser215Asp + Gly216Gln Val202Gln Thr212Asp + Asn217Gln Alal99Asn Asn211Glu + Thr214Pro Alal99Asn Ser210Asp + Tyr213Leu Pro200Asn Thr207Gln + Tyr213Asp Val204Gln Thr212Gly + Tyr213Cys Pro209Asn Thr214Gly + Leu216Met Pro200Gly Leu216Cys + Asn217Ser Val204Ser Asn211Asp + Leu216Met Gly201Asn Pro209Gln + Leu216Val Ser203Glu Tyr213Leu + Gly21δAsn Pro200Asn Thr214Ser + Leu216Cys Alal99Thr Ser210Glu + Leu216Cys Alal99Thr Tyr205Leu + Thr214Asp Gly201Asn Thr214Glu + Thr219Ser Gly201Pro Pro209Asn + Tyr213Ser Alal99Pro Thr207Gly + Ser210Asp Alal99Gly Tyr20δAsn + Leu216Thr

Ser203Glu + Asn217Glu Ser203Asp + Asn217Glu Gly201Ser + Asn217Asp Asn211Glu + Thr219Gly Alal99Pro + Leu216Glu Alal99Asn + Tyr213Ile Pro200Gln + Thr212Asp Ser203Asp + Tyr213Leu Alal99Ser + Gly216Asp Pro200Ser + Leu216Glu Alal99Asn + Asn217Asp Pro200Gly + Asn217Glu Gly201Asn + Leu216Glu Alal99Asn + Asn217Glu Tyr205Glu + Asn217Glu Gly201Gln + Ser215Glu Alal99Ser + Ser215Glu Asn211Ser + Gly2lδAsp Ser203Asp + Gly218Asp Val202Glu + Leu216Gln Val202Asp + Thr219Glu Ser203Asp + Thr214 sp Val202Asp + Leu216Ile Tyr208Gly + Gly218Asp Pro200Gln + Leu216Glu Gly201Gln + Asn217Asp Ser203Asp + Ser215Asp Gly201Gln + Thr214Glu Thr207Gln + Thr214Glu Tyr205Ala + Ser215Asp Alal99Gln + Thr219Glu Thr212Asp + Asn217Glu Gly201Asn + Leu216Glu Pro209Asp + Gly21δGln Gly201Pro + Thr219Asp Ser203Glu + Thr219Glu

TABLE26

Loop 6 - Quintuple Substitution Variants

Gly201Asn + Thr207Gln + Ser210Glu + Tyr213Ile + Val202Cys + Tyr205Ser + Thr212Pro + Tyr213Leu +

Gly21δAsn Alal99Ser + Pro200Ser + Tyr213His + Thr214Gln +

Leu2lβAsn Alal99Ser + Gly201Ser + Tyr206Ser + Ser210Glu +

Thr214Ser Alal99Gly + Gly201Asn + Val202Met + Tyr205His +

Thr214Gln Pro200Gly + Tyr213Leu + Thr214Pro + Asn217Ser +

Thr219Glu

Alal99Thr + Tyr205Gln + Thr207Gln + Thr212Asp +

Gly21δGln Val204Gly + Tyr205Ala + Tyr208Gly + Asn211Gln +

Gly218Asp Alal99Ser + Gly201Pro + Tyr205Gly + Ser215Glu +

Leu216Ser A.lal99His + Thr207Gln + Tyr20δThr + Gly21δGln +

Thr219Gln Alal99Gly + Pro209Gly + Asn211Ser + Thr212Ser +

Tyr213Ser Pro200Ser + Val202Gln + Thr207Asn + Tyr20δlle +

Thr219Pro Pro200Ser + Gly201Ser + Tyr20δGln + Ser210Glu +

Asn217Gln AIal99Gly + Gly201Ser + Val202Gly + Tyr205Asp +

Leu216Gly Aial99Gln + Pro200Ser + Gly201Ser + Tyr205Gln +

Thr214Asp Alal99Pro + Gly201Pro + Val204Asn + Tyr20δPro +

Thr214Gly Pro200Gly + Val202Gly + Ser203Asp + Asn211Ser +

Leu216Pro Tyr205Gly + Thr207Pro + Pro209Asp + Asn211Gln +

Asn217Ser Pro200Gln + Val204Ala + Tyr20δLeu + Thr212Glu +

Tyr213Met Tyr205Met + Asn211Gln + Tyr213Met + Thr214Asn +

Thr219Asp Ser203Asp + Val204Ser + Tyr213Gly + Thr214Asn +

Gly21δPro Gly201Gln + Val204Gln + Ser215Asp + Leu216Ala +

Thr219Ser Gly201Asn + Thr207Gly + Tyr208Gly + Thr212Gly +

Leu216His Gly201Asn + Tyr205His + Thr207Ser + Leu216Glu +

Gly21δSer Alal99Gln + Pro200Ser + Tyr205His + Thr214Ser +

Thr219Glu Pro200Gln + Gly201Gln + Leu216His + Asn217Asp +

Gly218Gln Alal99Gly + Val204His + Tyr205Met + Asn211Glu +

Thr212Pro Val204His + Tyr205Pro + Tyr208Ile + Gly218Glu +

Thr219Gly Pro200Ser + Val202Gln + Val204Gly + Tyr213Ile +

Ser215Glu Alal99Ser + Pro200Asn + Asn211Glu + Tyr213Thr +

Leu216Pro Ser203Asp + Val204Gly + Asn211Gln + Tyr213Ile +

Gly21δAsn Alal99Asn + Pro200Gln + Val202Ala + Val204Pro +

Thr207Pro Alal99Thr + Gly201Gln + Val202Gly + Ser215Asp +

Leu216Ala Thr212Gln + Tyr213Val + Leu216His + Gly218Gln +

Thr219Glu Alal99Gln + Pro200Ser + Gly201Asn + Tyr205Ala +

Thr214Gly Gly201Asn + Val204Gly + Thr212Gly + Leu216Val +

Thr219Pro Pro200Gly + Val202His + Thr207Gln + Tyr208Gln +

Asn217Asp Alal99Thr + Thr207Ser + Thr212Ser + Gly218Asp +

Thr219Pro Pro200Ser + Val204Pro + Tyr208Ile + Thr214Pro +

Asn217Ser Pro200Gln + Val202Ser + Tyr205Ser + Asn211Ser +

Ser215Asp Val202Thr + Thr212Gln + Ser215Asp + Asn217Ser +

Thr219Asn Val204Pro + Thr212Gly + Tyr213Val + Thr214Pro +

Ser215Asp Gly201Ser + Val204His + Tyr206Leu + Thr212Ser +

Gly216Glu Ser203Asp + Tyr205Gln + Thr207Gly + Thr212Gly +

Thr214Gln Pro200Gln + Tyr208Cys + Thr212Gly + Ser215Glu +

Thr219Gly Pro200Ser + Gly201Pro + Val202Ala + Tyr213Asp +

Gly218Pro Val204Gly + Ser210Asp + Thr212Asn + Thr214Pro +

Thr219Ser Pro200Gly + Ser203Asp + Pro209Asn + Leu216Cys +

Asn217Ser Tyr205Thr + Thr207Ser + Tyr20δSer + Pro209Ser +

Asn217Ser Gly201Asn + Tyr205Ile + Pro209Gln + Thr214Asn +

Leu216Val Alal99Asn + Ser203Glu + Val204Gln + Tyr213Leu +

Gly21δAsn Alal99Thr + Val202Met + Tyr205Leu + Asn211Ser +

Asn217Glu Alal99Pro + Gly201Pro + Pro209Asn + Ser210Asp +

Tyr213Ser Alal99Ser + Gly201Pro + Pro209Gly + Tyr213Pro +

Gly216Pro Pro200Ser + Pro209Gly + Thr212Pro + Thr214Pro +

Leu216Glu Gly201Ser + Thr207Gly + Ser210Asp + Asn211Ser +

Leu216Ile Alal99Ser + Gly201Gln + Val204Asn + Tyr205Leu +

Thr219Asp Thr207Pro + Tyr208Thr + Ser210Glu + Thr212Gly +

Asn217Ser Gly201Gln + Tyr205Asn + Thr207Asn + Pro209Asp +

Thr212Gly

Pro200Asn + Tyr205Glu + Tyr20δMet + Tyr213Val +

Leu21βlle Tyr205Ala ^■ > ^■ Tyr213His + Thr214Ser + Asn217Gln +

Thr219Gln Alal99Gln - Pro200Gly + Val202Met + Leu216Met +

Gly21δPro Pro200Ser + Val204Ala + Ser210Glu + Asn211Gln +

Gly21δSer Alal99Ser - Pro200Ser + Val202Asn + Leu216Val +

Gly218Pro Pro200Gln + Val202Gln + Tyr213Leu + Thr214Pro +

Gly218Glu Pro200Ser + Gly201Gln + Thr207Pro + Gly218Pro +

Thr219Pro Alal99Asn + Ser203Asp + Val204Asn + Tyr208Pro +

Leu216Pro Tyr205Ile + Tyr213Ile + Thr214Asn + Ser215Glu +

Thr219Pro Gly201Ser * Val204His + Asn211Gln + Thr212Gln +

Leu216Met Alal99Ser + Gly201Pro + Asn211Glu + Gly21δGln +

Thr219Ser Alal99Ser + Thr207Ser + Thr214Gln + Ser215Glu +

Leu2lβAsn Alal99His + Val204Cys + Asn21lSer + Thr214Gly +

Thr219Glu Alal99Asn + Val202His + Tyr206Asn + Leu216Thr +

Thr219Gly Ser203Glu - Thr207Pro + Tyr20δLeu + Thr212Ser +

Asn217Gln Pro200Ser -r Val202Asp + Val204Asn + Tyr208Met +

Thr212Gln Gly201Ser + Thr207Asn + Tyr208Leu + Ser210Asp +

Asn217Ser Val202Asp + Val204Gln + Tyr208Cys + Pro209Gln +

Tyr213Val Alal99Gly + Val202Thr + Thr207Gln + Thr212Glu +

Thr214Gln Gly201Pro + Val202Cys + Ser210Asp + Thr212Pro +

Tyr213Cys Alal99Asn + Gly20lSer + Val204Met + Pro209Asn +

Thr214Pro Gly201Asn + Ser203Asp + Val204Thr + Thr207Ser +

Leu216Ala Val202Thr + Tyr205Gly + Thr207Gln + Asn211Asp +

Leu216Ala Alal99Gln + Val204Gly + Thr207Pro + Asn211Asp +

Thr214Gly Gly201Pro + Val202Asn + Tyr205Ser + Asn211Glu +

Gly21δAsn Val204Gly + Pro209Asn + Asn211Asp + Asn217Gln +

Thr219Gly Val202Gly + Val204Gly + Ser210Glu + Gly216Ser +

Thr219Ser Alal99Ser - ¹ - Asn211Ser + Tyr213Met + Thr214Asp +

Thr219Pro Alal99Asn + Gly201Asn + Ser203Glu + Tyr213His + Alal99Gln + Pro2C0Asn + Thr207Ser + Pro209Ser +

Asn217Ser Tyr205Met + Thr214Asp + Ser215Glu + Leu216Cys +

Asn217Gln Alal99Pro + Val202His + Tyr20δMet + Thr214Glu +

Ser215Asp Pro200Asn + Pro209Glu + Ser210Asp + Thr214Ser +

Leu216Cys Val204Ser + Tyr20δPro + Leu216Met + Asn217Asp +

Gly218Asp Tyr205Pro + Thr207Pro + Leu ^* 216Asn + Asn217Asp +

Gly218Asp Pro200Gly + Tyr205Asn + Tyr20δGly + Asn217Glu +

Gly21δGlu Val202Thr + Val204Hιs + Pro209Gly + Asn211Glu +

Thr212Asp Gly201Gln + Val202Asn + Thr207Asn + Ser215Glu +

Leu216Asp Alal99His + Pro200Gly + Ser210Asp + Asn211Glu +

Thr212Pro Pro209Gln + Ser210Asp + Asn211Glu + Asn217Gln +

Thr219Pro Alal99Pro + Val202Ala + Ser210Glu + Asn211Asp +

Gly21δAsn Alal99Asn + Thr207Asn + Ser210Asp + Asn211Asp +

Thr212Gly Val204Ala + Tyr205Glu + Thr212Ser + Ser215Glu +

Asn217Gln Gly201Pro + Val202Asp + Thr207Gln + Thr212Ser +

Asn217Asp Thr207Ser + Pro209Asp + Ser210Glu + Asn211Asp +

Tyr213Asn Gly201Ser + Val202Glu + Ser203Glu + Thr214Pro +

Asn217Asp Ser203Glu + Thr207Asn + Thr214Gly + Leu216Glu +

Asn217Asp Gly201Ser + Ser203Asp + Tyr20δlle + Leu216Asp +

Asn217Asp Gly201Pro + Tyr208Gly + Leu216Asp + Asn217Glu +

Gly21δAsp Ser203Glu + Val204Pro + Thr207Gly + Tyr213Leu +

Ser215Asp Gly201Asn + Ser203Asp + Tyr20δVal + Asn211Gln +

Ser215Asp Alal99Asn + Pro200Ser + Ser203Glu + Tyr213Met +

Ser215Glu Alal99Ser + Ser203Glu + Asn211Gln + Ser215Glu +

Leu216Cys

Ser203Glu + Val204Gln + Thr214Gln + Ser215Asp +

Leu216Gly Aial99Pro -r Gly201Pro + Ser203Asp + Tyr205Val +

Ser215Glu Aial99Thr -r Ser203Asp + Val204Gln + Tyr213Gly +

Ser215Asp Val202Cys + Ser203Asp + Tyr205Met + Tyr213Gln +

Asn217Glu Ser203Glu + Val204Asn + Thr212Gly + Asn217Glu +

Thr219Gln Gly201Asn + Ser203Aεp + Tyr208Val + Pro209Ser +

Asn217Glu Ser203Asp + Thr212Pro + Tyr213Gly + Asn217Glu +

Gly21δSer Vai204Thr + Asn211Asp + Thr212Asp + Tyr213Asp +

Leu216Ser Val202Ala + Ser203Asp + Val204Gln + Tyr205Met +

Leu216Glu Ser203Glu + Val204Ser + Thr207Gly + Tyr20δVal +

Leu216Asp Pro200Gln + Pro209Gln + Ser210Glu + Thr212Glu +

Thr214Gly Pro209Glu + Asn211Asp + Asn217Gln + Gly216Gln +

Thr219Pro Pro200Ser + Tyr205Pro + Pro209Asp + Asn211Glu +

Tyr213Ile Alal99Asn + Ser203Asp + Tyr205Glu -r Thr207Asn +

Leu216Glu Ser203Glu + Tyr205Glu + Tyr213Met + Leu216Ile +

Asn217Gln Val202Glu + Ser203Glu + Asn211Gln + Leu216Met +

Gly21δGlu Alal99Ser + Val202Ser + Tyr205Asp + Tyr20δGly +

Tyr213Asp Pro200Ser + Thr207Pro + Thr212Asp + Thr214Asp +

Thr219Ser Pro200Gly + Thr207Ser + Thr214Gln + Ser215Glu +

Asn217Glu Pro200Gln + Thr207Gly + Ser215Asp + Leu216Gln +

Asn217Asp Alal99Asn + Tyr205Glu + Tyr206Ala + Ser215Glu +

Asn217Glu Gly201Pro + Val204Ser + Tyr205His + Asn217Glu +

Thr219Asp Alal99Ser + Tyr20δGln + Tyr213Asp + Thr214Pro +

Ser215Glu Thr207Asn + Tyr206Gln + Thr212Ser + Tyr213Glu +

Ser215Glu Gly201Gln + Tyr205Gly + Pro209Glu + Thr212Pro +

Tyr213Glu Ser203Asp + Pro209Ser + Thr212Gln + Ser215Asp +

Gly2lδAsp Ser203Asp + Pro209Gly + Ser215Glu + Leu216Ala +

Gly218Asp Prc200Ser + Ser203Asp + Thr207Gln + Leu216Gln +

Gly21δGlu Pro200Asn + Gly201Pro + Ser203Glu + Thr207Gly +

Gly21δGlu Val202Asp + Val2C4Cys + Tyr20δAla + Gly216Gln +

Thr219Glu Val202Glu + Val204Thr + Tyr208Gln + Asn217Ser +

Thr219Glu Pro200Ser + Gly201Pro + Val202Asp + Asn211Gln +

Thr219Glu Gly201Asn + Ser203Glu + Thr212Gln + Thr214Asp +

Thr219Ser Gly201Asn + Ser203Asp + Thr214Glu + Leu216Cys +

Thr219Gly Gly201Asn + Val202Cys + Ser203Asp + Thr214Asp +

Thr219Pro Val202Cys + Ser203Asp + Tyr208Thr + Thr214Asp +

Thr219Pro Val204Ser + Tyr20δLeu + Ser210Asp + Tyr213Asp +

Thr214Asp Alal99Hιs + Ser203Asp + Tyr206Ser + Tyr213Glu +

Thr214Asp Val204Cys + Ser210Asp + Asn211Ser + Tyr213Asp +

Thr219Gln Alal99Asn + Ser210Glu + Thr212Asn + Tyr213Glu +

Thr214Gln Ser203Asp + Tyr205Glu + Tyr208Gly + Tyr213Leu +

Gly218Asp Val202Glu + Tyr205Glu + Pro209Asn + Leu216Thr +

Gly21δGlu Tyr208Val + Ser215Asp + Asn217Ser + Gly218Asp +

Thr219Glu Tyr205Leu + Thr214Asp + Ser215Glu + Gly216Glu +

Thr219Asn Alal99Pro + Pro209Asp + Thr212Asp + Ser215Asp +

Gly21δSer Ser203Glu + Thr212Glu + Tyr213Ser + Thr214Glu +

Leu216Cys Ser203Asp + Tyr206Cys + Thr212Asp + Tyr213Ile +

Thr214Asp Val202Met + Val204Thr + Asn211Asp + Thr214Asp +

Ser215Asp Pro209Asn + Asn211Asp + Thr214Glu + Ser215Glu +

Leu216Gly Pro200Asn + Tyr205Glu + Thr212Asp + Tyr213Ala +

Thr219Pro Pro200Asn + Ser203Asp + Tyr205His + Thr212Asp +

Ser215Asp Ser203Asp + Val204Thr + Thr212Glu + Tyr213Ile +

Ser215Glu Tyr205Gly + Thr207Gln + Tyr20δlle + Thr212Glu +

Ser215Glu

Alal99Asn + Tyr205Asp + Ser210Glu + Thr212Glu +

Thr214Gly Val202Met + Tyr205Ala + Ser210Glu + Thr212Asp +

Ser215Asp Thr207Gln + Tyr208Leu + Asn211Glu + Tyr213Glu +

Leu216Glu Val204Met + Pro209Ser + Asn211Asp + Thr214Glu +

Leu216Asp Pro200Ser + Ser203Glu + Tyr213Glu + Leu216Gly +

Asn217Glu Alal99Gly + Tyr213Pro + Thr214Asp + Asn217Asp +

Thr219Asp Alal99Gln + Tyr208Ala + Thr214Glu + Asn217Glu +

Thr219Asp Alal99His + Val202His + Ser203Glu + Thr207Gly +

Thr219Asp Gly201Ser + Val202Gly + Ser203Glu + Tyr205Thr +

Thr219Asp Gly201Pro + Pro209Glu + Ser210Asp + Ser215Glu +

Leu216Met Tyr205Glu + Ser215Glu + Leu21βCys + Gly218Gln +

Thr219Glu Ser203Asp + Pro209Ser + Asn211Glu + Thr214Asp +

Leu2lβAsn Alal99Thr + Ser203Glu + Thr207Gln + Asn211Asp +

Thr214Glu Gly201Ser + Ser203Asp + Tyr205Gly + Pro209Asp +

Ser215Asp Pro209Glu + Thr214Asn + Leu216Glu + Asn217Asp +

Thr219Asn

TABLE27

Loop 6 - Sextuple Substitution Variants

Gly201Asn + Tyr205Gly + Thr207Gln + Ser210Glu +

Tyr213Ile + Thr219Gly Pro200Asn + Gly201Ser + Tyr20δGly + Asn211Asp +

Thr212Ser + Thr219Asn Gly201Pro + Ser203Asp + Val204Gln + Tyr205Pro +

Thr212Ser + Tyr213His Pro200Gln + Val204His + Thr207Ser + Thr214Pro +

Ser215Asp + Thr219Asn Alal99Pro + Pro200Ser + Gly201Pro + Val204Asn +

Tyr208Pro + Thr214Gly Pro200Gln + Val204Ala + Tyr208Leu + Thr212Glu +

Tyr213Met + Leu216Gln Alal99Pro + Val202Met + Ser203Glu + Tyr205Leu +

Asn211Gln + Tyr213Gly Gly201Gln + Val204Gln + Thr214Gln + Ser215Asp +

Leu216Gly + Thr219Ser Val204Thr + Tyr20δMet + Pro209Glu + Tyr213Gly +

Thr214Pro + Gly216Ser

Ser203Asp + Val204Met + Thr207Gln + Pro209Gln +

Asn217Ser + Thr219Asn Pro200Ser + Val202Met + Val204Gly + Pro209Gln +

Tyr213Ile + Ser215Glu Alal99Hιs + Ser203Asp + Val204Gly + Thr207Gly +

Asn211Gln + Thr214Ser Alal99Gln + Pro200Gln + Gly201Gln + Pro209Glu +

Leu216Ile + Asn217Ser Pro200Gln + Tyr206Ile + Pro209Gln + Thr212Gly +

Ser215Glu + Thr219Gly Pro200Asn + Ser203Asp + Val204Cys + Thr207Gln +

Pro209Ser + Thr214Asn Alal99Asn + Val202Gln + Val204Ser + Pro209Gly +

Thr212Asp + Asn217Gln Pro200Asn + Val204Met + Thr207Gln + Pro209Gly +

Thr212Gly + Tyr213Asp Pro200Gly + Val204Gln + Pro209Asn + Tyr213Ile +

Thr214Gly + Leu216Met Alal99Asn + Tyr205Thr + Tyr20δSer + Pro209Ser +

Thr214Asp + ^' Asn217Ser Tyr205Ser + Thr207Gln + Tyr20δCys + Pro209Asn +

Thr212Asn + Leu216Glu Alal99Asn + Pro200Ser + Ser203Asp + Tyr205Cys +

Tyr20δAsn + Thr214Gln Thr207Pro + Tyr213Ser + Thr214Asn + Asn217Ser +

Gly21δSer + Thr219Glu Alal99Pro + Gly201Pro + Thr207Gly + Pro209Asn +

Ser210Asp + Tyr213Ser Alal99Gln + Pro200Gly + Val202Met + Thr212Asp + Alal99Ser + Pro200Gly + Val202Asn + Asn211Gln +

Leu216Val + Gly218Pro Alal99His + Pro200Gln + Val202Gln + Thr214Gln +

Leu216Thr + Gly218Glu Pro200Ser + Gly201Gln + Thr207Pro + Tyr208Thr +

Gly216Pro + Thr219Pro Pro200Gln + Val204Met + Thr207Gly + Ser215Glu +

Leu216Met + Thr219Pro Thr207Ser + Pro209Asp + Thr212Gly + Thr214Ser +

Leu216Pro + Thr219Gln Pro200Asn + Gly201Ser + Thr207Asn + Tyr20δLeu +

Ser210Asp + Asn217Ser Alal99Asn + Val202Ser + Tyr205Cys + Thr207Gln +

Tyr213Leu + Asn217Ser Val202Ser + Pro209Gln + Tyr213Asp + Leu216Met +

Asn217Ser + Thr219Gly Gly201Gln + Val204Gly + Pro209Asn + Asn211Asp +

Asn217Gln + Thr219Gly Val202Gly + Ser203Glu + Val204Gly + Tyr213Met +

Gly21δSer + Thr219Ser Alal99Gln + Pro200Asn + Tyr205Thr + Thr207Ser +

Thr214Gln + Asn217Ser Alal99Ser + Gly201Asn + Val202Met + Val204Asn +

Asn211Gln + Gly218Glu Pro200Asn + Val202Pro + Val204Gln + Tyr20δPro +

Gly213Gln - Thr219Glu Thr207Gly + Tyr208Ile + Pro209Gly + Tyr213Gly +

Thr214Pro + Leu2lβAsn Val202Met + Ser203Asp + Val204Met + Thr212Asn +

Thr214Asn + Leu216Gly Alal99Ser + Val202Met + Val204Ala + Thr212Gly +

Tyr213Thr + Thr214Pro Tyr208Val + Pro209Gln + Thr214Ser + Leu216Cys +

Asn217Glu + Thr219Asn Pro200Gln + Gly20lGln + Tyr205Ala + Pro209Gln +

Leu2iβPro + Thr219Glu Alal99Gly + Pro200Gln + Gly20lSer + Tyr20δGln +

Ser215Asp -> ^■ Gly21δAsn Alal99Asn + Pro200Asn + Val202Asn + Val204Gln +

Thr214Gly + Asn217Glu Pro200Gly + Gly20lGln + Tyr213Gly + Thr214Asn +

Leu216Gln + Thr219Ser Gly201Gln + Val202Gly + Thr207Gly + Asn211Gln +

Thr214Gly + Ser215Asp Alal99Thr + Pro200Ser + Tyr20δAla + Thr212Ser +

Ser215Asp + Asn217Ser Alal99His + Val202Asn + Ser203Glu + Val204His +

Tyr213Ile + Asn217Gln Gly201Asn + Val202Cys + Tyr205Cys + Tyr213Ile + Alal99Thr + Gly201Ser + Val202Gly + Tyr205Pro +

Leu216Thr + Gly216Glu Alal99Ser + Val204Cys + Tyr206Cys + Pro209Gly +

Tyr213Ala + Leu216Asp Alal99Gly + Gly201Asn + Val204Met + Thr207Pro +

Thr214Glu + Leu216Met Pro200Gly - Tyr205Met + Tyr20δVal + Thr212Gly +

Tyr213Ser + Leu216Val Gly201Pro + Tyr208Val + Pro209Glu + Asn211Gln +

Tyr213Ala + Thr219Gly Alal99Ser + Pro200Ser + Val202Thr + Tyr205Gln +

Gly2lδAsp + Thr219Pro Alal99Gln + Ser203Glu + Val204Pro + Thr214Ser +

Gly21δGln + Thr219Pro Alal99Thr + Pro200Gly + Gly201Gln + Thr212Ser +

Leu216Val + Gly218Ser Pro200Ser + Val204Gln + Thr207Pro + Thr214Gly +

Ser215Glu + Gly21δGln Alal99Gly + Gly201Gln + Val204His + Tyr205Asp +

Pro209Gln + Thr212Ser Pro200Gln + Tyr208Asn + Asn211Ser + Thr212Ser +

Tyr213Thr + Leu216Asp Alal99His + Tyr208Leu + Thr212Pro + Ser215Glu +

Leu216Ser + Gly218Gln Gly201Pro + Val204Cys + Pro209Gln + Thr212Ser +

Tyr213Cys + Thr214Gln

Vai204Ser + Tyr205Ser + + Tyr208Cys +

Ser215Asp + Asn217Ser Pro200Asn + Tyr208Ala + + Thr214Glu +

Ser215Asp + Thr219Gly Val202His + Tyr208Met + + Thr212Asn +

Thr214Glu + Ser215Asp Pro200Gln + Gly201Ser + + Val204Ser +

Thr214Asp + Ser215Glu Alal99Gln + Gly201Ser + + Thr207Ser +

Thr214Asp + Ser215Glu Val202Asn + Tyr205Gly + + Thr212Ser +

Thr214Glu + Ser215Glu Pro200Asn + Pro209Glu + + Asn211Ser +

Thr214Ser + Leu216Cys Pro200Ser + Val204Pro + + Thr214Pro +

Asn217Asp + Gly21δAsp Gly201Gln + Tyr205Asn + + Thr212Gly +

Asn217Glu + Gly21δGlu Val202His + Thr207Asn + + Tyr213Pro +

Ser215Glu + Leu216Asp Alal99Asn + Gly201Ser + + Tyr205Thr +

Ser215Asp + Leu216Asp Pro200Gly + Val202Ala + + Ser210Glu +

Asn211Glu + Gly218Ser Val204Asn + Pro209Ser + + Asn211Asp +

Tyr213Asn + Leu216Met Alal99Asn + Val204Thr + + Asn211Glu +

Tyr213Leu + Thr214Pro Val202Cys + Val204Ala + + Thr212Ser +

Ser215Glu + Asn217Gln Thr207Pro + Ser210Asp + + Thr212Glu +

Asn217Ser + Thr219Ser Pro200Asn + Val202Asp + + Pro209Ser +

Asn217Asp + Gly218Pro Val202Gln + Pro209Glu + + Asn211Glu +

Thr212Gln + Asn217Gln Val202Asp + Ser203Asp + + Thr212Ser +

Tyr213Gln + Asn217Glu Alal99Asn + Ser203Glu + + Tyr208Pro +

Leu216Glu + Asn217Asp Val204Thr + Thr212Gly + + Ser215Asp +

Leu216Glu + Gly218Ser Alal99Gln + Ser203Asp + + Thr207Gly +

Ser215Glu + Leu216Gln Alal99Pro + Pro200Gln + + Asn211Ser +

Ser215Asp + Leu2lβAsn Ser203Glu + Tyr208Thr + + Thr214Gln +

Ser215Glu + Gly218Ser Alal99Thr + Pro200Gln + + Ser203Asp +

Ser215Asp + Gly218Ser Pro200Gly + Val202Cys + + Asn211Ser +

Ser215Glu + Leu216Pro Alal99Gln + Ser203Glu + + Tyr213Met +

Ser215Glu + Asn217Ser Pro200Ser + Gly201Asn + Ser203Asp + Tyr20δVal +

Pro209Ser + Asn217Glu Pro200Ser + Gly201Ser + Val202His + Ser203Asp +

Asn217Glu + Gly218Pro Val202Asn + Tyr205Glu + Thr207Gln + Tyr206Leu +

Thr214Asp + Gly218Pro Ser203Asp + Val204Thr + Tyr205Glu + Thr212Ser +

Tyr2I3Ile + Ser215Glu Alal99Asn + Pro200Ser + Tyr205Pro + Pro209Asp +

Asn211Glu + Tyr213Ile Gly201Pro + Val202Cys + Val204Ser + Pro209Glu +

Thr212Glu + Thr214Ser Alal99Asn + Val202Asp + Ser203Asp + Tyr20δCys +

Ser215Asp + Leu2lβAsn Alal99Asn + Ser203Glu + Tyr205Cys + Thr212Gly +

Thr214Glu + Ser215Asp Alal99Thr + Pro200Ser + Tyr20δMet + Thr214Ser +

Ser215Glu + Asn217Asp Alal99Pro + Tyr208Gly + Ser215Asp + Leu216Val +

Asn217Asp + Thr219Ser Pro200Asn + Pro209Gly + Tyr213Pro + Ser215Asp +

Leu216Gly + Asn217Asp Pro200Ser + Gly201Pro + Val202Ala + Asn211Glu +

Tyr213Asp + Gly218Pro Val202Met + Val204Ser + Tyr205His + Thr212Gly +

Asn217Glu + Thr219Asp Tyr205Val + Tyr208Pro + Ser215Glu + Leu216Met +

Asn217Asp + Gly218Asp Val202Ser + Tyr208Ala + Asn211Ser + Thr214Glu +

Leu216Asp + Asn217Aεp Gly201Pro + Tyr208Gln + Thr212Asp + Tyr213Asp +

Ser215Asp + Asn217Ser Alal99Gln + Gly201Gln + Tyr205Gly + Pro209Glu +

Thr212Pro + Tyr213Glu Ser203Asp + Pro209Ser + Asn211Gln + Thr212Gln +

Ser215Asp + Gly216Asp Ser203Asp + Pro209Gly + Tyr213Leu + Ser215Glu +

Leu216Ala + Gly216Asp Val202Glu + Val204Cys + Thr212Gly + Ser215Asp +

Leu216Gln + Gly21δGln Pro200Gly + Gly201Gln + Val202Glu + Val204Ala +

Thr207Pro + Ser215Asp Alal99Gln + Pro200Ser + Gly201Ser + Ser203Asp +

Gly21δGlu + Thr219Asp Alal99Gln + Gly201Ser + Tyr205Gln + Ser210Asp +

Thr212Glu + Thr214Asp Gly201Asn + Val202Pro + Ser210Glu + Thr212Glu +

Tyr213Thr + Thr214Glu Pro200Gln + Val202Met + Ser203Asp + Val204Gly +

Tyr20δlle + Gly216Asp Pro200Ser + Gly201Pro + Val202Asp + Tyr20δCys +

Asn211Gln + Thr219Glu

Ser203Asp + Tyr20δPro + Pro209Ser + Asn211Ser +

Thr214Asp t Thr219Pro Alal99His + Gly201Asn + Ser203Glu + Tyr213His +

Thr214Glu + Leu216Ile Ser203Glu + Tyr208Pro + Tyr213His + Thr214Asp +

Leu216Cys + Asn217Ser Val202Asp + Tyr205Met + Thr214Asp + Ser215Glu +

Leu216Cys + Asn217Gln Val202Cys + Val204Ser + Tyr206Leu + Ser210Asp +

Tyr213Asp + Thr214Asp Gly201Asn + Tyr205Gln + Pro209Asn + Ser210Glu +

Tyr213Glu + Leu21βAla Val202Cys + Pro209Asp + Tyr213Asp + Thr214Asn +

Ser215Glu + Asn217Gln Alal99Gly + Gly201Gln + Ser215Asp + Leu216Asp +

Gly218Asn + Thr219Glu Alal99Hιs + Thr207Asn + Asn211Ser + Leu216Glu +

Asn217Gln + Thr219Asp Val202Met + Thr207Gln + Pro209Gly + Thr214Glu +

Ser215Asp + Gly218Asp Pro200Gln + Val202Ala + Val204Pro + Thr207Gln +

Pro209Glu + Thr214Glu Gly201Pro + Tyr206Gly + Thr214Asp + Leu216Cys +

Asn217Glu + Gly21δAsp Alal99Gln + Thr207Pro + Tyr208Cys + Pro209Gln +

Tyr213Asp + Leu216Asp Ser203Asp + Thr214Gly + Ser215Glu + Leu216Ala +

Asn217Ser + Thr219Glu Pro200Gln + Ser203Glu + Val204His + Thr212Asp +

Thr214Asp + Gly218Gln Alal99His + Gly201Ser + Ser203Glu + Tyr205Ser +

Thr214Glu + Gly218Glu Gly201Pro + Ser203Glu + Thr207Gly + Tyr208Cys +

Thr214Asp + Gly21δGlu Ser203Glu + Tyr205Leu + Thr207Asn + Thr212Asp +

Tyr213Met + Ser215Glu Alal99Pro + Ser203Asp + Pro209Ser + Thr212Glu +

Ser215Asp + Gly218Gln Ser203Glu + Tyr205His + Thr212Asp + Tyr213Met +

Ser215Asp + Thr219Ser Ser203Asp + Val204Gly + Thr212Glu + Thr214Pro +

Ser215Asp + Asn217Ser Gly201Pro + Tyr205Gln + Thr207Gly + Asn211Glu +

Thr212Gly + Thr214Glu Pro200Asn + Val202Gln + Thr207Gln + Pro209Ser +

Asn211Asp + Thr214Glu Gly201Gln + Thr207Gly + Pro209Gln + Thr212Glu +

Ser215Glu + Gly218Gln Val202Met + Tyr205Ala + Ser210Glu + Thr212Asp +

Ser215Asp + Leu216Ala Gly201Gln + Pro209Gly + Asn211Asp + Tyr213Asp +

Thr214Gly + Leu216Asp Alal99Gly + Val202Glu + Ser203Glu + Tyr213Glu +

Thr214Pro - Thr219Ser Gly201Ser + Val2Q2Cys + Ser203Asp + Tyr205Asp + Pro200Ser + Ser203Glu ->- Tyr213Glu + Leu216Gly +

Asn217Glu + Thr219Gly Tyr205Asn + Pro209Asn + Thr212Glu + Tyr213Met +

Ser215Glu + Asn217Asp Alal99Gln + Tyr20δAla + Asn211Gln + Thr214Glu +

Asn217Glu + Thr219Asp Gly201Pro + Val202Gly + Ser203Glu + Tyr205Thr +

Tyr208Ala + Thr219Asp Pro200Asn + Gly201Pro + Ser203Asp + Thr207Gln +

Gly218Ser + Thr219Glu Gly201Asn + Ser203Asp + Val204Gly + Tyr20δVal +

Asn211Gln + Thr219Asp A.lal99His + Pro200Asn + Ser203Asp + Tyr205Thr +

Pro209Asp + Thr214Glu Gly201Pro + Val202Gly + Pro209Glu + Ser210Asp +

Ser215Glu + Leu216Met Alal99Gly + Val202His + Val204Ala + Pro209Asp +

Ser210Glu + Ser215Glu Gly201Asn + Val204Gln + Tyr20δLeu + Thr214Glu +

Ser215Glu + Thr219Glu Ser203Glu + Tyr205Gln + Tyr208Gly + Asn211Glu +

Thr214Glu -r Thr219Ser Gly201Ser + Ser203Asp + Tyr205Ala + Pro209Asp +

Tyr213Met + Ser215Asp Val202Asn + Pro209Asp + Asn211Glu + Thr212Ser +

Tyr213Leu + Ser215Glu Alal99Pro + Val204Thr + Tyr205Glu + Ser210Glu +

Asn211Asp + Tyr213Leu Val202Ala + Ser203Asp + Val204Gln + Tyr205Met +

Thr212Glu + Leu216Glu Gly201Pro + Ser203Asp + Tyr208Thr + Thr214Asp +

Asn217Gln + Thr219Asp Pro200Gln + Gly201Asn + Thr207Gln + Tyr213Glu +

Asn217Asp + Gly21δAsp Pro200Gln + Thr207Gly + Tyr208Ala + Thr212Glu +

Tyr213Glu + Asn217Glu Thr207Ser + Tyr20δSer + Pro209Glu + Thr214Glu +

Asn217Glu + Thr219Asn Alal99Gly + Tyr205Pro + Pro209Glu + Thr214Glu +

Leu2lβAsn + Asn217Asp Alal99His + Gly201Asn + Tyr205Pro + Pro209Glu +

Ser215Glu + Asn217Glu Pro200Gly + Pro209Asp + Tyr213Gln + Ser215Glu + Alal99His + Val204Ser + Thr207Gln + Pro209Asp +

Ser210Asp + Thr219Asp Alal99Thr + Thr207Pro + Tyr20δVal + Pro209Glu +

Ser210Asp + Thr219Asp Tyr208His + Pro209Glu + Ser210Glu + Tyr213Val +

Leu21βCys + Thr219Asp

Tyr205Met + Thr207Gly + Ser210Asp + Thr212Ser +

Asn217Glu + Gly218Asp Ser210Glu + Thr212Pro + Thr214Gln + Asn217Asp +

Gly21δGlu + Thr219Ser Pro2θ ^" θGln + Val204Thr + Tyr20δAsn + Asn211Asp +

Ser215Glu + Leu216Glu Gly201Pro + Tyr20δGln + Asn211Glu + Thr212Asp +

Asn217Glu + Thr219Ser Val202Asp + Ser203Asp + Asn211Glu + Thr212Pro +

Tyr213Leu + Thr214Asn Val202Glu + Ser203Asp + Tyr205Leu + Pro209Gly +

Thr212Asp + Thr219Ser Alal99Gly + Pro200Asn + Tyr208Leu + Ser210Asp +

Asn211Asp + Asn217Asp Pro200Ser + Gly201Ser + Val202Asp + Tyr208Gln +

Ser210Glu + Asn211Asp Val202Asp + Thr207Gln + Tyr20δThr + Ser210Glu +

Thr214Gln + Asn217Glu Thr207Gln + Asn211Asp + Thr214Glu + Leu21βHis +

Asn217Asp + Gly21δGln Ser203Glu + Thr207Ser + Pro209Gly + Asn211Asp +

Tyr213Gly + Ser215Asp Ser203Glu + Tyr205Val + Pro209Gly + Asn211Asp +

Ser215Asp + Thr219Gly

TABLE28

Loop 6 - Heptuple Substitution Mutation Variants

Alal99Ser + Pro200Ser + Val204Met + Tyr208Ser +

Tyr213His + Thr214Gln + Leu216Ala Alal99Ser + Gly201Ser + Val202Met + Thr207Gln +

Ser210Glu + Thr214Ser + Asn217Gln Gly201Gln + Val204Gly + Tyr205Ala + Thr207Gln +

Tyr206Gly + Asn211Gln + Gly216Asp Alal99Gly + Pro200Gly + Thr207Gln + Tyr208Thr +

Pro209Gln + Gly21δGln + Thr219Gln Alal99Gly + Pro200Ser + Val202Gln + Thr207Asn +

Asn211Ser + Thr212Ser + Thr219Pro Pro200Gln + Val204Ala + Tyr206Leu + Thr212Glu +

Tyr213Met + Thr214Asn + Leu216Gln Ser203Asp + Val204Met + Thr207Gln + Tyr206Met +

Pro209Gln + Asn217Ser + Thr219Asn Alal99Gln + Pro200Ser + Gly201Gln + Tyr205Ala +

Tyr213Cys + Thr214Gly + Gly21δGlu Alal99Thr + Pro200Ser + Thr207Gln + Tyr20δGln +

Thr212Ser + Gly21δAsp + Thr219Pro Gly201Ser + Val204Pro + Tyr20δSer + Thr212Gly +

Tyr213Val + Thr214Pro + Ξer215Asp Pro200Gln + Tyr20δCys + Pro209Gln + Thr212Gly +

Tyr213Pro + Ser215Glu + Thr219Gly Val204Met + Thr207Gln + Pro209Gly + Thr212Gly +

Tyr213Cys + Thr214Gly + Leu216Met

Pro200Gly + Ser203Asp + Thr207Asn + Pro209Asn +

Tyr213Ile + Leu21βCys + Asn217Ser Pro200Asn + Gly201Asn + Tyr205lle + Pro209Asp +

Tyr213Met + Thr214Asn + Leu216Val Alal99Asn + Ser203Glu + Val204Met + Pro209Gly +

Tyr213Leu + Leu216Ala + Gly218Asn Alal99His + Thr207Pro + Tyr213Ser + Thr214Asn +

Asn217Ser + Gly21δSer + Thr219Glu Alal99Ser + Gly201Pro + Val204Gln + Thr207Ser +

Thr212Asn + Tyr213Pro + Gly21δGlu Alal99Ser + Pro200Asn + Tyr205Met + Tyr20δMet +

Thr212Asn + Thr214Gly + Leu216Met Gly201Gln + Tyr205Asn + Thr207Asn + Pro209Asp +

Thr212Gly + Tyr213Val + Leu216Ile Pro200Asn + Tyr205Glu + Tyr206Met + Tyr213His +

Thr214Ser + Asn217Gln + Thr219Gln Alal99Gln + Pro200Gly + Val202Met + Tyr208Ile +

Thr212Asp + Leu216Met + Gly218Pro Alal99Ser + Pro200Gly + Gly201Gln + Val202Asn +

Asn211Gln + Leu216Val + Gly21δPro Alal99Asn + Val202Ser + Tyr205Cys + Thr207Gly +

Tyr213Leu + Thr214Asp + Asn217Ser Gly201Pro + Val202Asn + Val204Asn + Tyr205Ser +

Asn211Glu + Thr212Ser + Gly216Asn Alal99Ser + Val204Cys + Thr207Ser + Asn211Ser +

Thr214Gln + Ser215Glu + Asn217Gln Pro200Ser + Val202Met + Tyr205Gly + Tyr208Pro +

Ser210Glu + Thr212Ser + Tyr213Gly Pro200Ser + Val202Gln + Ser203Glu + Tyr205Cys +

Thr207Gln + Tyr208Ile + Asn211Ser Alal99Pro + Gly201Ser + Val204Ala + Thr207Ser +

Pro209Gln + Tyr213Asp + Leu216His Gly201Ser + Ser203Asp + Val204Gln + Tyr208Thr +

Asn211Gln + Leu216Ser + Thr219Ser Alal99Thr + Gly201Ser + Tyr205Pro + Tyr213Pro +

Thr214Gly + Leu216Thr + Gly21δGlu Pro200Asn + Gly201Gln + Tyr206Val + Thr214Gln +

Ser215Asp + Gly218Ser + Thr219Asn Pro200Gln + Val202Met + Val204Gly + Tyr208Gly +

Asn211Ser + Tyr213Pro + Gly218Asp Alal99Asn + Pro200Gly + Tyr205Thr + Tyr20δVal +

Thr212Gly + Tyr213Ser + Leu216Val Alal99Ser + Gly201Ser + Val202Gly + Tyr205Leu +

Thr214Asn + Leu216Ala + Gly21δGlu Alal99Ser + Gly201Gln + Val204Ser + Tyr206Asn +

Pro209Ser + Thr214Asn + Ser215Glu Alal99Pro + Gly201Asn + Tyr20δCys + Tyr213Gly +

Thr214Glu + Asn217Ser + Gly21δGln Alal99Pro + Val202His + Tyr20δMet + Pro209Asn +

Thr212Asn + Thr214Glu + Ser215Asp Alal99His + Val202Gly + Val204Ser + Thr207Gln +

Pro209Asp + Ser210Asp + Asn211Ser Alal99Gly + Pro209Glu + Ser210Glu + Thr212Gln +

Tyr213Ala + Leu216Pro + Thr219Asn Alal99His + Gly20lGln + Val202Met + Val204His +

Thr20 ^" 7Gly + Pro209Glu + Ser210Asp Alal99His + Val202Gln + Thr207Gly + Thr214Gln +

Leu21βThr + Asn21 ^~ ?Asp + Gly218Glu Giy201Gln + Val204Thr + Tyr205Pro + Thr207Gly +

^" l_eu2lβAsn + Asn217Asp + Gly218Asp Gly201Ser + Tyr20δThr + Pro209Gln + Tyr213Ser +

Asn217Asp + Gly21δGlu + Thr219Gly Gly201Ser + Val202Cys + Tyr205Val + Thr207Gln +

Tyr213Ile + Leu216Asp + Asn217Asp Gly201Asn + Val202Glu + Ser203Glu + Val204Gly +

Thr212Gly + Leu216Val + Thr219Pro Val202Asp + Ser203Glu + Val204Asn + Thr207Pro +

Tyr208Leu + Thr212Ser + Asn217Gln Gly201Gln + Tyr208Ser + Pro209Asn + Asn21lGln +

Tyr213Asp + Thr214Asp + Asn217Gln Alal99Asn + Pro200Asn + Val204Thr + Ser210Asp +

Asn211Glu + Tyr213Leu + Thr214Pro Gly201Ser + Val202Ala + Tyr208Met + Ser210Asp +

Asn211Asp + Thr212Asp + Asn217Ser Alal99Ser + Pro200Gly + Gly201Asn + Val202Glu +

Leu216Cys + Asn217Asp + Gly218Asn Alal99Asn + Pro200Ser + Ser203Asp + Tyr205Cys +

Tyr208Asn + Thr214Gln + Ser215Glu Alal99Gln + Val202Gly + Ser203Glu + Tyr205Gly +

Tyr213Met + Ser215Glu + Asn217Ser Gly201Asn -t- Val202Gly + Ser203Asp + Tyr208Ile +

Tyr213Pro + Ser215Glu + Asn217Ser Val202Met + Ser203Glu + Thr207Asn + Thr214Gly +

Ser215Asp + Leu216Glu + Asn217Asp Alal99Gln + Ser203Glu + Val204Cys + Asn21lSer +

Thr214Gly + Asn217Glu + Thr219Pro Alal99Gln + Ser203Asp + Tyr205Thr + Thr207Ser +

Thr212Asn + Asn217Asp + Gly216Asn Pro200Ser + Val202Ser + Ser203Asp + Val204Gly +

Thr212Asn + Tyr213Met + Asn217Glu Ser203Glu + Tyr205Met + Tyr208Thr + Pro209Gly +

Asn217Glu + Gly218Asn + Thr219Ser Alal99Gln + Tyr205Leu + Tyr208Pro + Asn211Glu +

Thr212Asp + Tyr213Asp + Thr214Gly Gly201Pro + Val202Thr + Val204His + Tyr205Met +

Asn211Glu + Thr212Asp + Tyr213Asp Gly201Pro + Ser203Glu + Tyr205Asp + Thr207Ser +

Ser215Asp + Leu216His + Thr219Ser Alal99Asn + Val202Gln + Val204Ser + Pro209Gly +

Ser210Glu + Thr212Asp + Asn217Gln Pro200Gly + Gly201Pro + Tyr20δVal + Pro209Glu +

Asn211Asp + Thr212Gln + Thr219Ser Alal99Asn + Pro200Gln + Val202Ala + Val204Pro +

Thr207Asn + Pro209Glu + Asn211Glu Alal99Asn + Ser203Asp + Thr207Gly + Asn211Ser +

Ser215Glu + Asn217Asp + Gly21δGln

Pro200Gln + Ser203Glu + Val204Gly + Tyr213Pro +

Ser215Glu + Asn217Glu + Thr219Gln Pro200Gly + Val202Asp + Ser203Asp + Thr207Ser +

Thr212 ^" Gly + Ser215Glu + Gly216Ser Val202Glu + Ser203Asp + Tyr208Ser + Asn211Gln +

Tyr213Ala + Ser215Asp + Thr219Ser Alal99Gln + Ser203Asp + Tyr205Met + Thr214Asp +

Ser215Glu + Leu216Cys + Asn217Gln Gly201Gln + Ser203Glu + Tyr205Glu + Pro209Gln +

Asn211Ser + Thr214Glu + Leu216Glu Gly201Asn + Val202Cys + Ser203Asp + Tyr205Asp +

~Thr214Asp + Leu216Gly + Thr219Pro Alal99Gly + Val202Met + Ser203Glu + Val204Pro +

Tyr205Glu + Thr207Ser + Thr214Asp Gly201Ser + Val204Met + Pro209Gly + Asn211Ser +

Tyr213Cys + Thr214Glu + Leu21βAsp Gly201Pro + Val202Asp + Thr207Gln + Thr212Ser +

Asn217Asp + Gly218Asn + Thr219Asp Pro200Ser + Val204Gly + Tyr205Gly + Tyr20δCys +

Thr214Ser + Ser215Glu + Asn217Glu Gly201Asn + Tyr205Ile + Tyr213Ile + Thr214Asn +

Ser215Glu + Asn217Asp + Thr219Pro Pro200Ser + Val202Gln + Tyr206His + Thr212Pro +

Thr214Ser + Ser215Glu + Asn217Glu Pro200Gly + Val204Gln + Thr207Ser + Tyr213Leu +

Thr214Gln + Ser215Glu + Asn217Glu Thr207Gly + Ser210Asp + Asn211Asp + Thr212Gly +

Tyr213Asp + Asn217Gln + Thr219Gln Ser203Glu + Thr207Asn + Tyr20δHis + Asn211Gln +

Thr214Asp + Leu216Glu + Asn217Glu Gly201Pro + Val202Met + Val204Ser + Tyr205His +

Thr212Gly + Asn217Glu + Thr219Asp Alal99Asn + Val204Cys + Tyr206Cys + Tyr213Ala +

Thr214Glu + Leu216Asp + Asn217Glu Pro200Gln + Thr207Gly + Tyr20δCys + Thr214Asp +

Ser215Asp + Leu216Gln + Asn217Asp Alal99Gln + Val204His + Asn211Ser + Thr212Ser +

Thr214Glu + Ser215Glu + Asn217Glu Val202Asp + Ser203Asp + Tyr205Ala + Thr207Gln +

Tyr213Met + Thr214Glu + Ser215Asp Alal99Gly + Pro200Gln + Val204Cys + Thr212Glu +

Thr214Glu + Ser215Glu + Leu216Gln Val204Ala + Thr207Gly + Ser210Asp + Asn211Asp +

Thr212Glu + Thr214Glu + Asn217Ser Gly201Pro + Pro209Ser + Ser215Glu + Leu216Glu +

Asn217Asp + Gly21δSer + Thr219Asp Pro200Asn + Gly2θ ^" lPro + Ser203Asp + Thr207Gln +

Asn217Asp + Gly21δSer + Thr219Glu Alal99Pro + Val202Gly + Ser203Asp + Pro209Gln +

Thr212Asn + Ser215Glu + Gly21δAsp Alal99Gln + Pro200Ser + Gly201Ser + Tyr205Gln +

Ser210Asp + Thr212Glu + Thr214Asp Alal99Pro + Pro200Ser + Thr207Gln + Ser210Glu +

Thr212Asp + Thr214Asp + Thr219Ser Alal99His + Ser203Asp + Val204Gly + Thr207Gly +

Asn211Gln + Thr214Ser + Gly218Glu Gly201Asn + Val202Asp + Val204Gly + Tyr208Val +

Asn211Gln + Asn217Ser + Thr219Asp Val202Asn + Tyr205Glu + Thr207Gln + Tyr20δLeu +

Thr214Asp + Asn217Asp + Gly216Pro Alal99Asn + Gly201Asn + Ser203Asp + Tyr20δAsn +

Thr214Glu + Leu216Cys + Thr219Gly Val202Cys + Ser203Asp + Val204Ser + Tyr208Thr +

Thr214Asp + Asn217Gln + Thr219Pro Ser203Asp + Tyr208Pro + Pro209Ser + Asn211Ser +

Tyr213Met + Thr214Asp + Thr219Pro Alal99Asn + Pro200Gln + Ser203Asp + Thr212Glu +

Thr214Asp + Ser215Glu + Asn217Ser Pro200Asn + Gly201Asn + Val202Glu + Ser203Asp +

Thr212Gly + Ser215Glu + Thr219Asp Alal99Gln + Gly201Gln + Pro209Gly + Ser210Glu +

Thr212Pro + Tyr213Glu + Leu216Ala Pro200Ser + Val204Ser + Thr207Asn + Tyr20δLeu +

Ser210Asp + Asn211Ser + Tyr213Asp Alal99His + Ser203Asp + Tyr205Asp + Thr207Asn +

Tyr20δlle + Leu216Ser + Gly218Asp Alal99Gln + Gly201Pro + Val202Glu + Tyr205Glu +

Pro209Gly + Thr214Asp + Leu216Gln Gly201Asn + Val204Pro + Tyr208Cys + Ser210Glu +

Asn211Glu + Thr212Pro + Thr214Asp Alal99His + Pro200Asn + Tyr20δVal + Thr214Glu +

Ser215Glu + Leu216Glu + Thr219Asp Pro200Gly + Val202Glu + Tyr205Pro + Tyr206Ala +

Thr214Glu + Asn217Asp + Gly21δAsn Val202Glu + Tyr205His + Pro209Gln + Thr214Glu +

Ser215Asp + Asn217Ser + Gly21δAsp Pro200Ser + Tyr205Leu + Thr212Gln + Thr214Asp +

Ser215Glu + Gly21δGlu + Thr219Asn Ser203Asp + Val204Gly + Tyr205Ser + Thr212Glu +

Thr214Pro + Ser215Asp + Asn217Glu Pro200Asn + Val204Gln + Pro209Glu + Ser210Glu +

Asn211Glu + Thr212Asn + Ser215Asp Alal99Gln + Thr207Pro + Tyr208Cys + Pro209Gln +

Tyr213Asp + Thr214Asn + Leu216Asp Alal99Gly + Pro209Glu + Ser210Asp + Thr214Glu +

Ser215Glu + Gly218Pro + Thr219Gly Alal99His + Thr207Ser + Tyr20δThr + Pro209Gly +

Ser215Asp + Asn217Gln + Gly21δGlu Alal99Ser + Ser203Glu + Tyr20δAla + Asn211Gln +

Ser215Glu + Leu216Cys + Thr219Asp Gly201Pro + Val202Gly + Ser203Glu + Tyr205Thr +

Tyr208Ala + Ser215Asp + Thr219Asp Pro200Gln + Val202His + Ser203Glu + Asn211Gln +

Thr212Asp + Thr214Asp + Thr219Gly Gly201Pro + Ser203Asp + Thr207Gln + Ser210Glu +

Thr214Glu + Ser215Asp + Thr219Ser

Alal99Ser + Tyr205Gly + Thr207Ser + Ser210Asp +

Tyr213Glu + Ser215Glu + Leu216Glu Val204Thr + Thr207Pro + Ser210Glu + Asn211Glu +

Thr212Glu + Gly21δAsp + Thr219Ser Val202Asp + Ser203Asp + Thr207Gly + Pro209Asn +

Asn211Glu + Asn217Asp + Gly21δSer Pro200Ser + Ser203Asp + Val204Pro + Tyr20δlle +

Thr214Asp + Asn217Ser + Gly21δAsp Pro200Ser + Gly201Pro + Val202Gly + Ser203Glu +

Tyr205Glu + Pro209Asp + Tyr213Glu Alal99Gly + Gly201Gln + Val202Ser + Asn211Asp +

Ser215Glu + Leu216Asp + Asn217Glu Alal99Thr + Tyr205Gln + Thr207Gln + Pro209Gln +

Thr212Asp + Ser215Glu + Gly21δGln Alal99Gln + Val204Ser + Tyr205Glu + Thr207Asn +

Ser210Glu + Thr212Glu + Asn217Gln Tyr205Val + Tyr208Pro + Pro209Gln + Ser210Asp +

Thr212Glu + Ser215Glu + Leu216Gly Alal99Pro + Gly201Ser + Thr214Glu + Ser215Glu +

Asn217Gln + Gly218Glu + Thr219Asp Ser203Glu + Ser210Asp + Asn211Gln + Thr212Glu +

Tyr213Ser + Thr214Glu + Leu21βCys Alal99His + Pro200Gln + Pro209Asn + Tyr213Gln +

Thr214Asp + Leu216Glu + Thr219Asp Alal99Asn + Ser203Asp + Tyr205Glu + Thr207Asn +

Pro209Asn + Asn211Glu + Leu21βGlu Alal99Thr + Gly201Pro + Ser210Glu + Thr214Asp +

Ser215Asp + Asn217Ser + Thr219Ser Ser203Glu + Thr207Prc + Tyr208Ala + Ser210Asp +

Ser215Glu + Asn217Asp + Thr219Pro Val202Asp + Val204Cys + Tyr205Asp + Tyr208Ala +

Pro209Gly + Gly21δGln + Thr219Glu Val202Ala + Val204Thr + Tyr206Leu + Asn211Glu +

Thr212Glu + Ser215Asp + Leu216Glu Pro200Asn + Val202Gln + Thr207Gln + Pro209Ser +

Asn211Asp + Thr214Glu + Leu216Asp Alal99Asn + Val202Met + Thr207Asn + Pro209Glu +

Tyr213Glu + Ser215Glu + Asn217Asp Alal99Gly + Val202Cys + Val204Gln + Tyr205Asp +

Ser210Asp + Asn211Gln + Thr214Glu Alal99His + Tyr20δThr + Pro209Asp + Ser215Asp +

Leu216Met + Asn217Asp + Gly21δAsp Alal99Asn + Pro200Asn + Gly201Gln + Pro209Asp +

Thr214Ser + Ser215Asp + Leu216Asp Alal99Asn + Pro200Gln + Gly201Ser + Val202Asp +

Asn211Asp + Ser215Glu + Leu216Asp Alal99Thr + Val202Asp + Val204Cys + Ser210Asp +

Tyr213Ile + Ser215Asp + Leu216Asp Gly201Ser + Val202Glu + Tyr205Gly + Tyr20δLeu +

Ser210Asp + Ser215Glu + Asn217Asp Gly201Gln + Thr207Ser + Tyr206His + Pro209Glu +

Asn211Glu + Tyr213Asp + Asn217Glu Pro200Gly + Gly201Asn + Ser203Glu + Tyr20δCys +

Thr212Asn + Asn217Gln + Thr219Glu Alal99Gly + Gly201Asn + Val202His + Val204Ala +

Pro209Asp + Ser210Glu + Ser215Glu Gly201Asn + Val202Glu + Pro209Gly + Ser210Glu +

Thr212Glu + Tyr213Thr + Thr214Glu Val202Cys + Val204Ala + Tyr205Glu + Thr212Ser +

Ser215Glu + Asn217Gln + Thr219Asp Gly201Ser + Ser203Glu + Ser210Glu + Asn211Ser +

Thr212Glu + Ser215Glu + Leu216Pro Alal99His + Ser203Glu + Thr207Gln + Ser210Asp +

Thr212Asp + Ser215Asp + Gly216Gln Alal99Gln + Pro200Asn + Gly201Gln + Val204Asn +

Thr214Asp + Ser215Glu + Thr219Glu Alal99Pro + Gly201Pro + Ser203Asp + Tyr205Val +

Pro209Glu + Ser215Glu + Thr219Pro Pro200Gly + Ser203Asp + Thr207Gln + Asn211Glu +

Tyr213Glu + Leu216Thr + Asn217Gln Val202Asp + Tyr205Glu + Tyr20δGly + Thr212Asp +

Tyr213Ala + Leu216Ile + Thr219Pro Pro200Ser + Val202Glu + Tyr205Asp + Asn211Glu +

Thr212Asp + Tyr213Cys + Thr219Gly Val204Pro + Tyr205Asp + Asn211Asp + Thr214Pro +

Ser215Asp + Asn217Ser + Thr219Pro Gly201Asn + Ser203Asp + Pro209Gln + Tyr213Thr +

Thr214Asp + Gly21δAsn + Thr219Glu Pro200Gln + Val204Thr + Thr212Asn + Tyr213Glu +

Thr214Pro + Asn217Asp + Gly21δAsp Alal99Gln + Gly201Gln + Val204Asn + Tyr206Met +

Ser210Glu + Thr214Glu + Asn217Ser Gly201Pro + Val204Thr + Tyr20δGly + Asn211Asp +

Thr214Asp + Asn217Glu + Gly21δAsp Pro200Asn + Pro209Glu + Ser210Asp + Asn211Ser +

Thr214Ser + Leu216Cys + Thr219Asp Ser203Asp + Thr207Ser + Tyr20δAsn + Pro209Asp +

Ser210Asp + Thr214Gln + Asn217Gln Val204Ser + Tyr20δPro + Pro209Ser + Asn211Asp +

Leu216Met + Asn217Asp + Gly2lδAsp Tyr205Ser + Tyr20δCys + Pro209Asn + Asn211Asp +

Thr212Asn + Leu216Glu + Asn217Asp Pro200Gly + Ser210Asp + Thr212Pro + Tyr213Val +

Asn217Ser + Gly21δAsp + Thr219Asp Val202Cys + Tyr205Ala + Thr207Asn + Asn211Asp +

Tyr213Val + Ser215Asp + Leu216Asp Alal99Gln + Val202Glu + Ser203Asp + Val204Thr +

Pro209Gln + Asn211Asp + Tyr213Pro Gly201Pro + Val202Glu + Ser203Glu + Val204Gly +

Pro209Gln + Thr212Glu + Leu216Ala Pro200Gln + Val202Asp + Ser203Asp + Tyr206Ala +

Thr212Glu + Leu216Val + Gly216Gln Alal99Thr + Gly201Gln + Val202Gly + Ser210Glu +

Asn211Asp + Ser215Asp + Leu216Ala Alal99Gly + Gly201Gln + Val204Asn + Ser210Asp +

Asn211Asp + Ser215Glu + Thr219Asn

Alal99Asn + Val202Ala + Ser203Asp + Val204Gln +

Ser210Glu + Thr212Glu + Leu21βGlu Val202Asp + Val204Asn + Pro209Ser + Ser210Asp +

Asn2IlAsp + Tyr213Asn + Leu216Met Val202Pro + Ser203Asp + Val204Ser + Thr212Glu +

Tyr213Asp + Thr214Asn + Gly218Asp Ser203Glu + Val204Pro + Tyr205Asp + Pro209Glu +

Thr212Gln + Tyr213Asn + Gly218Asp Pro200Ser + Thr207Gly + Ser210Glu + Thr212Gly +

Thr214Glu + Ser215Asp + Gly218Glu Val202Met + Val204His + Ser210Glu + Thr212Asp +

Tyr213Ile + Leu216Asp + Gly218Glu Ser203Glu + Tyr20δSer + Asn211Asp + Thr212Pro +

Thr214Asn + Ser215Glu + Gly218Pro Alal99Thr + Gly201Pro + Ser203Asp + Tyr205Gly +

Asn211Asp + Ser215Glu + Asn217Ser Pro200Gln + Gly201Asn + Ser203Glu + Pro209Asn +

Asn211Asp + Thr212Gly + Ser215Glu

TABLE29

Multi-loop Double Mutation Variants

Serlδ7Glu + Tyr213Ala Tyr205Cys + Asn211Asp Asnl62Gln + Tyr213Pro Serl56Asp + Leu216Gln Glyl56Asp + Val202Ala SerlOOAsp + Tyr205lle Gly 99Asn + Thr214Glu Thr 5δAsp + Glyl30Gln GlylδβGlu + Tyr208Pro Alal05Pro + Leu216Ile Serlδ7Asp + Tyr205Gln Leul25Asn + Serl87Glu Thr 65Gln + Thrl02Glu Tyrl03Ile + Tyr205Glu Thrl32Asp + Gly218Gln Asn211Ser + Thr212Asp Thr212Gln + Ser215Glu Thrl32Pro + Tyr205Gly Tyrl03Glu + Thrl32Ser GlylβδGln + Ser203Glu Gly βOPro + Thr 65Pro Gly 99Asn + Glyl53Asn Prol2δAsn + Val202His Alal05Glu + Glyl30Ser Thr212Gly + Gly21δGlu PhelδδVal + Tyr205Ser Asn217Glu + Thr219Gly Leu 95Gln + Ser215Asp Val 94Gln + Thr212Glu Asn βlSer + Ser203Asp Serl29Glu + Asnl54Gln Gly 62Glu + Thr219Asn Ser 97Glu + Tyr213His Gly 99Asp + Ilel64Thr GlylOlPro + Ser215Asp Thr 58Pro + Tyr205Gln GlylOlGln + Tyr20δGln Asn βlGln + Thr212Asp Thr212Asp + Leu21βlle Asn211Giu + Leu216His Glyl53Glu + Thrl63Gln Thrl32Asn + Tyr205Asp Asn βlSer + Ser203Glu Ser215Asp + Leu216Ser Gly βOAsp + Metl98Gln Serl58Glu + Tyr205Met Phel88Leu + Thr212Glu Glyl30Pro + Serl90Glu GlylOlAsp + Glyl26Ser Alal05Gly + Serl57Asp Asn βlSer + Serl31Asp Asn βlSer + PhelδδSer SerlOOGlu + Asnl62Gln

Thr 56Gly + Prol2δAsp SerlOOGlu + Tyr205Ala Glyl27Glu + Tyr213Thr Asnl54Glu + Leu216Gln Ser 97Asp + Alal99Pro Ser203Glu + Leu216Ser Glyl26Gln + Ser215Glu Gly βOSer + Asnl54Glu Thr 65Ser + Glyl56Asp Prol2δSer + Thrl63Gln Ser215Asp + Leu216Val Asn21lAsp + Tyr213Leu Val 94Ser + PhelδδVal Serl56Glu + Thr212Gln Gly 99Pro + Asnl54Gln Val202Ser + Tyr205Glu Tyr205Val + Asn217Glu Val 94Gly + Ser215Asp Tyr205Glu + Tyr213Pro Asn 96Ser + Glyl27Glu Tyr205Thr + Thr214Gly Gly 62Ser + Thr 65Ser Thr 65Glu + Prol2δAsn Ser215Asp + Leu216Gln Asp 59Glu + Gly βOPro Ser 97Glu + Thr212Gln GlylOlAsp + Val202Asn Serl29Asp + Val202Gly Tyrl03Ala + Thrl32Ser Leu 95Met + Leu216Asp SerlOOAsp + GlylOlPro SerlOOAsp + Thr219Pro Val 94Asn + Ser215Asp Glyl53Asn + Tyr205Asp Thr 56Glu + IlelOβCys Thr 65Glu + Tyrl03Pro Val 94Met + Ser 9δGlu Ser210Glu + Gly21δSer Leul25Pro + Alal99His IlelOβGly + Thr212Glu Tyr205Ile + Thr214Pro Glyl30Glu + Tyr20δHis Serlδ7Asp + Leu216Val Thrl02Glu + Thr219Gln Leul25Ser + Glyl53Glu Glyl53Asn + Val202Asp Thrl63Glu + Tyr205Gly Serl29Glu + TyrlββPro Glyl53Gln + Pro209Gln Thr212Ser + Ser215Glu Thrl63Asp + Tyr205His

Asnl54Glu + Tyrl ^ββ Thr Asn 96Ser + Alal05Gly GlylOlSer + PhelδδGly Glyl53Glu + Gly216Asn SerlOOAsp + Thr212Pro Glyl26Pro + Glyl56Asp Leul25Ser + Ser215Glu Asp 59Glu + Thr 65Gly Glyl56Pro + Glyl59Asp Val202Cys + Tyr213Ser Leu 95Met + Serl31Glu Ser 97Asp + Gly 99Ser Asn βlGln + Leu 95Asp Glyl26Asn + Tyr205Glu Glyl59Pro + Gly218Asn Leu 95Thr + Leu216Ala Alal05Glu + Leu216Thr Thrl02Glu + Glyl30Pro Gly 64Gln + Ser215Glu Tyrl03Asp + Glyl26Asn GlylOlGlu + Glyl27Gln Thrl32Asn + SerlβOAsp Thr 58Pro + Glyl53Asn Ser203Glu + Thr214Ser Tyrl03His + Serl29Asp Ser 96Asp + Pro209Ser Asp 59Glu + Tyr205Ser Gly βOGln + Ser215Asp Thrl32Asp + Val202Asn Gly218Ser + Thr219Gly Serl90Asp + Val202Thr Asp 59Glu + GlnlβlAsn Leu216Asn + Asn217Asp Val 94Asp + Tyr205Asn Tyrl03Ser + Ser203Asp Gly 99Pro + Phel8δGln Ser 97Asp + Leu216His Thrl63Asp + Leu2lβAsn Asn βlAsp + Thrl32Ser Leu 95Asn + Ser215Asp SerlOOAsp + Thrl32Gly TyrlββThr + Asn211Gln Thr207Pro + Leu216Thr IlelOβAla + GlylβδAsp Tyr205Asn + Ser215Glu Leu 95Asn + Val202Gln SerlβOAsp + PhelδδGly Gly 64Ser + Asn 96Gln GlylOlAsp + Asnl54Gln Gly 64Asn + Serl57Glu PhelδδAsp + Tyr213Asn Ser215Glu + Leu216Ile Tyrl03His + Serlδ7Glu

Asn 96Ser - Thr214Asn Gly 62Asr. ~ Ser 98Asp IlelOβSer * 3erl57Glu Thr 65Glu - Thr212Gly Glyl27Ser + Ser215Asp Glyl56Ser + Asn217Glu Ser 98Asp + Tyrl03Thr Glyl27Asn + GlylβδPro Glyl53Glu -r Leu216Met Serl29Asp + Glyl65Pro Asn βlAsp + Thrl63Gln Glyl26Asn + Tyr205Met GlylόSGln -- Ser215Glu Thr βSGiu * GlylOlPro Val 94Gln -r GlylβδGlu Tyrl03Ile -r Serl90Glu Asn217Gln + Gly218Ser Ser 98Glu i- Gly 99Asn Thrl02Asp ÷ Thr212Ser Thr212Ser Ser215Asp Glyl30Glu + Pro209Asn Prol28Glu * Glyl65Pro Thr 58Asn - Leul25Glu Leul25Hιs * Thrl63Ser Thr212Asp - Tyr213Ala Thr 65Pro + Ser210Glu Val 94Thr + Thrl32Gly Tyr205Cys + Thr214Gln Glyl27Gln + AlalδβGln Glyl27Glu + Tyr205Cys Ser 98Glu + Glyl30Ser Glyl27Glu + Phel8δTyr PhelδδAsp + Asn211Ser Gly 64Asn -r Leu216Asp Ser203Glu + Thr212Ser

Ser 98Glu + GlylOlPro Leul25Cys + Ser215Asp Leul25Cys + Glyl26Ser Gly 99Gln + Ser210Asp SerlβOAsp + PhelδδLeu Val204Gln + Ser215Glu Asn βlGln + Leu216Asp Leul25Gly + Serl55Asp AlalδβPro + Val204Gln IlelOβMet + SerlβOAsp Leul25Ile + Leu216Ser Gly βOSer + Ser 97Glu Glyl65Asp + Tyr213Gln Prol2δGlu + Asnl54Gln Gly βOAsp + Glyl26Pro

Ser203Glu -r Leu216Ala Glvl2oGlu + Val202Gly Thrl32Asp + Tyr213Ser Serl57Asp + Thr212Ser Glyl30Ser + Ser203Glu Gly 99Asn + Glyl30Ser Asp 59Glu + Glyl59Asn Glyl53Asn + Thr212A.sp Glyl53Asp + Tyr205Ser SerlOOGlu + Tyr205Thr Glyl26Asn + Asnl54Glu Serl90Asp ^■ > ^■ Tyr205Val Thrl02Glu + TyrlββThr Serl57Glu + Pro209Gly SerlOOAsp + Tyr205Leu Serl31Glu + Thrl32Asn Glyl27Asp + Leu216Thr Ser210Glu + Asn217Gln Asn βlAsp + IlelOβGln Alal05Pro + Tyr205Pro Serl90Asp + Leu216Cys Ser203Glu + Tyr205Thr Leu 95Asp + Asn 96Ser Tyr205Ala + Thr212Glu Thrl63Gln + PhelδδGlu Tyrl03Ala + Thr212Asp Serl55Glu + Gly21δAsn Val 94Thr + Thrl63Ser Thrl02Glu + Asn217Gln Glyl59Ser + Val202Glu Asnl54Ser + TyrlββAla Asp 59Glu + PhelδδGly Thrl63Gln + Gly21δSer Glyl26Ser + Thr212Glu Ser203Asp + Leu2lβAsn Glyl26Glu + Thr212Pro Val 94Pro + AlalδβGlu Serl29Asp + Glyl53Asn GlylβδSer + Ser215Asp Gly βOGln + Serlδ7Glu Ser203Asp + Leu216His GlylOlGlu + IlelOβMet Val 94Glu + Glyl27Gln Val202Gly + Leu216His Asp 59Glu + Thrl32Asn Val202Asn + Tyr205Pro Leu 95Ile + Ser215Glu Prol2δGlu + Leu216Pro Ser 97Asp + Tyr213Met Serlδ7Glu + Leu216Cys Asnl54Ser + Glyl56Gln

Leul25Gly + Glyl26Ser Asnl54Ser -r Leu216Asn

SerlβOAsp + Asn21lSer Ser210Glu - Gly218Gln Tyr205Ala - Thr212Asp Serl58Asp + Leu216Ser Thr212Gly + Ser215Glu Alal99Ser + Val202Ala Serl29Glu + Tyr205Asn Asnl54Gln + Gly20lGln Serl90Glu + Thr212Ser Thr 58Pro + SerlβOAsp Glyl27Glu + Glyl30Ser Asnl54Glu + Thr212Gly Glyl53Ser t GlnlβlAsn Tyr205Thr + Ser210Glu Leul25Gly + Pro200Gln Tyrl03Val + Thrl32Gly Glyl26Asp + Leu216Ala SerlOOGlu + Glyl27Asn Serl29Asp + Alal86Ser SerlβOGlu + Tyr205Ser Thrl63Glu ^ Ilel64His Glyl59Ser + GlnlβlGlu Asp 59Glu + Glyl59Pro GlylOlAsp + TyrlββCys Thr 58Asn -^ Serl29Glu Asn βlGlu + Thr2145er Glyl27Ser + Ser203Glu Glyl27Pro + Glyl30Asp Glyl27Glu -i- Val202Gly PhelδδMet + Ser203Glu Ser 97Glu + TyrlββAsn Gly βOGlu + Gly 99Asn Asnl62Glu + Asn217Ser Thrl02Asp -i- Leu216Gly Gly 99Gln + Alal05Thr Glyl53Ser + Ser203Glu Tyrl03Pro + SerlδδGlu Serlδ7Glu + Tyr205Pro SerlβOAsp + Val204Thr SerlOOAsp + Leu216His GlylOlAsn + Ser203Asp Asn 96Ser + Tyr205Pro Thrl32Gly + Thr212Pro IlelOβPro + Val202Cys Gly 62Ser + Pro209Glu PhelδδCys + Leu216Asp Leu 95Cys + Serl04Glu Serl04Asp + Tyr205Met

Tyr205Glu -- Thr214Gly Glyl53Glu + Leu2lβAsn Val 94Thr + SerlOOGlu Thr 65Asp + Val202His Val202Gln Ser215Asp Val 94Ser + Alal05Glu Thr 58Pro + Glyl53Asp AlalOSAsn + Ser203Glu

TABLE 33

Multi-loop Triple Mutation Variants

Gl 99Pro -r Asnl54Gln 4 Ser203Glu T rlόβAsp ^■ + ^■ Tyr205Thr - Thr214Gly Gly 625er * Thr 65Ser + Asn211Glu Thr 65Giu + Prol28Asn + Leu216Gln Val 94Thr Thr212Gln Leu216Met Serl29Asp + Thrl32Ser + Val202Gly Leu 95Met + Tyrl03Ala + Leu216Asp Val 94Met * Ser 98Glu 4 Tyrl03Pro Leul25Pro 4 Aial99His + Ser203Glu IlelOβGly + Tyr205Ile 4 Thr214Pro Serl29Glu + Tyrlβ ^β Pro + Tyr205Gly Prc209Gln 4 Thr212Ser 4 Ser215Glu Asnl54Glu 4 TyrlββThr + Tyr205His Asn 96Ser 4 GlylOlSer + Alal05Gly Glyl53Glu PhelδδGly + Gly21δAsn Glyl26Pro 4 Glyl56Asp + Thr212Pro GlylδβPro + Glyl59Asp + Val202Cys Leu 95Met 4 Ser 97Asp + Gly 99Ser Glyl26Asn 4 Glyl59Pro + Gly21δAsn Thr β5Gln * Leu 95Thr + Leu216Ala Tyrl03Asp + Glyl26Asn + Glyl27Gln Thr 58Pro 4 AlalOSGlu + Glyl53Asn Gly όOGln Tyr205Ser + Ser215Asp Thrl32Asp 4 Gly218Ser + Thr219Gly Tyrl03Ser 4 Phel88Gln 4 Ser203Asp Ser 97Asp + Gly 99Pro + Leu216His Asn βlAsp 4 Thrl32Ser + Leu216Asn Leu 95Asn 4 Thrl32Gly + Ser215Asp TyrlββThr 4 Asn211Gln + Leu216Thr IlelOβAla + Glyl65Asp + Thr207Pro Leu -95Asn + Val202Gln + Ser215Glu Gly 64Ser 4 GlylOlAsp + Asnl54Gln Thr 58Asn 4 Gly 64Asn 4 Serl57Glu Gly 62Asn Asn 96Ser 4 Thr214Asn Glyl30Asp + PhelδδAsn + Gly218Pro Gly 99Ser 4 Prol28Glu + Leu216Gly Val 94Ala + Pro209Ser + Ser215Asp Leu 95Thr + Glyl26Gln + Leu216Gly Asp 59Glu Glyl59Ser + Tyr205Pro Val202Gln + Ser203Glu + Tyr205Asn Thr 5δSer Serl90Glu + Tyr205Pro Leu 95Asn + Tyr205Ala + Pro209Glu Thr 58Asn 4 Glyl53Pro + Ser210Glu Serl31Glu + Thrl32Asn + Glyl65Asn Giyl27Ser 4 Thr212Asn + Ser215Asp Thr β5Gly 4 Thrl02Asp + Glyl27Asn Gly βOAsn 4 Tyr205Thr + Asn217Gln Alal99Ser Val202Ala + Thr212Gly Thr 58Pro + Glyl30Ser + SerlβOAsp Glyl53Ser + Asnl54Glu + Thr212Gly Leul25Gly + Tyr205Thr + Ser210Glu Tyrl03Val + Thrl32Gly + Pro200Gln SerlOOGlu 4 Glyl27Asn + Leu216Ala

SerlβOGlu AlalδβSer 4 Tyr205Ser GIyl59Ser ~ Thrlβ3GIu Ilel64His Phel88Met + Val202Gly + Ser203Glu Thrl02Asp + Alal05Thr Leu216Gly Thr 58Asp 4 Asn 96Ser 4 Tyr205Pro Ser 97Asp ^■ - Thrl32Gly 4 Thr212Pro IlelOβPro 4 Val202Cys + Ser215Asp Leu 95Cys 4 Phel88Cys + Leu216Asp Thr 65Asp 4 Val 94Thr Val202His Thr 58Pro 4 AlalOSAsn 4 Glyl53Asp Serl04Asp Alal05Gly + Ilel64Val Glyl26Gln + Ser203Asp Leu216Hιs SerlβOAsp + Gly218Gln + Thr219Asn Gly 62Ser 4 Alal05Glu + Prol2δSer Thr 58Gly ^ IlelOβGly + Leu216Asp Ser 97Asp + Thrl63Gln + Alal86Ser Thr 65Glu + Glyl26Gln 4 Leu216His Phel88Ile + Ser203Glu + Leu216Thr Thrl32Gln - GlylβδAsp Thr212Ser Serl29Asp + Asn211Ser 4 Leu216Thr Leul25Ile + Asnl54Ser Leu216Met Thr 65Pro + GlylOlPro + Thr212Asp Thrl02Asn + Thr212Gly + Leu216Asp Thr 65Gln + Tyr205Gln Leu216Met Leul25Asn + Pro209Gly + Thr214Glu Leul25Pro 4 Val202His + Ser215Asp SerlβOGlu + GlnlβlAsn + Tyr205Val Glyl26Ser + Thr212Glu + Gly218Asn Val 94Thr + Serl55Asp + Thr207Gly Serl57Glu + Tyr208Gln 4 Thr212Gln Tyr205Met + Thr212Gln 4 Ser215Glu Prol28Gln + Thrl32Pro 4 Tyr213Asn Gly βOSer + Glyl5βAsp + Pro209Asn Asnl62Asp + Thrl63Asn 4 Alal99Gly GlylβδSer + Tyr205Thr 4 Ser215Glu SerlOOAsp + Glyl56Pro Tyr205Gln Alal05His + Serl57Glu + AlalδβAsn Asnl62Gln + Serl90Glu + Val202Ser Gly 99Gln + Glyl26Gln 4 GlnlβlAsp Glyl26Asp + Glyl59Ser + Thr212Gly Gly 62Glu + Asnl54Gln + Leu216Gln Asnl54Asp + Tyr205Ser + Tyr213Met Asn βlAsp + Leu 95Ser Val204Gln Thrl02Gln + Glyl59Gln + Serl90Glu Prol28Asn + Glyl59Asn + Asn211Ser Glyl59Ser + Thrl63Gly + Alal99Thr Asn211Gln + Tyr213Gln + Ser215Glu Gly 99Ser + Leul25Gly + Ser215Glu

Thr 65Gly + Serl90Glu + Thr219Ser Val 94Met + Asnl62Ser + Leu216Met Leu 95Ser + Tyrl03His Glyl56Glu Thr 58Gly + Asn211Glu + Leu216Ala

Alal05Gly + Thr212Pro + Gly21δGlu

Serl58Asp + Glyl59Ser + Tyr205Pro Thr 58Gln + Serl58Glu + Thrl63Gly Thr 65Pro 4 Leul25Met + Thrl63Gly Gly 62Asn + Glyl26Pro + Ser210Glu Leu 95Met Thrl32Asp + Asn211Ser Tyrl03lle Glyl59Asp + Val202Gly GlylOlGln TyrlββPro + Asn211Gln Prol2δGly * Thrl32Pro + Thr214Gln Pro200Ser + Thr212Asp + Leu216Gly Thr212Asp + Leu216Met + Gly218Gln Thrl63Gln 4 Tyr205Cys + Thr212Pro Thr 58Gln + Ser 98Glu + Thr212Pro Thrl02Pro 4 Serl55Asp + Tyr205Asn Gly 62Ser 4 Gly 99Glu + Val202Ser Leu 95Asp + IlelOβGln + Glyl26Asn SerlOOGlu + GlylOlGln + Val202Ala Thr 58Ser SerlOOGlu + Ilelβ4Leu GlylOlSer - Phel88Asp Tyr213Gly Serl04Giu 4 GlylSβGln + Thr212Gly Thr 65Asp + Val 94Gly + Tyr205Met Ser203Glu 4 Thr212Ser + Tyr213Val Thr 65Ser 4 Leul25Glu + GlnlβlSer Thr 65Gly + Tyr205Asp + Tyr213Thr GlylOlPro + Serl 04Asp + Thrl63Gln Aial05Gly + Asnl54Asp + Pro209Gly Glyl26Gln 4 GlnlβlSer + Leu216Ala Glyl53Gln TyrlββCys + Giy218Asp Gly 99Gln 4 Val204Pro + Thr219Glu Thr 58Ser + Ser 97Asp + Thr212Pro A-snl62Ser + Metl98Gly + Val202Thr Val 94Ser + Leul25Asp + Tyr205Thr Serl57Asp + Tyr205Ser + Leu216Thr Glyl65Asn + Ser215Glu + Leu216Pro GlylδβAsn + Thrl63Glu + Alal99Ser Prol28Ser + Glyl56Glu + Thr219Gly Asn 96Gln + Glyl26Gln + Ser203Glu Glyl59Asp + Glyl65Pro + Tyr213lle Thr 58Gln + Glyl53Ser + Asn217Gln Prol28Ser 4 Thrl32Glu + Thr212Gly Glyl56Gln + Tyr205Pro + Ser215Glu Gly 62Ser Tyr205Ile + Leu216Asp Leul25Pro 4 GlyI59Glu + Glyl65Pro Thr 58Gln + Ser203Asp + Thr212Ser Thrl02Gln + Val202His + Thr214Ser Gly 99Glu + IlelOβGly + Gly218Asn Ser 97Asp + Leul25Val + Tyr213Ile IlelOβLeu + PhelδδGlu + Thr212Asn Thr 5δPro + Glyl53Pro + Asnlβ2Gln Glyl27Ser 4 Prol2δGln + Thr212Pro Tyrl03Pro + Ser203Asp + Tyr205Ile Thr 5δGln 4 Thrl63Ser + Tyr205Ser Val 94Cys + Ser215Glu + Gly21δPro Thrl02Pro + Glyl65Pro + Val202Thr

Thr 58Glu - Tyrl03Asn 4 Pro200Gln

PhelδδThr - Val202Asn 4 Asn217Ser

Gly 99Asn - Tyr2055er 4 Ser215Glu

Tr.rlOlGly - Glyl65Asp * GIy218Gln

Gly ^β OAsp - AlalββPro - Tyr2C5Asn

Tr.rl02Asn -r IlelOβAsn * Serl58Glu

Gly β2Ser ^■ - ^■ Ser 97Glu + Gly218Asn

IlelOβHis Serl29Glu + Thr212Asn

Glyl26Glu + Prol28Asn 4 Val202Asn

Asp 59Glu + Gly 64Asn 4 Tyr213Ser

Thrl63Pro + Val204Thr + Tyr205Cys

Serl29Glu - Asnl62Ser 4 Leu21βCys

GlylSβAsn PhelδδAla + Tyr205Hιs

Val 94Ser GlylOlSer 4 Leul25Asp

Asn 96Glu TyrlββAla + Leu21βCys

Tyrl03Gln Phel88Met 4 Ser210Glu

GlylOlSer + Thr212Pro + Thr219Pro

Serl04Glu + Glyl59Gln + Thr212Pro

Gly 62Asp Glyl26Ser + Leu21βPro

GlylOlAsp Gly218Pro 4 Thr219Asn

Serl57Glu + Tyr205Pro + Thr212Ser

Prc209Glu Ser210Glu 4 Gly21δSer

Asnl54Glu + Serl55Glu 4 Gly218Pro

Asr.lllGlu * Thr212Asp 4 Leu21βHis

Tyr205Gly - Ser215Glu LeullβAsp

Val202Ser 4 Ser215Asp + Leu216Asp

Thr 58Asp Asp 59Glu 4 Thr212Ser

Tyr205Glu Tyr213Pro 4 Ser215Asp

GlylδβSer Tyr205Asp 4 Ser215Glu

Gly βOGlu Ser 97Glu 4 Gly 99Asn

Serl55Glu + Asnl62Ser + Serl90Glu

Leu 95Asn Ser203Asp 4 Ser215Asp

Ser203Asp + Ser215Asp + Thr219Gln

Gly β2Asn Ser203Glu 4 Ser215Glu

Ser203Glu + Tyr205Pro + Ser215Asp

PhelδδAsn + Ser203Asp 4 Ser215Asp

Gly 62Asn 4 Ser203Glu 4 Ser215Asp

Ser203Glu + Ser215Asp + Leu216Ser

Glyl56Gln + Ser203Glu 4 Ser215Asp

Ser203Asp + Tyr205Ala 4 Asn217Glu

Tyr205Ala + Ser210Glu 4 Thr212Asp

Ilel64Cys + Ser210Glu 4 Thr212Asp

Serl5δAsp + GlnlβlGlu + Tyr205Pro

Asp 59Glu Asn βlGlu 4 Glyl30Ser

Gly 64Gln + Ser 9δGlu + SerlOOAsp

Ser 97Glu + Gly 99Asp + Tyr213Hιs

Asnl54Gln Serl57Glu + Serl90Asp

SerlδδAsp SerlβOGlu 4 Tyr205Cys

Gly201Pro + Ser203Glu + Tyr205Asp

Gly 99Gln + Serl29Asp + TyrlββGlu

SerlβOAsp + Asnl62Glu 4 PhelδδGly

Serl29Asp + Serl31Asp + Gly21δAsn

Asp 59Glu + Glyl65Asn 4 Pro209Glu

Val 94G17 Ser215Asp + Asn21 ^'7 Glu Vai202Glu + Tyr205Gln + Leu216Glu GiylSoGIu + 5erl58Glu + Tyr205Met Asn βlAsp + Asn 96Glu + Glyl27Ser Glyl59Asn + Phel88Asp + Gly218Asp Tyrl03Hιs + Asnl62Glu + Serl90Asp Ser 97Asp + SerlOOGlu + Gly201Asn Serl57Glu + SerlβOGlu + Tyr205Pro Glyl53Asp + Val202Ser 4 Gly218Asp Serl57Glu + Glyl59Glu + Serl90Asp Asp 59Glu + Asn 96Glu + Tyr205Ser Giyl53Ser + Asnl54Asp + GlylόδAsp AsnI54Glu + Glyl65Asp + Asn21lSer Serl55Asp + Glyl56Asp + Thr219Asp Ser210Asp * Tyr213Asp + Thr214Glu Asn βlGlu + AlalδβGly + Pro209Asp Asnl54Asp + Serl57Glu + Thr214Ser SerlOOGlu Leul25Glu 4 Tyr213Hιs Gly βOGlu -r Leu 95Glu + IlelOβLeu G y 99Glu -r GlylOlGlu 4 Tyrl03Glu Thr 58Glu 4 Th 65Glu IlelOβCys Ser 97Glu 4 GlylOlAsp + Val202Asn Gly 62Asp + Ser210Asp * Leu216Ala Asn βlAsp SerlOOGlu + Asnl62Ser Glyl27Glu + Asnl62Glu + GlylβδGlu IlelOβAsn 4 Glyl56Glu + Glyl59Glu SerlOOGlu + Glyl26Glu + AlalδβGln Gly 62Asp + PhelδδLeu + Thr212Glu IlelOβGln Ser215Glu + Gly218Glu Tyrl03Hιs + Serl55Glu Gly218Glu Alal86Glu + Val202Glu + Ser215Asp GlyI27Glu 4 Asnl54Glu + Tyr213Thr Serl57Glu + SerlβOAsp + Serl87Asp Giy β2Asp Leu21βGlu 4 Thr219Gln Thr 58Asp + Ser210Asp 4 Thr212Glu Serl55Glu Serl57Asp + Gly21δAsp Prol28Asp + Asnl62Glu + Asn217Ser Glyl56Asp + Glyl65Glu + Tyr205Asn Sei:158Glu + PhelδδAsp + Val202Met Thrl02Glu + Serl04Asp + Leul25Glu Ser 9δAsp + SerlOOAsp + Serl04Glu Gly 62Asp + Pro209Asn 4 Asn211Asp SerlβOGlu + Serlδ7Asp + Serl90Asp Glyl59Asp + Val202Asp + Ser203Asp Prol28Glu + Thr212Asp + Tyr213Asp Leu 95Glu + Tyr205Asp + Ser215Asp Asp 59Glu + Tyr205Glu + Ser215Glu Gly βOSer + Thr β5Glu + Leu21βAsp GlylOlSer + Asnl54Glu + Serl58Glu Prol28Asp + Ser203Asp + Ser215Asp Ser203Asp + Ser210Glu + Ser215Glu Serl04Glu 4 Ser203Asp + Asn217Glu SerlβOAsD + Ser203AsD 4 Leu216Asp

Ser203Glu - SerliOAsp + LeullβAsp Glyl27Glu - Serl29Glu + Ser215Glu Prcl28Glu 4 AlalδβGlu + Phel88Asp Asp 59Glu + Asn βlGlu + Ser203Asp Ser 97Asp + Gly 99Asp + Ser210Asp Asn 96Glα - SerlOOAsp + Thr212Glu Thr 65Asp + Thrl63Gln + Ser215Asp Ser 98Asp ~ Thrl02Glu 4 Leu216Gln Thri32Glu ->- Serl57Asp + Serl90Glu Serl29Glu "- 5erl57Asp + Serl90Glu Serl90Glu + Ser203Glu + Tyr205Glu Asn βlAsp Ser 98Asp + Tyr205Asp Serl29Asp + PhelδδGlu + Serl90Asp SerlOOGlu + Serl04Glu + Tyr205Met Glyl27Glu + Serl90Asp + Tyr205Leu Asp 59Glu 4 Ser203Glu + Pro209Asp GlnlβlGlu + Ser215Glu + Asn217Asp Asp 59Glu - Ser 97Glu 4 Serl57Asp Leul25Glu - GIyl53Glu 4 Ser203Glu Serl04Glu - Seri57Asp + SerlβOAsp Asp 59Glu - Asn 96Glu 4 Serl87Glu Serl29Asp + Thrl32Glu + Asnlβ2Glu Serl31Glu + Ser203Glu + Gly218Glu GlylδβAsp + Ser203Asp + Thr214Asp SerlβOGlu + Thrl63Asp + Ser215Asp Thr 58Asn 4 Gly 99Glu + AlalOδGlu Asp 59Glu + Phel88Leu + Thr212Asp Serl04Asp 4 Serl31Glu + Tyr205Glu Gly 62Asp + Serl04Asp + Serl31Asp Serl04Glu + Serl31Glu + GlylδβGlu Giy βOAsp + GlylOlAsp + Metl98Gln Ser 9δGlu + Ser215Glu + Gly21δAsp Tyrl03Asp + Serl29Asp + Tyr205Glu SerlOOAsp + IlelOβAsp + Ser215Asp Thr 65Asp + SerlOOGlu + Alal05Glu Asp 59Glu 4 SerlOOAsp + Tyr205Ile Glyl53Glu + Asnl62Asp + Ser203Glu Ser 98Glu + Thr212Glu + Ser215Glu Serl31Asp + Thr212Glu + Ser215Asp Alal05Asp + Thr212Glu Ser215Asp Serl58Glu + Thr212Asp + Ser215Asp Serl57Asp Ser203Glu + Thr219Asp Gly 62Gln Thr212Glu Leu216Asp Ser 97Glu + Serl87Glu 4 Asn217Asp Serl55Glu + Asn217Asp + Gly21δAsn Glyl59Glu + Serl90Glu + Asn211Glu Prol2δAsp + Serl90Glu + Ser215Asp Asp 59Glu + Glyl56Asp + SerlβOGlu Gly βOAsp + SerlOOGlu + Ser215Glu Glyl30Glu + Glyl53Glu + Tyr205Asn Thrl02Glu + Glyl53Glu + Asn217Asp Serl29Glu + Serl55Glu + GlnlβlAsn Tyr205Glu + Asn211Glu + Leu216Cys

TABLE 34

Multi-loop Quadruple Mutation Variants

Val202Gln Thr212Gly + Leu216Gln GlylOlAsp + IlelOβAla + Tyr205Pro Gly 64Gln + Thrl32Gly + Glyl65Gln Tyr205Glu + Tyr213Ala + Thr219Asn Val202Ser + Tyr205Cys + Asn217Glu Asnl54Glu + TyrlββThr + Tyr205His Alal05Gly + PhelδδGly + Gly21δAsn Val202Gln + Tyr205Thr + Thr212Ser Serl90Glu + Pro209Asn + Leu216Val Glyl27Ser + Asnl54Ser + Tyr205Leu Ser203Asp + Tyr213Met + Leu216Val Asn 96Glu + Ilel64Asn 4 Leu216Ile TyrlββGln + Thr212Asn + Leu216Val TyrlββAla + Tyr20δPro + Leu21βlle Ser 98Asp 4 Glyl30Pro + Tyr205Val Phel88Asn + Leu216Gly + Gly218Pro Glyl26Gln + Pro209Ser 4 Leu216Gly Ser203Glu + Tyr205Asn + Gly218Asn Leu 95Asn + GΪyl53Pro 4 Ser210Glu Serl31Glu + Thrl32Asn 4 Glyl65Asn Glyl27Asn + Thr212Gln + Thr214Glu Glyl26Asn + Glyl27Ser 4 Val202Asn SerlβOAsp + Asn211Ser + Thr212Gly Alal99Ser + Val202Ala + Tyr205Ser Thrl32Gly + Pro200Gln Tyr205Thr Thrl63Glu + Ilel64His + Tyr205Ser PhelδδMet + Val202Gly + Ser215Glu Asn211Gln + Thr212Pro + Ser215Asp Pro200Ser + Thr214Gln + Leu216Gly Thr212Pro + Leu216Met + Gly218Gln Val202Ser + Tyr205Asn + Gly218Asp IlelOβGln + Glyl26Asn + Val202Ala Ser203Asp + Thr212Asn + Leu216Asn Ser210Glu + Thr212Pro + Thr219Pro Glyl59Gln + Thr212Pro + Leu216Pro AlalδβGly + Thr212Gly + Ser215Asp Thrl32Pro + Leu216Pro + Asn217Gln PhelδδThr + Tyr205Gln + Ser215Asp Gly βOAsp + Val202Cys + Thr212Pro Val202Asp + Thr212Ser + Thr214Gly GlylβδGlu + Tyr205Ser + Tyr213His TyrlββAsn + Ser203Asp + Leu216His

;-. / 62Giu - Leu 95Met + Alal05Ser - Glyl25Ser

IlelϋβGln -- Serl87Glu * Tyr205Gly - Thr219Pro

-erlόOGlu * Tyr205Ser + Thr212Pro - Leu216Thr

Glyl CGlr. + Serl31Glu ->- Asnl62Gln + Leu21βAsn

; /-. t ON.:.- ^{, ■} Tnrlβ3Ser 4 Asn211Ser - Thr212Asn

Giyi65Gln ^■ - Phel88Leu 4 Val202Hιs + Thr212Glu

Val 94Cys + Prol2δAsp 4 Val202Thr + Tyr205Ala

Leu 95Thr 4 Glyl5βSer 4 Thr214Glu Leu216Ala

Giy 99Pro + Tyrl03Glu 4 GlylβδPro + Leu216Cys

Gly 64Asn ^ Thr 65Glu 4 TyrlββAla 4 Phel88Tyr

Tyrl03Gln + Glyl56Pro 4 TyrlββAsn + Ser215Glu

3iyl26Gln - Prol28Gln 4 Val202Thr - τhr212Glu

Gly 64Ser Val 94Met 4 Glyl30Asp Tyr213Gly

Gly 99Asr. - Val202Pro ÷ Thr212Asn Ser215Glu

Glyl'-lFro Asnl54Ser - Leu216Hιs + Asn217Glu

Tyrlύ3Asp Glyl53Pro + AlalδβAsn ^ Val202Ser

Leul25Thr + GlnlclAsn 4 Thr212Gly 4 Leu216Asn

Giy 64Pro + Val 94Hιs -r Ser210Asp Thr212Giy

Asn όlSer + Thr 65Gln 1- Serl90Asp + Val202Gly

Aial05Thr + Glyl53Gln 4 GlylδβGln + Asn211Gln

Asn βlGln + Tyrlββlle 4 AlalδβAsn + Ser215Asp

Gly βOGln + Tyrl03Gly 4 Glyl30Asn + Asn217Asp

Leu 95Prc ^■ - Tyr205Leu Asn211Gln + Ser215Glu

Tnrl' 2Asp 4 Glyl26Gln 4 Val202Asn + Thr212Gly

Val 94Thr + Glyl53Pro + AlalSβPro + Tyr205Glu

Ξerl04Asp 4 Alal05Asn Thrl63Ser + Tyr205Ala

Giy 64Ser 4 Val 94Asn 4 Glyl30Asp + Leu216Cys

GlylOlAsn + Leul25Asn 4 Gly201Pro + Leu216Ser

Giyl59Pro + Glyl65Pro 4 AlalδβThr + Thr212Pro

Prol28Gln + Serl29Asp + AlalδβSer + Asn217Ser

Gly βOAsn + Glyl59Ser 4 PhelδδVal + Ser203Glu

Ser 96Glu + Gly 99Asn Thrl02Asn + AlalSβPro

Thr 65Asn + Gly 99Gln 4 Thr212Ser + Leu216Asp

Val202Gln + Tyr205Thr 4 Tyr213Val + Ser215Glu

TnrI32Ser + Glyl56Ser Phel88Thr + Asn211Glu

Leui25Hιs 4 GlylδβSer ^ Thr212Glu + Leu21βAsn

Glyl26Ser + Tyr205Asp + Thr214Gln + Leu216Asn

AlalδβGly + Ser215Glu 4 Gly21δPro + Thr219Gln

Thr 5δGly + Gly βOAsn 4 Leul25His + Leu216Gly

Asp 59G1U + Val 94Gly + Tyr205Gln + Tyr213Ile

Serl55Glu + Metl9δThr 4 Val202Met + Tyr205Thr

Glyl27Asn + TyrlββPro 4 PhelδδLeu + Thr214Glu

Leu 95Cys + Glyl30Gln + Thr212Ser + Leu216Val

Asp 59Glu + Thr β5Gly + Leu 95Gly -^ Leu216Asn

Serl31Glu + Gly201Ser 4 Val202Gln + Tyr205Gly

Leul25Val 4 Tyr205Gln 4 Ser215Asp + Gly218Gln

Val 94Gly + Thrl02Ser 4 Ser203Glu + Leu21βAsn

Asp 59Glu + Gly βOGln 4 Thr212Gly + Leu216Cys

Alal86Gln + Gly201Gln 4 Val202Pro + Ser203Glu

Thr 65Ser + Ser 9δAsp + GlnlβlSer + Tyr205Cys

TyrlββAla + Serl87Glu 4 Tyr205Gly + Leu216Val

Gly 99Asp + Metl98Cys + Pro209Gly + Thr212Asn

Thr 65Pro + IlelOβGlv + Tvr205Gln + Leu21βGlu

_ tr « 95Ser -> ^. Thrl02Asn 4 Leu216Asp + Asn217Ser

Tr.r 58Gly - Glyl27Ser ~ Tyrl ^ββ Giu + Val202Asn

Val 94Pro - Tyr213Thr 4 Thr214Glu + Asn217Gln

Ser 97Giu + GlnlβiAsn -r TyrlββGly 4 Val202Ser

7yrl03Met + Thrl32G.y 4 TyrlββMet + Ser210Asp

Leu 95Met + GlylSβAsn 4 Yal202Glu + Leu216Gln

Ser 97Asp 4 Metl98Aia 4 Val204Thr + Tyr205Thr

GlylOlSer + Glyl53Pro + Asn217Glu 4 Thr219Pro

IlelOβMet + Thrl63Glu + Tyrlββlle + Thr212Ser

Glyl65Ser + Serl90Asp + Pro200Ser + Val202Met

Giy 62Ser + Glyl53Asp + Thrl63Ser + Gly201Gln

GlylOlGln + Thrl02Ser + Glyl53Ser + Val202Asp

Val 94Ala + Leu 95Gly + Leul25Thr + Glyl30Asp

Asn 96Asp + Tyr205AIa + Thr212Pro + Thr219Gly

Glyl53Gln + Tyr205Ile + Thr212Gly + Ser215Glu

IlelOβCys + Asnl54Glu + Tyr205Ala + Leu216Ala

Asn 96Asp + Gly 99Pro + Tyr205Val + Asn217Gln

Alal05Thr + Leul25Thr + Val204Aia + Tyr205Met

Thrl02Prc + Glyl56Pro + Tyr208Val + Thr212Asn

Giy βOSer + Phelδδlle + Thr212Ser + Leu216Asp

SerlOOAsp + Tyrl03Gln + Glyl59Gln + Leu216Val

Prol28Gly + Glyl65Gln + Ser203Glu + Leu216Ser

5erl29Glu + Thrl32Gln + Metl9δThr + Thr219Gln

Val 94Pro + Gly 99Asn + Thrl32Gly 4 Giyl59Asp

Serl55Asp 4 Glyl55Ser 4 Tyr205His + Thr212Gly

Gly 99Asp 4 PhelδδVal 4 Thr207Asn 4 Leu216Pro

Thrl02Gln + Glyl2βAsn + Glyl59Asn + Leu216Asp

Asp 59Glu 4 ThrI32Pro + Thrl63Pro + PhelδδThr

GlylOlSer + Alal05Glu + A.lalδβPro + Thr212Gly

Serl29Asp + Glyl53Asn + Glyl59Pro + Val202Met

Gly βOPro t Asn 96Asp + Glyl2βGln + TyrlββAla

Thr 65Gln + Gly 99Pro + Tyr205Ala + Ser215Asp

Val 94Gln + GlnlβlAsp + Tyr205Pro + Leu216Met

Asp 59Glu + Giy βOGlu + AlalδβGly + Gly218Asn

Ser 98Asp + Gly 99Glu + Phel88Met + Tyr213Ala

SerlβOAsp + GlnlβlAsp + Tyr205Ser + Thr219Gln

Alal05Gly + IlelOβCys + Tyr205Asp + Ser215Asp

Pro200Asn + Tyr205Asp + Asn211Gln + Ser215Glu

Tyr205Asp + Thr207Ser + Thr214Ser + Ser215Asp

Asn βlAsp + Thr βSGln + Ser 97Asp + Tyr205Ala

Leul25His + Ser203Giu + Ser215Glu + Leu216Asp

Thr 65Asn + Ser203Glu + Tyr205Cys + Ser215Asp

Giy 64Pro + AialδβSer + Ser203Asp + Ser215Asp

Prόl28Ser + Ser203Asp + Tyr213Ala + Asn217Glu

Val202His + Tyr205Vaϊ + Ser210Glu + Thr212Asp

Gly 99Asn + Glyl30Ser 4 Ser210Glu + Thr212Asp

Tyr205Ala + Ser210Glu + Thr212Asp + Gly218Gln

PhelδδAla + Ser203Asp + Tyr205Asp + Thr212Ser

Asnl54Glu + Serl55Asp + Thrlβ3Asp + Tyr213Thr

Gly 99Gln + Serl29Asp + TyrlββGiu + Leu216Thr

Thrl02Gly + Alal05Thr + Serl55Glu + Serl57Asp

Glyl27Pro + Serl57Glu + Glyl59Asp + Glnl ^β lAsp

GlylOlAsn + Asnl54Asp + Thrlβ3Gly + Gly21δGlu

Serl58Asp -r Thrl63Asp 4 Asn217Ser Thr214Asn 4 Ser215Asp 4 Asn217Asp Asnl54Asp + Serl57Asp + Serl90Glu Glyl53Glu 4 Thri63Asp Thr214Pro Serl57Glu - Glnl ^β lGlu 4 Tyr205Cys Ser18 ^" 'Asp + Serl90Glu + Thr212Gln Serl55Asp + Serl57Asp 4 Serl87Glu Ser203Asp Ser215Glu + Gly218Asp Serl37Asp - Serl90Glu + Thr212Ser Serl87Glu - Val202Asn ^ Leu21όSer GlylβδAsp + Thr212Gln + Leu216Cys Serlδ7Asp + Pro200Ser + Tyr213Val Thrl02Asp + AlalOSPro + Tyr213Val Glyl5βGlu + Asnl62Asp + Thr212Ser SerlOOAsp + Tyr205Val + Leu216Gly Asnl54Glu + Serl57Asp 4 Asnl62Glu Thr212Glu + Ser215Glu + Leu216Glu Gly 62Glu - Ser 9δAsp + Thr214Gln Ser 98Glu -r Glyl30Ser 4 Val202Pro Tyr205Met 4 Thr212Asp + Thr214Gly PhelδδAsp + Val202Asp + Asn211Ser Pro209Ser 4 Asn211Asp + Thr212Glu Glyl26Gln + Glyl27Asp + Serl55Asp Val 94His + Asnl54Ser + Ser210Glu Glyl26Glu + Serl55Asp + Gly201Pro TyrlββPro + Thr212Glu + Ser215Glu Ser215Glu + Leu216Ala + Thr219Ser GlylOlSer 4 Ser210Asp 4 Thr212Glu Glyl26Asp Glyl27Asn + Leu216Pro Gly 99Asp + ThrlC2Asp + Serl04Asp Thr212Asp + Tyr213Pro 4 Ser215Glu Prol2δSer + AlalδβGlu + Ser203Asp Thrl02Asp ^ Serl29Asp + Leu2lβAsn Leu 95Asp + Glyl59Asn + Leu216Gly Ser 97Asp + Ser210Glu + Thr212Gln Tyr205Pro 4 Thr212Ser + Thr219Glu Tyrl03Pro 4 Ser215Asp 4 Asn217Asp Thrl63Glu + Serl87Glu + Asn211Gln TyrlββGly 4 Serl87Glu + Leu216Met Glyl59Glu + SerlβOAsp 4 Pro209Gln Serl29Glu -r SerlδδAsp + Glyl59Asp Serl31Asp + Serl55Glu + Thr212Asn Asnl54Glu + Serl55Asp + GlnlβlAsn Glyl30Glu + Glyl56Glu + GlnlβlSer SerlOOAsp + Ser203Glu + Leu216Ser Serl29Asp + Val202Asn + Ser215Glu Serl04Glu + Thr212Pro + Ser215Glu Serl57Glu + Serl56Asp + Ser215Asp Asn 96Asp + Ser 97Asp + Serl87Asp Ilel64Gly + Ser215Asp + Leu216Asp Ser215Asp + Leu216Glu + Gly218Gln Serlδ7Asp + Ser215Glu 4 Leu216Glu Serl04Asp Thr212Glu 4 Tyr213Glu

Thrl02Glu + Serl ^β OGlu 4 GlnlδlAsp Leu216Val Asn βlGiu + Gly 62Glu 4 Ser203Glu Thr212Gly Tyr205Val + Ser2lOAsp 4 Asn21lGlu Leu216Glu Asp 59Glu + Glyl26Ser Tyr205Glu Ser215Glu r- j 23Asn 4 Serl29Asp Tyr205Asp Ser215Asp

GlylOlGlu 4 Tyr205Glu Ser215Asp Leu216His

Serl55Glu 4 ilel64Ser Tyr205Asp Ser215Asp

Tyrl03Ala + TyrlββGlu Tyr205Glu Ser215Glu

Giy βOGlu + Ser 97Glu Gly 99Asn Asnlβ2Glu

Serl87Glu + Ser203Glu Tyr205Asp Tyr213Pro

Serl57Glu + Ser203Asp Tyr208Pro Ser215Glu

Ser 98Glu + Ser203Glu Tyr205Val Ser215Glu

Glyl27Gln + SerlβOGlu Ser203Asp Ser215Glu

Val 94Glu 4 Ser203Asp Ser215Asp Leu216Pro

SerlOOAsp + Ser203Glu Ser215Glu Leu216Thr

Se l57Glu Phel88Thr Ser203Asp Ser2I5Asp

Val 94Gly 4 Leu 95Glu Ser203Glu Ser215Asp

Phel88Val 4 Serl90Asp Ser203Asp Ser215Glu

Serl58Asp + Ser203Asp Ser215Asp Asn217Ser

Serl57Asp + Ser203Asp Ser215Asp Thr219Gln

Glyl26Pro + Glyl53Glu Ser203Glu Ser215Glu

Serl31Glu + Glyl65Asn Ser203Glu Ser215Asp

Tyrl03Glu + Glyl53Asp Serl90Glu Tyr205Val

Glyl59Glu 4 Ser203Glu Leu216Gln Asn217Glu

SerlOOAsp + Glyl53Gln Thrl63Asp Serl90Asp

Thrl02Asp + Glyl27Glu Serl55Asp Tyr213Pro

Asn βlGlu + Asnl54Glu Serl90Asp Val204Met

A.snl54Asp + GlnlβlSer Serl90Asp Ser203Glu

Thr 65Gly 4 Glyl27Asp GlylβδAsp Ser203Asp

Asn 96Asp + Gly 99Glu GlylOlAsn Serl87Glu

Thrl02Asp + Alal05Glu GlnlβlSer Pro209Asp

Glyl27Glu + Serl29Glu Glyl30Ser SerlβOAsp

Thrl02Glu 4 Serl04Glu Pro200Asn Thr212Glu

Ser 96Asp 4 SerlOOGlu Prol2δGly Glyl59Asp

Glyl59Asp + GlnlβlAsp Val204Gly Ser215Glu

Serl55Glu 4 Phel86Glu Tyr205Asn Ser215Glu

Serl29G1U 4 Thrl32Asn Glyl65Asp Gly218Asp

SerlOOGlu 4 Glyl53Asp Glyl65Gln Thr219Asp

Glyl26Asp 4 Serl31Glu Thr207Gly Gly218Gln

Asn βlAsp + Gly 99Glu Tyrl03Thr Serl31Asp

Gly 99Asp 4 Ser203Asp Tyr205Asp Thr212Pro

Serl90Glu + Ser203Glu Tyr205Asp Leu216His

Gly βOPro + Serl57Asp Ser203Glu Tyr205Asp

Serl57Glu Thrl63Glu Pro209Ser Ser215Glu

Gly βOAsn Serl57Asp Thrlβ3Glu Asn211Asp

Thrl02Glu Glyl27Asn Glyl30Glu Tyr205Gln

Asn βlGlu Glyl53Ser Pro209Asp Ser215Glu

Asp 59Glu Asn βlGln Leu216Asp Gly216Asp

Leul25His Serl29Asp Serl31Asp Val202Glu

IlelOβCys Tyr205Asp Ser210Glu Leu216Asp

Ser 97Glu Glyl56Asp Thrl63Asp Thr212Gln

Serl31Asp Asnl54Gln Ser215Glu Asn217Asp

Serl04Glu Thr212Ser Ser215Asp Asn217Glu

SerlΞ7As? -r SerI87Asp + Ser203Asp -r Thr212Gly

Asn βlAsp + Asn 96Asp 4 Alal05His + Asnl62Asp

Asn βiA≤p + Asr. 96Glu 4 Glyl27Ser -r Glyl30Asp

Thr 58Gly + Serl58Glu Asn211Asp ->- Tyr213Asp

Serl58Asp + AsnI62Asp + Thr212Asp - Leu216Thr

Gly 99Ser + SerI57Asp + Asnl62Glu + Leu216Asp

Glyl59Gln 4 Asnlβ2Giu 4 Serl90Asp 4 Ser215Glu

Ser 97Asp + SerlOOAsp + Glyl26Asn 4 SerlβOAsp

Gly βOGlu + Ser 98Glu 4 Glyl53Gln + AlalδβAsp

Serl57Asp + SerlβOAsp + Thr212Asn 4 Ser215Glu

Gly 62Pro + Serl31Glu + Glyl65Glu + Thr219Asp

Seϊ:190Asp + Val202Glu ^■ >- Ser215Glu + Leu216Ala

Asn 96Giu + Glyl53Asp + Glyl56Asp + GlnlβlAsn

Tyrl03Glu + Serl31Asp + Ser203Asp + Leu21βCys

G!V130G1U + Serl57Asp + Serlδ7Asp -t Tyr20δHis

Asn βlAsp + Thrl63Gln + Ser203Asp + Thr214Glu

Serl55Asp + Serl5δAsp + Ser203Asp + Tyr205Thr

Serl55Asp + Glyl56Ser + Ser215Glu + Thr219Asp

Gly βOGin 4 Thr 65Gln + Asn 96Asp + Leul25Glu

Asp 59Glu 4 Asn 9βGlu + Ser203Glu + Thr214Asn

Asp 59Glu + Asn 96Asp + Ser215Asp + Leu216Ile

Gly 62Asp + Ser 97Glu + Alal05His + Serl31Glu

Leul25Asp 4 Glyl65Asp + Ser203Asp + Tyr213Gln

Leu 95Glu + TyrlββAsn ^ Ser203Glu 4 Thr214Glu

GlylOlAsp + AsnIβ2Asp + Glyl65Glu 4 Leu216Asn

Gly 99Glu + Tyrl03Glu + Serl29Glu + Tyr205Asn

Asp 59Glu + Asn 96Ser + Thr212Glu + Ser215Asp

Asn βlGlu + SerlOOAsp + AlalδβGly + Thr212Glu

Thr 65Giy + Ser 97Glu + GlylOlGlu + Glyl30Glu

AlalδβGlu + PhelδδTyr + Thr212Asp + Asn217Asp

Asn βlGln + Glyl56Glu + Ser215Glu + Thr219Glu

Gly 99GIn 4 Serl29Glu 4 Serl55Glu + Gly21δGlu

SerlβOGlu + Thrlβ3Asp + Thr212Ser 4 Ser215Asp

Asn 96Asp + Thrl02Asp + Serl57Glu + AlalδβHis

Gly 99Glu + Alal05Asp + Thr214Asn + Leu216Val

Asnl62Gln + AlalδβAsp + Tyr205Ser + Leu216Asp

Ser 97Glu + Glyl56Glu + Glyl59Asp + Val202Asn

IlelOβCys + Serl29Glu + Serl55Glu + AlalδβGlu

SerlOOGlu + Leul25Gly + Glyl27Glu + Ser203Asp

Thr 65Asp + Serl31Asp + Ilel64Asn + Thr212Asp

Ser 97Glu + Thrl02Glu + GlnlβlSer + Tyr205Leu

Asp 59Glu + Gly 99Glu + Ser203Asp + Tyr205Asn

Asp 59Giu + Gly 99Asp + Glyl26Ser + Thr214Glu

SerlβOAsp + Pro209Asn + Ser215Asp + Gly218Glu

Serl55Giu + Glyl59Asn + Pro209Glu + Gly218Glu

Serl55Asp + Asn211Asp + Tyr213Met + Gly21δAsp

Serl29Asp + Thrl32Glu + SerlβOGlu + AlalδβSer

Val 94Glu + Ser 97Asp + GlylOlPro + Ser203Glu

GlylOlAsp + Glyl26Asp + Thrl32Asp + Thr212Asn

Ser 98Glu + Glyl27Glu + Thrl63Asp + Thr212Asn

Gly 99Asp + GlylOlPro + Thrl02Glu + Ser203Glu

Glyl59Asp + Thr212Asp + Ser215Asp + Asn217Ser

Serl5δGiu + Asnl62Gln + Thr212Glu + Ser215Asp

^' al 94Pro - Serl55Asp 4 Thr212Glu 4 5er215Glu

Gly 99Gln - IlelOβAsp 4 Thr212Glu 4 Ser215Glu Thr 58Asp + Tyr205Pro + Thr212Glu 4 Ser215Asp Asp 59Glu + Ser 9δGlu 4 Ser203Asp 4 Tyr205Gly Asp 59Glu + SerlOOAsp Serl04Asp + Alal05Pro Asp 59Glu ^■>■ SerlOOAsp ^■ >- Serl04Asp 4 Tyr205Ile Prol23Glu ⁺ Asnl62Asp + Thr212Glu + Leu216Cys Asn 96Gln + Thrl02Asp + Serl29Glu + Asnl62Glu Ser 97Glu + Glyl30Asn 4 Serl55Asp + AlalδβGlu Alai05Glu ^■ + ^• Serl55Glu 4 Glnl ^β lAsp + Tyr205Thr Gly 62Asp 4 Serl58Asp + Ser215Asp + Leu2lβAsn ThrI02Asp -r Glyl26Asp + Serl58Asp + Thr212Ser Thr 58Asn 4 Gly 64Pro 4 Thr 65Glu + Val 94Asp Serl90Glu 4 Ser203Glu + Tyr213Glu + Leu216His Glyl59Asp + Ser203Asp + Tyr205Cys + Tyr213Asp IlelOβAsp 4 Glyl30Asp + Ser215Asp + Asn217Gln Ser 98Glu + Thrl02Asp + Ser203Asp + Thr214Gly Gly 99Gln 4 Serl04Asp + Glyl30Asp + Asnl54Glu Serl04Glu + Glyl26Ser + Glyl30Asp + Asn217Glu Asn 96Gln + SerlOOGlu + Serl04Glu + Tyr205Asp Asn βlGln + GlylOlAsp + Serl57Asp + GΪyl65Glu Thrl02Ser + Val202Cys + Thr214Glu + Gly21δGlu Asn βlGln + Serl04Glu 4 Glyl27Glu + Glyl65Prc Thr 65Gly Tyr205Glu + Asn211Glu + Leu216Cys Gly 99Gln + Glyl59Ser 4 Pro209Asp + Leu216Asp Asp 59Glu + Serl5δAsp + Tyr205Pro + Thr212Glu Asp 59Glu + Serl31Glu 4 Thr212Glu + Leu216Ile Alal05Asp + Leul25Glu + Asn211Glu + Leu216Ile Thr 58Asp + Glyl56Ser 4 Asn211Asp + Ser215Glu Serl57Glu 4 Glyl59Ser + Glyl65Pro + Thr219Glu Asnl62Asp Tyr205Gln + Thr212Glu 4 Leu216Asp SerlOOGlu + Metl96Gln 4 Thr212Asp 4 Leu216Glu Gly 64Gln + Prol2δGlu + Thrl32Asp + Thrl63Gly Gly β2Gln 4 Prol2δAsp + Thrl32Asp 4 Leu216Thr GlylOlAsp 4 IlelOβMet + Leul25Gly + Prol2δGlu Thr 65Glu + Asn 96Glu + Glyl56Asp + Leu216Cys Serl04Asp + Leul25Gln 4 Serl29Asp + Asnl62Gln Asp 59Glu + Glyl30Glu + Glyl53Glu + Tyr205Asn Serl29Asp + Serl55Asp + Tyr205Val + Leu216Asp Serl29Glu + Serl55Glu + GlnlβlAsn + Ser210Glu Gly βOAsp + Glyl27Asp + Asnl62Glu + PhelδδAsn Gly 62Gln + Ser 9δGlu + Serl87Asp + Ser203Glu Serl04Asp + Glyi30Gln 4 Serl87Asp 4 Ser203Glu Asn βlAsp + Tyrl03Ser 4 Thr212Asp + Thr219Asp Asp 59Glu + GlylOlAsp + Glyl59Pro + GlnlβlGlu Gly βOGlu + Leul25Ser + Glyl53Glu + Val202Asp Thrl32Glu + Serl57Glu + Thr212Gly + Thr219Asp GlylOlGlu + LeuI25Cys + Glyl30Glu + Thr219Asp Gly βOAsp + Prol2δGlu + Serl57Asp + Tyr205Cys Prol2δGlu + Asnl54Gln + Serl57Asp + Ser203Asp Ser 97Asp + Alal05Asp + Ser203Asp Thr212Gln SerlOOAsp + Serl5δGlu 4 TyrlββAsp 4 Phel88Pro Tyr205Thr + Asn211Glu 4 Ser215Glu + Leu2lβAsn

TABLE 35

Multi-loop Quintuple Mutation Variants

Tyrl03Cys + Glyl26Gln + Glyl56Glu 4 Val202Gln +

Thr212Gly Val 94Mef + GlylOlAsn 4 Thrl02Asn Tyr205Leu 4

Gly218Asp Ser 98Asp + Tyrl03His 4 Leul25Cys 4 Glyl59Asn 4

Metl98Cys Leu 95Met + GlylOlPro + Tyrl03Ala + Glyl65Ser

Leu216Asp Thr 65Asp + Val 94Thr + Thrl32Gly + Asnl54Ser +

Thr219Gly Val 94Thr + Thrl32Gly + AlalδβGln. + Tyr205Cys +

Thr214Gln Thrl63Gln - Tyr205Cys 4 Thr212Pro + Leu216Met +

Gly216Gln Leu 95Asp + GlylOlGln + IlelOβGln + Glyl26Asn 4

Vai202Ala Gly 99Glu + Glyl26Ser + TyrlββAla + Asn211Gln +

Leu2lβAsn Gly βOAsn + Glyl59Glu + GlnlβlSer + Asnl62Gln 4

Leu216Ser Alal86His + Val202His + Thr212Pro + Ser215Glu +

Leu216Thr Gly 99Gln + Leul25lle 4 Glyl27Ser 4 Tyr205Val 4

Thr212Asp Thr 58Gly + Val 94Gly + TyrlββCys + Ser203Asp +

Tyr205His Val 94Asp + Gly 99Asn + GlnlβlSer + Gly201Ser +

Thr212Asn Glyl26Asn + Glyl27Pro + Glyl53Asp + Glyl59Pro +

Ilel64Ala Asn βlGlu + Thrl02Asn + Glyl65Gln + PhelδδLeu +

Val202His Val 94Cys + Glyl56Ser 4 Val202Thr + Tyr205Ala 4

Leu216Ala Tyrl03Gln 4 Glyl26Gln 4 Glyl56Pro 4 TyrlββAsn 4

Ser215Glu Gly 99Asn + Val202Pro 4 Thr212Asn Tyr213Gly +

Ser215Glu GlylOlAsp + Leul25Thr 4 GlnlβlAsn + Thr212Gly +

Leu2lβAsn Asn βlSer + Glyl53Gln + Glyl56Gln + Val202Gly +

Asn211Gln Asn βlGln + Alal05Thr + Tyrlββlle + AlalδβAsn +

Ser215Asp Alal05Gln + Leul25Gly Thrlβ3Ser Val202Gln +

Asn217Asp Pro200Ser + Val202Ser + Ser203Glu + Thr207Gly +

Thr214Gly

Val 94Asn 4 Glyl30Pro + Tyr2C5Ser + Ser215Asp 4

Thr219Pro Thrl63Asn + AIal99Gln + Tyr205Asn ^■ > ^■ Thr212Ser +

Ser215Glu Leu 9fMet - GiyI5βAsn ->- Val202Cys Leu216Gln +

GIy218Glu Val 94H15 + Gly 99Giu -r Asn21iSer + Tyr213Hιs +

Leu216Met Gly 64Ser ^■>■ Val 94Gln 4 Serl57Asp + Tyr205Ser +

Thr207Pro IlelOβAla + Glyl65Asn 4 Serl90Glu + Alal99Asn 4

Tyr205Asn Asp 59Glu + Glyl59Ser 4 Glylβ5Asn + Phel88Thr 4

Asn217Gln Glyl30Pro + Serl55Glu + Glyl59Asn + Tyr205Pro 4

Thr212Ser Tyrl03Gln 4 Serl55Glu + Val202Asn + Thr212Ser 4

Thr219Asn Giy βOPro + Val 94Ser + SerlOOGlu + Tyrl03Leu 4

Tyr205Ser Gly βOGln + Leu 95Val + Prol28Gly + Serl5δAsp +

Tyr205Thr Thrl32Gln + Asnl62Ser 4 Glyl65Asp + Tyr205Gly +

Tyr20δCys Gly 62Asn + Val 94Thr 4 Alal05His + Prol26Glu 4

Tyr205His Thr 58Pro 4 Glyl56Pro + Val202Pro + Thr212Glu 4

Leu216Thr Leu 95Met Gly 99Gln 4 Tyrl03Cys + Asnl54Gln 4

Thr212Gln Ser 98Glu + Tyr205Asn + Pro209Gln + Tyr213His +

Thr219Ser Gly 62Ser + Val 94His 4 Tyrl03Gln + Serl87Glu ^■ + ^■

PhelδδTyr Gly 62Gln + Asn 96Ser 4 Glyl65Glu + Val202Gly +

Tyr205Thr Gly βOPro + IlelOβThr 4 SerlβOGlu + Thr214Gln 4

Leu216Ile SerlOOAsp + Alal05Thr + Tyr205Ile + Thr214Gly +

Leu216Cys Leu 95Pro + Alal05Asn + PhelδδLeu + Thr212Pro +

Leu216Val Asp 59Glu + Gly βOSer + Glyl27Pro + Tyr205Thr 4

Leu216Ala Alal05Asn + Serl31Asp + Thrl63Pro + Leu216His +

Gly21δAsn Leu 95Cys + Tyrl03Met + IlelOβCys + AlalδβAsp +

Tyr205Met Leul25Cys + Tyr205His + Leu216Asn + Asn217Ser +

Thr219Asn Asn 96Ser + Glyl53Glu + Asnl54Gln + AlalδβGln 4

Thr212Asn GlylOlGln + Alal05Pro Glyl30Asp + Asnl54Gln +

GlylδβAsn Thr 58GΪV + Glyl59Pro + Thrl63Ser 4 Tyr205Gln +

Thr212Asn Tyr205Pro - Thr212Asn + Tyr213Gln 4 Ser215Asp 4

Leu216Gin Glyi30Pro + Glyl53Pro + Glnl ^β lGlu + Tvr205Met +

Thr212Gln Alal05Gly + Serl57Asp + TyrlββAsn 4 Val202Gln +

Thr212Ser Thr 58Gln ⁺ Asn 96Ser Ser203Asp 4 Thr207Gly 4

Leu216Ser SerlSoGIu - Val202Ser + Thr212Asn 4 Thr214Gln 4

Thr219Asn Asn όlSer ^ SerlOOGlu + Tyr205Leu 4 Thr212Ser 4

Asn217Ser Giy βOGln + Gly 99Glu 4 Thr212Ser 4 Tyr213Gln 4

Leu216Thr Leul25Asn Serl29Asp + Asnl62Ser + Alal86His +

Tyr205Gln Gly 64Gln + Leu 95Glu * Alal99Asn 4 Val204His +

Leu216Met Leu 95Hιs GlylOlPro + Val204His + Tyr205Asn 4

Leu216Gln Thrlβ3Ser + GlylβδAsn - Pro209Gly + Tyr213His 4

Leu216Asp Glyl27Gln Serl31Asp + ThrI32Pro + Tyr205Cys +

Leu2lβAsn Asn βlGln + GlylOlPro + Serl90Asp + Tyr205Pro +

Thr214Gly Glyl26Asn ^■>■ Pro200Gly Val204Gly + Tyr205Asp +

Thr212Asn Asn βlGln + Val 94Gln 4 Glyl26Asp + Tyr205Val +

Thr212Gln G-.7 βOGln 4 IlelOβPro 4 Serl31Glu + Ilel64Ala 4

Val202Pro GlylOlSer 4 Tyrl03Glu 4 Serl04Asp + Glylβ5Asn 4

Pro209Gln Thr 58Glu + Asp 59Glu 4 TyrlββHis + Val202Cys

Thr219Asn Glyl2βPro + AlalδβHis + Tyr205Asp + Tyr213Val +

Ser215Asp Gly βOAsp + Ser 97Glu + Alal05Pro + Leul25Cys +

Glyl53Pro Asnl54Ser + Pro209Ser 4 Thr214Glu + Ser215Asp +

Leu216Glu Thrl63Pro + Ser203Giu 4 Val204Thr + Tyr205Cys 4

Ser215Asp Gly 62Ser + Metl9δGln + Ser203Glu + Ser215Asp +

Leu216His Glyl56Asn + Ser203Asp + Tyr205Met + Ser215Asp +

Gly216Pro Gly βOGln + Prol26Ser + Ser203Glu + Ser215Glu +

Leu216Pro

TyrlββCys - Tyr205Giu + Pro209Asn + Ser215Glu 4

Leu216Asp TyrlJ3Leu - Leul25Gly + Glyl53GIu + Serl90Glu 4

Leu21βlle Gly 99Pro * IlelOβThr 4 Glyl53Asp + Serl55Asp +

Serl90Asp Thr 65Pro 4 Leul25Hιs + Ser210Glu + Thr212Asp + Gly ό45er - Thr + Pro209Glu 4 Leu 95Asn 4 GlylOlAsn + Asnl54Asp + Glyl56Glu +

TyrlββGln Serl55Glu + ThrIβ3Asp + Tyr205Cys + Thr212Pro + Glyi59Pro Thrl63Ser Ser203Asp + Tyr205Asp +

Thr212Ser Thrl02Gln 4 Glyl26Asp + Glyl53Asp + PhelδδThr 4

Tyr205Hιs Asr. βlSer - Val202Glu + Ser203Glu + Tyr205Asp +

Thr212Pro Tr.r 56Gln - Thrl02Pro 4 Tyr213Cys + Ser215Glu 4

Asn217Asp Gly βOAsp - Gly 52Glu ^■ >- Tnr 65Glu 4 Glyl26Asn +

Leu216Thr AlalOSGly 4 Glyl26Pro * Glyl53Glu + Leu216Thr +

Gly2lδAsp Tyrl03Gln 4 Alal05Thr 4 Val202Asp + Tyr205Hιs +

Ser215Glu Val202Glu 4 Tyr205Thr + Pro209Asn + Ser215Asp + Val 94Asn - Alal05Asn 4 IlelOβVal + Ser203Glu +

Gly218Asp A-sn 96Ser - 5er203Asp 4 Tyr205Ile + Thr212Gln +

Gly218Asp Asn βlAsp + Gly 62Gln 4 Thr 65Asp + Glyl26Asn +

Glyl59Pro Glyl53Gln + Alal86Glu + Tyr205Thr Leu216Thr +

Asn217Asp Leul25Asp + Glyl26Asp + Alal86Ser + Gly21δGlu +

Thr219Pro Thr 58Glu ^■ - Thr 65Glu 4 Val 94Gln 4 GlylOlPro +

Glyl65Gln Glyl53Giu Serl58Glu + Thrl63Glu + Glyl65Pro +

Leu216Met Glyl59Asp 4 Thrl63Glu + TyrlββThr + AlalδβHis +

Pro200Ser Leul25Thr -^ Glyl30Ser + Asnl54Asp + Serl57Asp +

AlalδβGlu Asp 59Glu + Asn 96Glu 4 GlylOlGlu + Prol2δSer +

Thr212Asn Asp 59Glu + Asn 96Asp + GlylOlAsp + Tyrl ^β βAsn +

Thr219Asn Gly 99Glu + GlylOlGlu 4 Glyl27Glu + TyrlββLeu +

Asn217Gin Alal86Gln + Serl87Asp + Leu216Pro + Gly218Glu +

Thr219Gln Thr 58Pro ^■ > ^■ Leu 95Ile + Glyl26Glu + Glyl53Glu +

Phel88Asp Thr 58Glu 4 Asn 9βGlu + Giy 99Asp 4 Phel88Met +

Gly218Gln Giy 62Glu 4 Asnlβ25er + Val202Met + Thr212Asp +

Leu216Met Giy 62Pro 4 Gly 99Gln + Serl04Glu 4 Serl31Asp +

Tyr213Thr Gly 62Glu + Glyl30Ser + Tyr205Glu + Ser215Glu +

Leu2lβAsn Glyl26Gln + Glyl27Glu 4 Asnl54Glu + Tyr213Pro +

Leu216His Serl29Glu + Asnl54Glu + Asnlβ2Ser GlylβδAsp +

Leu216Cys Thr 58Gly 4 Leul25Asn 4 GlylδβAsp + GlnlβlGlu +

Tyr205Giy Glyl26Ser + Glyl53Gln + GlylδβGlu + GlnlβlAsp +

Tyr205Pro Thrl63Gln + Val202His + Tyr205Asp + Pro209Gly +

Thr212Asp Gly 62Gln + Val202Met + Asn211Asp + Thr212Asn +

Thr214Glu Thrl02Gln 4 Glyl26Pro 4 Thr212Asp + Ser215Glu +

Leu2lβAsn GlylOlSer 4 GlnlβlAsn Thr212Glu + Ser215Asp 4

Leu216Gln Leul25Gln 4 AlalδβGlu + Val202Cys + Ser203Asp +

Ser215Asp Serl29Glu + Serl55Glu 4 Thrl63Asp + AlalδβSer +

Tyr20δSer Thr 5δAsn + Leul25Glu + Prol26Glu + Thrl63Ser +

GlylβδPro Asn βlGlu + Gly 62Asp + Glyl30Ser + Asn211Asp 4

Thr212Gly Thr 65Gln + Asnl54Asp + Ser215Glu + Leu21βHis +

Asn217Glu Asp 59Glu + Ser 97Glu + Thrl02Gln + Ser210Asp +

Leu216Thr Glyl53Glu + Ilel64Thr + Val202Gln + Ser203Glu +

Asn217Glu Val 94His + Leul25Cys + Glyl26Ser + Serl55Glu +

GlnlβlGlu Serlδ7Glu + Tyr205Thr + Thr212Asn + Tyr213Gly +

Asn217Glu Gly 99Ser + Glyl30Glu + Asnlβ2Asp + Thrl63Asp +

Tyr205Asn Prol28Gln + GlylSβAsp + Alal86Glu + Ser203Glu +

Tyr213Gln Gly 62Asp + Glyl56Asn + AlalδβPro + Thr212Glu +

Leu216Asp

Giyl26Ser + TyrlββGly + Ser203Asp + Pro209Asp +

Ser215Asp Tyrl03Val + Proi28Asp + Glyl53Asn + Asnl62Ser 4

Serl90Asp GlyI30Prc ->- Glyl5βAsn GinlόlAsp ⁺ Thrl63Glu +

Serl87Glu Leul25Pro + Ser203Asp + Pro209Gln + Thr212Glu +

Leu216Asp Leu 95Asp + Glyl2βGlu + Prol26Gly + Glyl59Gln +

AlalδβHis Giy 62Ser + Serl90Asp + Thr214Gly 4 Ser215Asp 4

Gly2lδAsp Gly 64Asn + Tyrl03Ile + Leul25Asp + Ser203Asp +

Asn217Glu Glyl2βSer ^ GlnlβlAsp + Asnl62Ser 4 Thr214Glu +

Ser215Glu IlelOβLeu + GlyI26Asp 4 Glyl27Glu 4 Giyl30Gln +

Val202Glu Asp 59Glu 4 Thrl02Ser + AlalOSGly + Glyl59Glu +

SerlβOGlu Leu 95Asp + Asn 96Asp + TyrlββLeu + Val204Ala +

Gly216Glu GIyl30Glu 4 Serl31Asp + Glyl53Gln + Serl5δGlu +

Alal99Pro Gly βOGlu 4 Asn βlAsp ->- GlnlβlAsn + Pro209Gln 4

Ser215Glu Thr 65Asn 4 Gly 99Gln + Asnl54Asp + Pro209Asp +

Ser210Glu Asnl54Glu + Serl55Asp + Tyr213Thr + Ser215Glu +

Gly21δPro SerlOOAsp + Asnl54Glu + Serl55Asp + Pro209Ser +

Thr214Gly Gly β2Asn + Glyl53Asp + Asn211Glu + Thr212Asp 4

Gly216Gln Tyrl03His + Serl29Glu + Tyr205Pro + Asn211Asp +

Thr212Glu Serl29Asp + Asnl62Gln 4 Tyr205Leu + Asn211Asp +

Thr212Asp SerlOOGlu + Glyl53Pro Thrl63Pro + Val202Glu +

Ser203Glu Thr 56Asp + Thr 65Pro 4 Val202Glu + Ser203Asp +

Leu2lβAsn Thr 65Pro + Glyl26Asn 4 Serl57Glu + SerlδδAsp +

Ser215Asp GlylOlAsn + Serl57Asp + Serl5δGlu + Ser215Glu +

Leu216Ile Asn 96Glu + Ser 97Glu 4 Glyl59Asn + PhelδδHis +

Ser210Glu Thr 65Asn + Ser 98Asp + Glyl59Asn + Ser215Asp +

Leu216Asp Asn 96Glu + Prol2δSer + Tyr205Cys + Ser215Glu +

Leu216Glu IlelOβGly + Serl55Glu + Thr212Asn + Ser215Glu +

Leu216Asp Leul2ΞGl 4 Thrl63Asn + Val202Thr Ser215Asp 4 GlylOlGln + Giyl30Glu + Tyr205Ala Ser215Glu + Prcl28Giy + SerllOAsp 4 Thr212Ser ^■ >- Ser215Glu +

Leu216Glu Serl04Asp + Alal05Glu + Metl98Pro 4 Ser203Glu +

Leu216Ser Ser 97Giu ^■ *- Ser 98Glu + Alal05Gly + Glyl53Asp +

Asn217Gln Glyl53Gin + SerllOAsp + Asn211Asp 4 Thr212Ser 4

Ser215Asp Thr 65Giu + Leul25Cys + Glyl26Ser 4 Val202Glu +

Ser215Asp Thrl02Gin + Glyl65Asn Ser203Glu 4 Tyr205Glu +

Pro209Asp Leu 95Glu + Asn 96Gln 4 Tyr205Glu 4 Pro209Ser +

Ser215Glu GIyl26Asn + Tyr205Asp + Ser210Asp 4 Ser215Glu +

Leu216Met Gly 62Gir. + Leu 95Pro + SerlδδAsp + Tyr205Asp +

Ser215Glu Asn βlGlu + GlylOlGlu + Glyl2βSer 4 Leu216Met +

Thr219Ser GlylSβAsp + AlalδβGln 4 Serl90Glu + Ser203Glu +

Thr212Pro Gly βOGlu 4 Ser 97Glu + Gly 99Asn 4 Asnl62Glu +

Asn2175er Thr 58Pro + SerlβOAsp + Serl90Asp + Val202Asn +

Thr212Asn GlnlβlAsn + Ser203Asp 4 Asn211Glu 4 Tyr213Cys +

Ser215Glu Prol28Glu + Glyl65Gln 4 AlalββHis 4 Ser203Glu +

Ser215Glu GlylOlPro + Thrl32Ser 4 Serl5δGlu + Ser203Glu +

Ser215Glu Prol2δSer + Alal86Gly + Ser203Glu + Ser210Glu +

Ser215Asp Asn βlGln + Serl58Asp + AlalδβSer 4 Ser203Asp +

Ser215Glu Leu 95Val + SerlOOAsp 4 TyrlββMet 4 Ser203Glu +

Ser215Asp _-_yl53Glu + Alal86Gln 4 Ser203Glu Ser215Asp 4

Thr219Gly SerlβOAsp + Ser203Glu 4 Val204Gln 4 Thr212Pro +

Ser215Glu Thr 58Pro + Leu 95His + Serl55Asp + Ser203Asp +

Ser215Asp Asnl54Gln + Serl57Asp + Ser203Glu + Thr212Ser 4

Ser215Glu Leu 95Gln + SerlOOAsp + Ser203Glu + Tyr205His +

Ser215Glu

Asnl54Asp + TyrlββHis + Ser203Asp 4 Tyr205Ala 4

Ser215Glu Ser 98Asp + Ser203Glu + Asn211Ser + Ser215Asp +

Leu216Ser Asnl62Glu + Ser203Asp + Tyr208Asn + Thr212Ser +

Ser215Asp GlnlβlAsp + Thrl63Gln + Ser203Glu + Ser215Glu +

Leu216Thr Leu 95Cys + Serl29Glu + Ser203Asp + Asn211Gln +

Ser215Asp ThrI32Asn Asnl54Glu + Ser203Asp + Thr212Asn 4

Ser215Asp Prol28Gln 4 Serl31Asp + GlylδβAsn + Ser203Glu +

Ser215Asp Thr 58Glu 4 Asn βlSer + Gly 99Pro + Ser203Glu +

Ser215Asp SerlOOAsp + Glyl53Ser + Ser203Asp + Tyr205Val +

Ser215Glu AlalOSGiu Ser203Asp + Thr212Asn + Ser215Glu +

Gly218Asn Thrl02Asn + Serl04Asp + Glyl27Asn + Ser203Asp +

Ser215Asp Gly 62Asp + Leul25Thr + Glyl53Glu + Glyl65Ser +

Serl90Glu Glyl53Glu + PhelδδHis + Serl90Glu + Ser203Glu +

Leu216Gly Thr 5δAsp + PhelδδAsn + Ser203Glu + Tyr205Leu +

Asn217Asp Thr β5Gly + Thrl02Ser 4 Glyl26Asp + Thr212Ser +

Gly2lδAsp Asp 59Glu 4 Thrl63Glu + Serl90Glu + Thr212Gln +

Leu216Ser TyrlββVai + Ser203Glu + Ser210Asp + Thr212Ser 4

Leu216Asp Glyl59Asp + Ser203Glu + Thr207Pro + Thr212Asn +

Leu216Asp Serl57Asp + Asnl62Gln + GlylβδGln + Ser203Glu +

Leu216Asp Leu 95Thr + Serl31Glu + Val202Ser + Ser203Asp +

Leu216Glu SerlOOAsp + Glyl27Asn + Ser203Asp + Tyr20δVal +

Leu216Asp Thrl02Gly + Serl29Glu + Ser203Asp + Tyr205Ala +

Leu216Asp Thr 65Gln + Ser 9δAsp + Thrl02Pro + Glyl26Asp +

Glyl65Asp Thr 65Asn + Glyl27Glu + Serl29Asp + Tyr205Ile +

Ser215Glu Gly βOAsn + Ser203Glu + Tyr205Val + Ser210Asp +

Thr212Glu Val 94Cys + Ser 9δGlu + Pro209Ser + Ser210Asp +

Thr212Glu Gly βOGln 4 Tyrl03Asn 4 Prol2δGlu + Serl57Asp +

Glyl59Asp Ser 98Gl - SerlOOAsp 4 Ser203Glu + Thr212Gln +

Leu216Thr Gly βOGlu - Thr 65Asp + Thrl32Gln + Asnl54Ser + Asn 96Asp - SerlOOAsp 4 Thrl02Pro + Tyr205Asp +

Thr212Gln T rl02Gly - Phel88His + Giy201Asn + Tyr205Glu +

Gly218Asp Asnl54Gln - Serl55Asp + Tyr205Gln 4 Pro209Glu +

Thr212Asp Tyrl03Vai ^■ >• Glyl53Asn 4 Pro209Asp + Thr212Asp +

Gly21δGlu Thr 65Glu + IlelOβMet Leul25His 4 Tyr205Leu +

Ser215Asp Leul25Me 4 Glyl56Asp - TyrlββSer + Serl87Glu +

Ser215Asp Thr 58Giu - Gly βOGlu 4 Serl31Glu Tyr205Val 4

Gly218Pro Thr 58Asp - Gly όOAsp ÷ Asn βlSer AsnlβlGln +

Ser203Asp Serl57Glu * Ser203Asp 4 Tyr205Asp + Thr212Ser +

Leu216Cys Thr 58Asn - Thrl02Gln 4 Serl55Glu + Serl57Asp +

Thr212Glu Alal05Gln ~ Serl55Asp + Serl57Asp 4 Ser203Asp 4

Thr212Gly Thrl63Glu ^■ > ^■ Val202Asp ^■ > ^■ Tyr205Cys + Leu216Pro +

Gly21δGlu Thr 58Gln 4 Asn βlGlu + IlelOβThr 4 Pro209Asp +

Ser215Asp Gly 62Glu - Ser 97Glu + Asnl54Ser + Tyr205Asp +

Tyr213Vai Leul25Gln + Glyl30Asp + Leu216Glu + Gly218Asp +

Thr219Gly Glyl26Ser -•- Serl31Glu + Tyr205Ala + Leu216Asp +

Gly216Glu IlelOβAla + SerlβOGlu + Asnl62Glu + AlalδβGly +

Ser203Asp Asp 59Glu * Thr 65Asp + Asnl62Asp + Leu216Ile +

Asn2175er Asn 96Gln + Thrl02Pro + Glyl27Glu + AlalδβPro +

Serl90Asp Gly 99Asn + Leul25Thr + Serl57Glu + Glylβ5Glu +

Thr212Gln Val 94His + Glyl26Asn 4 Serl57Glu + Glyl65Glu +

Tyr205Val Asp 59Glu Asn βlSer 4 Val202Asn + Thr207Pro +

Asn211Asp Asp 59Glu + Tyrl03Ala ^■ >- Serl55Glu 4 Pro209Glu +

Thr212Asn GlylOlGln + Glyl27Glu + Glyl53Ser + Asnl54Glu +

Serl58Asp

Ser 97Glu Thr212Ser 4 Ser215Asp Leu216Cys +

Asn217Glu Gly 99Gln * SerlΞ ^{^} Giu + Ser215Asp 4 Leu216Ser +

Asr.217Asp SerlllGlu - Glyl59Asn 4 Tyr205Met 4 5er2I5Glu ^■ +

Asn217Glu Val 94Ala + GlylOlAsp 4 GlylδβGln 4 Ser215Asp 4

Asn217Asp Asn βlAsp + Asn 96Glu 4 Pro209Ser 4 Ser215Glu 4

Leu216Ala Asn βlGlu + Asn 96Clu + Glyl27Ser 4 Ser203Glu 4

Thr214Ser Gly 99Pro + SerlOOAsp + Thrl02Glu + AlalOSGly +

Asn211Glu Serl55Glu + AlalSβPro 4 Tyr205Asp + Leu216Cys +

Gly216Glu PhelδδAsp + Ser203Asp 4 Tyr213Ser 4 Thr214Glu 4

Gly216Asn Serl29Asp + Thrl63Gly + Glyl ^β δGlu + Ser203Asp +

Tyr205lle Serl04Asp + SerlδδGlu 4 Asnl62Glu + Val202Gln 4

Leu216Val Ser 98Asp 4 Serl58Asp 4 Asnl62Glu 4 Leu216Ile +

Thr219Gly Prcl28Gly + Serl5SAsp + Asnl62Glu 4 Val202Gln 4

Asn217Glu Thrl02Glu + Alal05Asn 4 Serl29Asp + Tyr208Thr 4

Leu216Gln Thr 65Asn + Serl57Asp 4 Asnl62Glu 4 Ser203Asp 4

Tyr205Gly Alal05Gly + Serl57Asp 4 SerlβOGlu + AlalδβHis 4

Ser210Asp Ser 98Glu + Glyl53Asp + Val202Ser 4 Tyr205Asn 4

Gly218Asp Gly 62Asp + Gly β4Asn 4 Gly 99Pro + Glyl27Asp +

Glyl53Glu Thr 5δSer + Val 94Met + Serl04Glu 4 Glyl53Asp 4

TyrlββAsp Asn βlAsp + Thr 65Gly + AlalδβGln + Ser210Asp +

Thr214Asp Glyl27Asn + Val202Asp + Ser210Glu + Ser215Glu +

Asn217Ser Gly 62Asn + GlylOlAsp + Tyrl03Met + Serl04Glu +

Ser203Glu Giyl59Asp + Asnlβ2Glu + Ilel64Ala + Thr212Asn +

Ser215Asp Ser 98Asp 4 GlylOlGlu + Asnl54Gln + Tyr205Gln 4

Ser210Asp Leu 95Glu + TyrlββAsn + Phel88Met + Ser203Glu 4

Thr214Glu GlylOlPro + Serl29Glu + Ser203Asp + Thr214Glu +

Gly21δGln Asn 96Gln + Asnl62Glu + Glyl65Glu + Val202Asp +

Tyr205Met Thr ^β ΞGln 4 Val 94Asp + Serl04Giu Leul25Asn +

Thr212Asp Tnrl02Prc 4 SerI55Asp ⁺ Serl87Glu 4 Tyr205Val +

Ser215Glu Asn 96Glu 4 ThrI02Gln + AlalδβAsp + PhelδδGln +

Serl90Asp Thr 58Glu 4 Asn βlGlu + Gly 64Asn 4 Alal05Pro 4

Asn217Glu Val 94Ala + Glyl26Asp + Asnl54Glu + TyrlββPro 4

Thr212Glu Thr 58Asn 4 Asn βlSer - Glyl30Glu 4 PhelδδGlu 4

Asn217Asp Thr 58Asp - Ser 97Asp 4 IlelOβLeu 4 Glyl53Gln 4

Glyl56Asp Thr 58Asp + Ser 97Glu Thrl02Pro 4 Alal05Ser +

Serl5δAsp Tyrl03Glu 4 Glyl27Glu 4 Asnl54Gln 4 Serl57Asp +

Thrl63Ser Asn 96Gln ⁴ Prol2δGlu 4 Glyi30Gln 4 Serl31Glu +

Serl57Asp Glyl27Glu 4 Glyl53Asn + Pro209Asp + Thr214Glu +

Thr219Ser SerlOOAsp ⁴ HelOβMet Prol2δAsn + Serl90Asp +

Gly216Glu Thr 58Asp + Asn 96Asp Glyi27Asp + TyrlββAla +

Alal86Ser Asn 96Glu + Thrl63Pro + PhelδδAsp + Gly201Gln +

Val202Glu Ser 9δGlu 4 Glyl27Asp 4 Serl31Glu 4 Val202Ala +

Thr214Ser Gly 99Asn 4 Giyl27Glu 4 Serl31Glu + Glyl56Gln +

Asn211Glu Val 94Asp + Prol26Asp + Serl31Asp + Tyr205Thr +

Leu216Ser GlylOlGlu + Glyl26Ser 4 Glyl27Asp + Tyr205His +

Ser215Asp SerlβOAsp + Tyr205Glu 4 Asn211Gln + Thr212Asp +

Thr219Gly Gly β2Asp + Glyl27GIn + Glyl53Pro + Serl87Glu +

Leu216Glu Gly βOAsp + Glyl27Asp + Glyl59Pro + Ser210Asp +

Gly218Asn Asn βlSer + Gly 99Asp 4 Thrl02Asp + Ser203Asp +

Thr212Gln Glyl26Pro + SerlβOAsp + Tyr205Met + Thr212Glu +

Ser215Asp Thr 65Ser + Glyl27Asp + Thrl32Ser + Thr212Asp +

Ser215Asp Asnl54Ser + Serl87Glu + Thr212Glu + Tyr213Gln +

Ser215Glu Serl29Asp + Glyl53Glu + SerlδδGlu + Glyl65Asn +

Leu216Thr

Ilei^'όHis - Leul25Pro ^■>■ Serl29Glu + AlalδβGlu 4

3er203Glu Glv βOPro ⁴ Giy 62Gln 4 Tyr205Thr Ser215Glu 4

Thr219Glu Asp 59Glu - Val 94Pro + Glyl30Gln + Serl87Asp 4

Thr219Asp Glyl53Ser ⁴ SerlΞ5Glu + SerlβOAsp + Asnl62Ser +

Thr212Pro SerlΞSAsp - GinlβlGiu + AlalδβThr Thr214Gly 4

Ser2I5Glu Serl04Asp 4 Glyl30Asn + Serl87Glu + Thr212Gin +

Asn217Glu Alal05Asn Ser203Glu + Thr212Glu + Asn217Ser +

Thr219Asp Leul25His ⁴ GlylβSGln - Phel8δAsp + Ser203Glu +

Tyr205Leu Asn 96Asp 4 Serl04Asp + Gly201Pro 4 Val202His +

Leu216Ser Gly 52Asp - TyrlC3Val + Serl90Glu 4 Asn211Glu 4

Thr219Ser Giy 62Asp 4 Serl90Glu + Val202Ser 4 Asn211Glu +

Thr214Gln SerlOOAsp 4 Prol28Asp Tyr205lle 4 Thr212GIn +

Leu216Hιs Thrl32Gly + GlylSβAsn + Glyl59Glu 4 Serl90Asp +

Ser203Glu Asn 96Ser Ser 9δAsp + Glyl59Asp + Serl90Asp 4

Leu216Met Val 94Asp + Ser 9δGlu + Thrl32Asn 4 PhelδδCys +

Thr219Asp GlylOlSer - Tyrl03Glu + Glyl53Glu + TyrlββHis 4

Serlδ7Asp Gly 99Ser + SerlβOAsp + Serl90Glu + Val202Glu +

Tyr205Ser Asn βlAsp + Gly 99Gln + AlalδβGlu + Thr212Gln +

Leu216Asp AlalδβHis + PhelδδGln + Serl90Glu + Pro200Ser +

Asn217Asp Asn βlSer + Thr 65Asp + Serl29Glu + Tyr205Glu +

Tyr213Gly Asn βlAsp 4 Gly 62Ser Ser203Asp + Asn211Gln +

Tyr213Glu Thrl02Asn * Glyl30Asp 4 TyrlββSer 4 Tyr205Asp +

Gly2lδAsp Asn βlGlu + Asnl54Gln 4 GlnlβlAsn + Tyr205Asp +

Gly216Glu Gly 99Glu + Glyl27Asp + Thrl63Pro + Tyr213Ile +

Asn217Glu GlylOlGln + Glyl2βAsp + Serl31Asp + Glyl56Asn +

Ser203Glu Thrl02Asp + Glyl30Glu + Pro209Asn + Thr212Ser +

Ser215Asp Thr 5δSer + SerlOOGlu + Serl04Glu + Ilel64Leu +

Ser215Glu Asp 59Glu + IlelOβVal Glyl27Glu 4 TyrlββAsn +

Serl90Glu Gly 99Ser 4 Prol28Asp 4 GlylδβAsp + Tyr205Cys +

Asn217Asp Thr β5Asp Leu 95Glu 4 Gly 99Pro 4 Serl58Asp +

Tyr205Gln Ser 97Asp 4 Aial05His 4 Serl55Glu Tyr205Asn 4

Pro209Glu Giy 62Asn 4 Thr β5Asp 4 Ser 97Giu 4 Serl31Giu 4

Leu216Gly Glyi30Pro 4 Glyl53Glu + Asnl54Gln 4 GlnlβlAsp +

Pro209Asn Giyl59Ser 4 TyrlββGiu + Tyr205Asp 4 Thr212Pro +

Thr219Glu Gly 62Asp 4 Leu 95Cys + SerlOOGlu Thrl63Pro +

Serlδ7Asp Tr.r 58Gly 4 Glyl59Glu + Serl87Asp + Ser203Glu +

Thr219Asn Thr 58Pro + TyrlββAsp + Val202Met + Pro209Asp +

Ser215Asp Leu 95Asp + Thrl02Pro + Alal05Gly + Glyl27Glu +

Leu216Glu Prol26Glu + GlnlβlAsp + PhelδδGln + Ser203Glu +

Asn217Gln Leul25Pro 4 Prol2δGlu + Thrl32Asn + GlnlβlGlu +

Leu216Asp Ser 97Glu 4 Thrl02Asp + Val202Thr 4 Ser215Glu +

Gly21δSer Asp 59Glu 4 SerlOOAsp 4 PhelδδGln + Thr212Ser 4

Ser215Glu GlylOlGln + Thrl02Asn 4 GiyI30Glu 4 Thrl63Glu +

Ser210Asp Asn βlGlu 4 GlylOlAsn 4 Serl29Glu 4 Tyr205Thr +

Leu216Glu Leu 95Glu + Ilel64Val + Thr212Glu + Tyr213Gly +

Leu21βAsp Thr 56Gln 4 Glyl26Glu 4 GlylδβGln 4 Thr212Asp +

Leu216Asp Leul25Met + Glyl26Ser + Serl31Asp + Thr212Asp +

Leu216Asp Gly βOAsn 4 Asn 96Gln + Serl04Glu + Thr212Asp +

Leu216Glu Thr 65Asp 4 Glyl26Gln + SerlδδAsp + Glyl65Asp +

Leu216Val Serl5δAsp + Glyl65Glu + Ser203Asp + Leu216Ser +

Gly216Gln GlylOlAsp + Leul25Gly + Prol2δGlu + AlalδβGly +

Thr219Gln Thr 65Glu 4 Asn 96Glu + Tyrl03Val + Serl55Glu +

Metl96Ala Thr 65Asp + Asn 9βGlu + Leul25His + Asnl62Glu

TyrlββHis

Asn βlSer 4 SerlOOAsp 4 PhelδδAsp 4 Ser203Glu +

Gly21δSer . Serl04Gl 4 GlyiS ^β Gln 4 Phelδ8Asp 4 Ser203Glu +

Thr212Gly Gly 99Asp ~ TyrlOSAsp 4 Pro209Asp + Thr212Pro 4

Tyr213Met Thrl02Asp * Asnl54Seι + TyrlββGiu + Ser203Asp +

Gly218Ser GlylOlGlu 4 Serl29Asp 4 Serl57Asp + Thr214Ser 4

Leu2lβAsn Asp 59Glu + GlylOlAsp + Glyl27Ser + Glyl59Pro 4

GlnlβlGlu Gly βOAsn - Glyl27Asp 4 Thrl32Asp + Asnl62Gln +

Ser203Glu Thr 58Asn * Gly βOAsp + Glyl30Glu + Val202Ser 4

Thr212Glu SerlOOAsp - TyrlββAsp 4 AlalδβHis Tyr205His +

Leu216Thr Ser 97Asp 4 GlylOlSer + Alal05Glu + Tyr205Met +

Ser215Glu Ser 97Glu + AlalOδGlu + Leul25Ala + Serl58Asp +

Tyr205Cys SerlOOGlu ⁴ IlelOβHis + Glyl27Ser + Serl58Glu +

TyrlββGlu ThrlC2Asn 4 Glyi30Ser + Serl31Asp + Asn211Asp +

Ser215Glu Giyl27Gln 4 Serl31Glu 4 Asn211Asp + Tyr213His +

Ser215Glu 5erl29Glu + Serl58Glu + Val202Gly + Ser215Asp +

Leu216Gin Leul25Pro + Prol2δSer + Serl57Glu + Ser203Glu +

Gly21δSer Thr 65Gln 4 GlnlβlGlu 4 Ser210Glu 4 Thr212Gln +

Ser215Glu Val 94Met + GlylOlGlu + Alal05Asn + AlalδβHis 4

Thr212Asp

TABLE 36

Multi-loop Sextuple Mutation Variants

Asn βlSer Giy 64Gln GlylOlAsp + Thrl32Gly +

Glylβ5Gln ⁴ Tyr205Pro Asn 96Ser + GlylOlSer + Alal05Gly + Asnl54Glu +

TyrlββThr + Tyr205His Val 94Ala + Leu 95Thr 4 Glyl2βGln + Pro209Ser 4

Ser215Asp 4 Leu216Gly Thr 65Gln + Leul25Asn + Asnl62Asp + Tyr205Gln 4

Pro209Gly 4 Leu216Met Thrl63Gln + Tyr205Cys + Thr212Pro + Leu216Met +

Asn217Gln Gly21δGln Gly βOGln + Val 94Cys + Asn 96Ser + Glyl27Pro +

Glyl59Asp Asnl62Ser

1-1'/I59? o - Ilei6 la Val- 3 1y≤ - Irill-Asp ~ GlylΞβSer ⁴ Val232Thr ⁴

Tyr205Ala + Leu216Aia Asn βlGln 4 AlalDSThr ⁺ Glyl53Gln + GiylSβGln 4

Tyrlββlie + A.sn211Gln Gly βOAsn Serl04Asp + Alal05Asn + GlnlβlSer 4

Thrl63Ser + Tyr205Ala Gly 64Ser Val 94Asn + GlylOlAsn 4 Leul25Asn +

Giyl30Asp + Leu216Cys Glyl59Pro ⁺ Glyiβ5Pro AlalδβThr + Gly201Pro +

Thr212Prc - Leu216Ser Gly 99Asn Giyl59Pro + Ser203Glu + Tyr205Leu +

Asn211Gln 4 ^" τhr212Ser Thr 58Gln 4 LeuIlSPro 4 Thrl32Asp + Glyl53Asn 4 IlelOβAla Asnl54Glu + Asnl62Gln + Metl98Thr +

Alal99Asn 4 Tyr205Asn Asp 59Glu 4 Glyl59Ser 4 Glyl65Asn + Phel38Thr 4

Pro200Gln ⁴ Asn217Gln Gly 99Ser GlyllβAsn + Thrl32Gly + Serlδ7Asp +

Val202Ser 4 Tyr205His

/ 1 -. '03Gin 4 GlyI30Pro 4 Serl55Glu + Glyl59Asn 4 :20SPro - Thr212Ser

Val 94Ser + SerlOOGlu + Tyrl03Leu 4

Tyr205Ser 4 Thr219Pro Thr 58Gly + Leul25Ala + Val202Cys + Tyr205Asn +

Ser215Asp + Leu216Ala Val 94Thr 4 Serl04Glu Glyl30Pro + Thr212Pro +

Leu216Asn + Gly218Ser Ser 97Glu + Thrl32Gly 4 Pro200Gly + Tyr205Ala +

Asn211Ser 4 Tyr213Gln Gly 62Asn Leu 95Asp TyriββVal + Tyr205Cys +

Asn211Ser 4 Leu216Vai GlylSβAsn 4 Glyl65Pro 4 Gly201Asn + Tyr213Ala +

Thr214Gln 4 Asn217Glu Gly 62Ser Ser 9δAsp + Glyl2βSer + Tyr205Pro +

- Jhr212Ser + Tyr213Thr Alal05Gln + Glyl27Ser + GlnlβlAsp + Thrl63Gly +

Tyr205Cys + Leu216Pro Val 94Ala + Glyl30Gln + Alal86His + Ser203Asp +

Tyr205Cys + Thr214Gln Thr β5Gln 4 Leul25Gly + Serl87Asp + Phel8δGln +

Val202Asn + Thr212Asn Thr 58Ser + IlelOβPro 4 Thrl32Gly + GlnlβlAsn +

Tyr205Asn + Thr212Asp SerlOOAsp Alal05Thr + Leul25Pro + Tyr205Ile +

Thr214Gly + Leu216Cys Thrl02Ser + Glyl27Pro + Prol28Gly + Ser203Glu +

Tyr20δHis + Tyr213Thr Thrl02Ser + GlnlβlGlu + Asnl62Asp + Thrl63Gln +

AlalδβHis + Phelδδlle Gly 62Pro + Prol28Asp + Serl29Asp + Glyl56Asn 4

Tyr2I3Cys - Gly2I8Asn Thr ^' 58Gln + Glyl27Pro 4 AlalδβGln 4 Val202Asp + Giyl5βSer + PhelδδGly + Ser203Glu + Tyr2I3Gln 4

SerllSAsp - Leu216Asp GlylOlGln A-ial05His + Asni62Gln 4 Alal86Gly 4

Ser203Glu - Ser215Glu Thr 58Pro 4 τhrlβ3Asn + Ser203Glu + Tyr205Ile +

SerllSGlu - Leu216Gin Thr β5Asn t Giyl27Glu + Glyl56Asn - TyrlββGiu 4

Phel88Met + ^" τ r212Pro Leu 95A.SP + SerlOOGlu 4 GlylOlGln IlelOβGln 4

Ilel54Leu 4 Val202Ala Tyrl03Pro GIyl27Glu + Serl29Asp + Glyl30Glu 4

Asnl62Gln 4 Leu216His Gly βOGln 4 Glyiό5Ser + Ser203Glu + Tyr205Asp +

Thr2I4Glu Ser215Asp Leu 95Asn Leul25Pro + Ser203Asp + Tyr205Thr

Thr214Giu Ser215Asp Gly 62Asn 4 Tyrl03Ile + Leul25Cys + Glyl2δGlu +

Glyl53Asp 4 TyrlββCys Giy 9 ^" 9Ser + Prol28Glu + Glyl30Asp + PhelδδAsn +

Leu216Gly + Gly21δPro Asp 59Glu + Gly βOAsp 4 Thr β5Asp + Asnl62Ser +

Thrlβ3Gln + TyrlββGln PhelδδGlu + Tyr205Thr + Thr212Asn + Tyr213Gly +

Asn217Glu Gly21δAsp Gly 62Gln + Ser203Glu + Tyr205Ser 4 Thr212Ser +

Ser215Asp 4 Gly218Asp Serl87Glu + Serl90Asp Tyr205Val + Pro209Ser +

Thr214Ser 4 Thr219Ser Thr 58Ser + Asp 59Glu + As 96Asp + Asnl54Ser

AlalδβHis 4 Leu216Ser Thr 65Gly + Thrl02Gly 4 Leul25Asp + Glyl65Asp 4

Alal99Ser 4 Val202Thr Leul25Asn + SerlδδGlu + Glyl59Asp + Serl90Glu +

Thr212Gly + Leu216Val Thr 5δPro + Asn βlAsp + Val 94Glu + Asn 9βAsp 4

Glyl26Ser + Glyl65Pro Alal.δ ^' βGlu + Val202Glu + Tyr205Ala + Tyr213Val 4

Ser215Asp + Asn217Glu Tyrl03Val + Ser203Glu + Tyr205Asp + Thr212Glu +

Ser215Asp + Leu216Ile Gly 62Ser + IlelOβLeu + Prol2δSer + Asnl54Asp +

Serl57Glu + Thr214Ser Val 94Asp + Gly 99Asp + Prol2δGln + Alal99Gln +

Tyr205Gln + Leu216Ile Asn βlGln + Thrl02Gln + Glyl26Asp + Serl55Glu +

Serl90Asp + Val202Ser Leul25A.sp + Glyl26Asp + AlalδβSer + Tyr205Gln +

Gly21δGlu 4 ^" τhr219Pro Asn βlGlu + SerlOOAsp Thrl02Asn GlylβδGin +

PhelδδLeu 4 Val202His

Tvrlj3Gly 4 Tvr205Leu + Thr212Glu 4 Ser215Glu 4 Leu216Glu - ^" Asn217Asp ιa Pro 4 SeriJ4Glu ⁴ IlelOβThr 4 Serl3IAsp

GlylOlGln - Tnr214Ser Le _^ 9Ξlle 4 Glyl2δGlu * Giyl53Glu 4 Serl8 Asp +

Pr.elcSA.sp - Asnl11Gin Giy 52Asn ⁴ Leu 95Ser 4 Glyl53Ser 4 TyrlOSAsp 4

Thr212Giu * Leu216Asp Alal05Gly + ϋelOβCys ⁴ Serl55Asp + TyrlOSAsp 4

Ser215Asp + Leu216Asp Tyrl03Ala + Serl55Glu + TyrlββPro + Ser203Glu 4

Tyr205Glu 4 Ser215Glu Prol28Gly + Asnl62Glu 4 Glyl65Asn + Ser203Glu +

Tyr205Glu - Ser215Glu Giy 99Pro 4 LeullSGln 4 Glyl30Asn 4 AlalδβAsp 4

PnelδδSer - Ser203Glu Ilel ..βAsn 4 GlylSβGlu + Glyl59Asn 4 PhelδδAsp +

Ser215Glu * GlyllSAsp GlylSβPro + Asnlβ2Asp 4 Ser203Glu - TyrlOSGln 4

Ser21SAsp ⁴ Asn217Glu GlylS3Asn + serl55Glu + Serl57Asp + Serl90Glu 4

Thr212Asp ⁴ Leu216Ile Leu 95Glu + Gly 99Glu + Leul25Glu + prol28Gly +

Asnl54Glu 4 Thr219Gly Gly βOAsp + IlelOβMet + AsnlβlSer 4 ser203Glu 4

Tyr205Asp 4 Leu216Asp Ser 97Asp + Asnlβ2Ser 4 Thrl63Gln 4 Ser203Glu 4

Tyr205Asp 4 Leu216Asp Leui25Ile + Glyl59Glu + Ser203Glu + Thr214Glu +

Ser215Glu 4 Leu216Gln Glyl5βSer + Serl58Glu + Tyr205Asp + Tyr213Glu +

Ser215Asp + Leu216Cys Gly β2Gln + Asnl54Asp + Serl55Asp + SerlβOAsp -

Giyl65Glu + Tyr205Met Gly 64Pro + AlalδβGlu + Serlδ7Asp + Ser203Glu 4

Tyr205Hιs 4 Ser215Glu Tyrl03Asp + TyrlββSer 4 Alal86Asn + Val202Glu 4

Ser203Asp 4 Tyr205Glu Leu 95Gln + Glyl26Gln + Glyl27Asp + Prol28Ser 4

Serl55Asp + Serl5δGlu Asp 59Glu + IlelOβHis + Tyr205Ala + Tyr213Glu +

Ser215Glu + Leu216Asp Serl5δAsp + SerlβOGlu + Thrlβ3Asp + Pro209Gln 4

Thr212Ser + Ser215Glu Gly 62Asp + Tyrl03Met + Val202Ser + Asn211Glu +

Thr214Gln Leu216Ala Asp 59Glu + Ser 97Asp + Ser 9δAsp + Glyl53Ser +

GlnlβlAsn 4 Ser203Glu LeuI25Asn + Glyl53Glu + Serl57Asp + Thrlβ3Glu +

Val202Asp + Thr214Pro Leu 95Glu + SerlOOAsp + Leul25Asp + Prol2δAsn +

Serl31Asp + Tyr205Gly Glyl26Gln + Serl56Glu + Ser203Asp + Ser215Glu +

Leu216Thr - Gly218Asp GlylOlSer 4 ProI28Asp Serl58Glu 4 Glyl59Asp -

Alalδ ^β Giy - PhelδδVal Se l Giu + Ser203Asp 4 Tyr205Cys Pro209Gln ⁴ G_y 62Ξer 4 Serlδ7Giu - Metl98Gln ⁴ Ser203Glu 4 ThrI32Asn + GlylβδPro 4 Serlδ7Glu + Ser203Glu ⁴

Tyr205His 4 Leu216Asp Asn 96Ser + Serl31Glu + Glyl59Asp + SerlβOAsp

Thr214Gln 4 Leu216Gly Glyi30Glu + Serl58Asp + Glyl59Glu + Thrl63Gln

Tyr205Thr + Thr207Asn Giyl30Asp + Serl31Glu GlylβδPro 4 Serl90Glu +

Val202Gly 4 Asn217Ser Leu 95Thr + Glyl30Glu Serl31Glu + Tyr205Ala +

Ser210Glu + Gly218Gln Thr 56Gly + Alal05Glu 4 IlelO ^β Gly + Tyr205Val +

Ser215Glu + Leu216Asp Thr 58Asp 4 Asp 59Glu + Leul25Asn + Ser203Glu +

Tyr205Cys + Thr212Ser Thr 58Glu + Asp 59Glu 4 Tyrl03Asn + Pro209Gln +

Thr212Asn 4 Ser215Glu Thr 65Ser 4 Ser 97Asp + Ser 96Glu + Serl04Giu 4

Thrl63Gln + Leu216Ser Ser 97Giu + Ser 9δGlu 4 GlylOlGln + Thr212Asn 4 GlylOlAsp + Thrl02Glu 4 IlelOβVal + Thrlβ3Pro +

Tyr205Glu + Ser215Asp Giy βOPro + Serl29Glu 4 Val202Glu + Thr214Asp +

Ser215Asp + Leu216Ile Giy β2Asp + Gly 99Glu 4 Tyr205Asn + AsnlllAsp +

Tyr213Met ⁴ Thr214Asp Thr ^~ 58Gly + Glyl27Glu 4 Serl31Glu + Serl55Asp 4

GlyiβSPro + Tyr205Cys Glyl59Asp + TyrlββGly + Tyr205Asp + Tyr213Asn 4

Ser215Asp + Leu216Thr Gly 62Glu + GlylOlSer 4 Thrl32Gly + Ser203Glu +

Asn211Asp + Tyr213Glu Asn -96Asp + Ser 97Asp + Leul25Pro + Glyl27Gln +

Ser203Asp + Ser215Asp Asp 59Glu + Val 94Glu + Ser 9δAsp + Serl04Glu +

Leul25Asn + Tyr213Gly Serl55Asp + Glyl56Gln + Serl90Asp + Tyr205Glu

Thr212Ser 4 Leu216Ala Val 94Met + Gly 99Pro 4 IlelOβThr 4 Serl55A.sp 4

Serl90Asp + Thr212Glu GlnlβlSer + Thrl63Glu 4 Val202Ala + Ser203Glu +

Asn211Ser + Ser215Asp GlylδβGlu + AlalδβAsn + Ser203Asp + Thr212Gln +

Ser215Glu + Leu216Gln Ser 98Glu + Ser203Asp + Val204Cys + Ser215Asp +

Asn217Ser + Thr219Gln

Asn όlGln Thr 65Ser 4 Serl31Asp 4 Ser203Asp + IlelOβVal ⁴ Thrl32Ser + Serl58Glu + Ser203Glu 4

Thr214Ser + Ser215Glu Val 94Thr + Glyl53Glu + Alal86Gln + Ser203Glu +

Ser215Asp + Thr219Gly Val 94Met + Thrl02Asp + Prol28Glu + Serl31Asp 4

GlylβSAsn + Thr219Ser SerlSSAsp + GlnlβlAsp + Serl90Glu + Ser203Glu 4

Thr212Ser 4 LeullβCys Asn βlGiu ~ SerlOOAsp 4 Thrl32Pro + AlalδβGly 4

SerllOGiu 4 Thr212Glu AialOδGlu + Thrl32Ser + GlylOlSer 4 Ser203Glu +

Thr212Pro + Asn217Asp Thr 58Gly + Gly 62Pro + Alal05Asp + Ser203Asp +

Tyr206Ile + Leu216Glu Asn 9βAsp + Gly 99Asp + IlelOβGly + Asnl54Gln +

GlnlβlAsn + ^" τhr214Glu Asn 96Glu + Gly 99Glu 4 Thrl02A.sn 4 Glyl27Ser 4

Ser203Glu + Asn217Gln Asp 59Glu + GlyI27Asp 4 SerI29Asp 4 Tyr213Hιs +

^' Leu216Val ^{A "} τhr219Gly Thr βΞGin Tyr205Ser + Pro209Gln 4 Ser210Glu +

Thr2I2Glu + Thr219Asp Thr 53Asp + Asn 96Ser + Serl58Glu + GlnlβlAsp +

GlylOlGln + Tyr205Val Asp 59Glu + Asn βlGlu + TyrlββHis + PhelδδVal +

Thr212Gly + Ser215Asp Thrl02Glu + Glyl26Pro + Glyl27Asn + Val202Asn +

Prc209Asp + Asn211Glu Asn βlGln 4 Glyl59Gln 4 Serl87Asp 4 Pro209Asp +

Asn2iiAsp Glyl18Ser Ser 9^Asp + Ser 98Glu 4 GlyiSβAsp + Serl58Glu +

ThrI63Gln 4 Pro209Gln Gly βOGin + Glyl30Asn 4 Serl55Glu + PhelδδGlu +

Tyr205Asn 4 Ser215Glu Val 94Asp + IlelOβGlu + Prol2δAsp + Serl31Asp +

Tyr205Thr + Leu216Ser Gly βOPro + Ser 9δAsp + SerlOOGlu + Prol2δGly +

Serl57Asp + Glyl59Asp Thr 5δAsn + Gly 99Asp + Thrl02Gln 4 Pro209Asp +

Thr212Glu + Tyr213Met Thr 65Glu 4 Tyrl03Gln + GlylδβPro + TyrlββAsn

PhelSBTyr 4 Ser215Glu Val 94Asp + GlylOlGlu + Ilel64Gly + Alal86Pro +

Ser203Asp + Leu21βPro Gly βOAsn + Asn βlGlu + Glyl53Ser + Pro209Asp +

Asn211Aεp + Ser215Glu Thrl32Gly + Serl57Glu + Serlδ7Asp + Ser203Asp +

Tyr205Met + Ser215Glu Asn βlAsp + Asn 96Glu + GlylδβAsn + Ser203Glu +

Thr212Gln + Ser215Glu Gly 99Pro 4 Serl53Glu + SerlβOGlu + Tyr205His +

h l l l S e : 3 e r 2 1 5 u C I Pro 4 5erl57Glu - Ser203Asp 4 Tyr205Asp

ThrlllSer - Leu2IβCys Val 94Ala 4 Prol2δSer ^" 4 Glyl56Gln + Serl90Asp 4

3er203Asp ⁴ Tyr205Glu IlelOβMεt Leul25Ile 4 SerlβOAsp + ser203Asp *

Tyr2o5Glu + LeullβSer Leu ^* 95Glu 4 Ser 97Glu 4 Thrl02Glu 4 Giyl59Asp 4

Tyr205Leu - Leu216Ala Leul ^" 25Cys + GiyllβSer 4 Glyl59Asp + GlnlβlGlu 4

3er215Glu 4 Asn217Giu SerlOOGlu 4 Thrl02Gly + Alal05Thr Serl55Glu 4

Serl57Asp - Asnl62Ser Ser 9SGlu + Glyl27Asn 4 5erl57Asp + GlnlβlAsn 4

Asnl62Asp ⁴ Glyl65Glu Asn βlGin + SerlOOAsp 4 Serl04Glu + IlelOβAsp +

3er215Asp ⁺ LeullβAla Seri31Giu ⁴ Glyl59Asn + Val202Cys + Ser215Asp 4

LeullβAsp * Thr219Giu LeullSCys 4 GinlβlAsp Asnl62Glu + Ser203Giu 4

LeullδThr 4 GlyllδGlu GlylOlAsp 4 Alal05Asp 4 Giyl26Asn 4 GlnlβlSer 4

AlalSβPro 4 Thr214Glu SerlOOGlu + Thrl02Glu 4 Tyrl03Thr + Glyl26Asp 4

SerlβOGlu + Leu216Met Glyl27Gln 4 Asnl54Ser + Serl57Glu + Thrl63Glu 4

Ser2I5Glu + Asn217Glu Leu 95Ile + Asn 96Glu Thrl02Gly + Tyrl03Glu 4

GlyllβGlu 4 Glyl65Glu 5erl29Asp + Serl31Asp + Thrl32Asn + Glyl59Ser 4

Tyr213Gln ⁴ Thr214Glu GlylOlPro + Glyl2βGlu + Serl29Asp + Tyr205Giu +

Ser215Glu + Leu216Gly Leu 95Met + Prol28Gly + Serl29Glu + Glyl53Glu +

Thrl63Gly + Leu216Ser Gly βOGlu + Val202Met + Tyr205Ile + Thr212Glu 4

Ser215Glu + Leu216Asp Thr 5δSer + SerlδδAsp 4 GlnlβlAsn + Thrl63Asp 4

Leu216Asp 4 Gly21δAsp IlelOβSer 4 Serl58Asp 4 Serl87Asp + Serl90Asp +

Tyr205Met 4 Ser215Asp Serl67Giu + Val202Gln + Tyr205Asn + Thr214Glu

Leu216Glu + Gly21δSer Asp 59Glu + SerlOOAsp Glyl26Pro + Glyl27Asp +

TyrlββGiu + Thr212Asn Asp 59Glu + Gly 62Pro + Glyl27Asn + AlalδβPro +

Val202Glu + Pro209Asp Asp 59Glu + Asn 96Glu 4 GlylOlGlu + Thrl02Gln 4

Prol2δSer Leu216Glu Glyl59Asp + Asnlβ2Asp AlalββGln + Val202Gln 4

Ser203Glu + Ser215Asp Glyl59Ser + AlalδβAsn Ser210Asp + Tyr213Pro

Ser215Asp + Asn217Asp

Serl04Glu 4 Serl31Asp 4 Thrl32Gln + Asnl62Ser 4

GlyiβδAsp 4 TyrlOδCys Giy-IΞβGlu 4 SerlSBAsp 4 Thrlβ3Gly + Ser203Asp 4

Thr212Asn 4 Leu216Ser Gly →. Ε s. - Tr.r βSGlu + AialCSAsp 4 VallClThr 4

SerllΞAsp - Leu216Glu Thr ≡SPro 4 Gly 99Ser + SerlOOAsp + GlylOlSer 4

ThrlOlAsp + Asn211Glu Gly ό2Glu 4 Asn 96Ser 4 SerlOOAsp + GlylOlSer

^{^} SerllOAsp + Tyr213Ala Gly βOPro 4 GlnlβlAsp Glyl65Asp + PhelδδVal +

^* Serl90Glu 4 Ser210Glu Glyl53Gln 4 Serl55Asp + Glyl59Asn 4 Pro209Glu +

Thr212Asp ÷ Ser215Asp Thr 5SGin 4 Thrl02Glu 4 ^" SerI29Glu 4 GlylSOAsp 4

Tyr205Aia - Ser215Asp TyrI03Ser 4 Asnl54Asp 4 Serl87Giu 4 Val202Asn +

Ser203Glu - Leu216Ser Leul25Pro 4 Prol28Ser + Serl57Glu 4 GlnlβlGlu +

Ser203Glu + Gly21δSer Ser 97Glu + Gly 99Asp + Alal05Ser + Leul25Asp +

Glyl65Gln 4 Tyr205Asp Thr 6~5Asp 4 Leu 95Cys + Ser 97Asp + Asnl62Ser +

Thr2I2Glu Ser215Asp Ser 97Glu SerlOOGlu 4 GlylδβAsn 4 Serl57Glu +

SerlδOGlu 4 Tyr205Pro Leu 95Asp 4 Aial05Asp + Serl57Glu + Val202His +

Tyr205lle Leu216Met Leu 95His 4 GlylδβPro 4 Serl57Asp 4 Asnlβ2Glu 4

Serl67Glu + Leu216Asp SerlOOGlu + Leul25Gly + Glyl27Glu + Ser203Asp +

Tyr20δVal + Leu216Asp Asnl54Ser + Glyl65Asp + Serl90Glu + Thr212Gly 4

Ser215Asp + Gly21δPro Thr 56Gln 4 Ser 9δGlu + Glyl53Asp + Val202Ser +

Tyr205Asn 4 Gly216Asp Asn βlGln 4 Thrl32Gly 4 Glyl56Glu + SerlSβGlu 4

. Ser215Asp 4 Thr219Glu Val 94Glu + Ser 97Glu + Alal05Asp + Glyl65Pro +

Leu216Met + Thr219Asp Val 94Glu + Ser 97Glu + Alal05Asp + Asnl54Gln +

GlnlβlAsp + AlalδβAsn Glyl30Gln + Serl55Glu + SerlδδGlu + AlalδβGln +

Ser203Asp + Leu216Cys Asn βiGln + SerlβOAsp + Ser203Glu + Val204Gln +

Thr212Glu + Ser215Glu Thrl02Glu + Alal05Asp + Serl31Glu + Ser203Glu +

Tyr205Aia 4 Thr214Asn Thr ^' δδPro 4 Serl55Glu + GlyI59Asn + Pro209Glu +

Thr212Glu + Gly21δGlu Asp 59Glu + Gly 99Asp + Glyl26Ser Metl96Thr +

Thr212Asp + Gly21δPro Thrl32Ser + Asnl54Ser + Serl57Asp + Val202Glu +

Ser215Glu * Leu216Gly Val 94Asp Giy 99Asn + SerlOOAsp + Glyl303er 4

Asnl54Gln - Ser215Asp ProllSGlu + Glyl565er 4 SerlβOGlu + Serl87Aεp

Serl90Glu - Thr2I2Ser Asn βlGln + ser 98Glu + IlelOβCys + Ser203Asp +

Tyr205Gly 4 Gly21δAsp Alal05His + SerlδδGlu 4 Serlδ7Glu + Ser203Glu

Tyr205Leu 4 Ser215Glu Asn 96Ser 4 Gly 99Gln 4 Glyl53Glu + AlalδβGlu +

Tyr205Glu ⁴ ser215Glu Asp Ξ9Glu + ser 9δGlu 4 Giyl53Glu + Serl55Glu 4

PhelδδPro ⁴ LeullβHis AsnlβlAsp + GiylβSAsp 4 Val204Gin + Tyr205Thr 4

SerllOAsp 4 Thr212Pro

;ι ^{■ •} IC-lPro + GlylδδAsp + TyrlββAsp + Serl90Glu

Tyr205Asp + Thr212Asn GlyllβGlu + Thrl32Asp + Serl55Asp + Serl57Asp +

Val202Gly 4 Tyr213Ser Gly βOAsp + Asn βlAsp + Asnl54Glu + Serl5δGlu 4

GlylβδGln 4 Tyr213Gln Gly 6 ^~ 2Glu + Leul25His + SerI31Asp + TyrlββLeu 4

Ser203Glu 4 Ser215Asp Asp 59Glu + Val 94Asp 4 Thrl02Gly + Ser203Glu 4

Tyr205Asp 4 Thr219Gly Serl04Glu + TyrlββCys + Val202Thr + Thr212Asp +

Ser215Asp + Asn217Glu SerlOOAsp + Glyl26Pro + Asnl54Asp + Gly201Ser 4

Ser203Glu 4 Thr219Asp GlylOlAsp 4 Prol2δAsp + Serl55Asp + Serlδ7Asp +

Thr207Gly + Thr212Gln IlelOβSer + SerlβOGlu 4 Serl90Asp + Ser203Asp +

Ser215Glu + Leu216Val Gly 99Gin 4 Glyl53Asp + SerlβOAsp + PhelδδLeu 4

Serl90Asp + Ser210Asp SerlδδAsp + GlylβSAsn 4 Serl90Glu 4 Val202His 4

Tyr205Asp 4 Tyr213Leu Thr 65Asp + Leu 95Thr 4 Serl31Glu + Val202Ser 4

Ser203Asp + Leu216Glu Asn -βlGlu + Glyl65Ser + Metl9δGln + Val202Glu +

Tyr205Glu 4 Leu216Asn Val 94Ala + Glyl26Asp + Asnl54Glu + TyrlββPro +

Val202Gln + Thr212Glu Ser 97Glu + Leul25Glu + Serl55Glu + Glyl59Asn 4

TyrlββAsp + Leu216Val Asn 96Glu + Ser 97Asp + Thrl02Gln + Leul25Asp +

Leu216Glu + Thr219Gln Tyrl03Cys + SerlβOGlu + Thrl63Asp + Tyr203Ser

Leu216Met + Gly216Glu Val 94Cys + Leul25Glu + Serl55Asp + Glyl56Asp +

Gly201Gln + Ser215Glu Asn βlSer + Thr 65Glu + Val 94Asp + Tyrl03Pro +

Ser215Asp 4 Asn217Asp

Giy 99Gln 4 Glyl27Asp + Thrl32Pro + Serl55Glu +

Serlδ7Glu + Ser203Asp Ser.9δGlu ⁴ Serl29Glu 4 Glyl53Glu + Serl90Asp +

Tyr213Val + Thr214Pro Asn βlSer 4 Glyl30Glu + PhelδδGlu + Tyr205Asp 4

Thr212Pro + Asn217Asp Serl53Glu Giyl59Glu + Val202Ser + Tyr205Met 4

Pro209Giu + 5er215Glu Asn βlAsp 4 Gly 99Asn 4 GlylOlAsp + Ser203Asp 4

Tyr205Asp + Thr212Pro Ser ^~ 98Asp 4 SerlOOAsp + Serl04Glu + Glyl53Glu +

PhelδδGly + Gly21δAsn Leu 95Val + Gly 99Asp + Tyrl03Val + Alal05Asp +

Tyr205Asp + Ser215Asp Asp 59Glu + Asn βlAsp + SerlβOGlu + Pro200Asn +

Asn211Ser Thr212Asp Gly 62Glu ⁴ Thr 65Gly + ser 97Asp + Thrl02Pro +

Serl31Giu + Leu21βAsp GlyllβPro - Prol23Giu -< GlylδβGlu + SerlδδAsp +

Tyr205Asn 4 Asn217Asp Asp 59Glu 4 Glyl30Asn 4 Serl55Asp 4 Ser210Asp 4

Tyr213Gly + Gly216Asn Asn 96Gln 4 Gly 99Glu 4 SerlOOAsp 4 Prol26Asp 4

Tyr205Cys + Asn217Glu Prol28Gly + Serl29Asp + Asnlβ2Glu + Glyl65Asn +

Ser203Asp + Ser215Asp Asn 96Ser + SerlOOGlu + Tyrl03Glu + Tyr205His + Asn 9βGlu + Ser 98Glu + AlalδβAsp + Val202Asn 4

Tyr213Met + Leu216Glu Gly 99Asp + Serl56Asp + Asnlβ2Asp + Thrl63Asn +

Serlδ7Glu + Alal99Gly Leu 95Ser + Ser 97Asp + Ser 96Glu + GlylβδGlu +

Leu216Ile + Gly21δGlu GlylOlAsp + Leul25Asp + GlnlβlAsn + Tyr205Asp +

Thr212Gly + Leu216Asn SerlβOAsp + Val202His + Ser203Asp + Thr212Asp +

Tyr213Val + Leu216Asp Leu 95Val + Tyrl03Glu + Alal05Glu + Thrlβ3Ser +

Thr212Asp + Leu216Glu Serl29Asp 4 Glyl30Ser 4 Serl57Asp + GlylβδAsp +

Tyr205Glu + Leu216Gln Thrl32Asn + GlylδβGlu + TyrlββThr + Ser203Glu +

Thr212Glu + Asn217Glu Asn βlGln + Ser 97Glu + Serl87Glu + Ser203Glu +

Pro209Gln + Ser215Glu GlylOlGln + Alal05Asp + Serl87Glu + Ser203Asp +

Ser215Glu + Leu216His Gly 99Asn + Serl04Asp + Thrl32Gly + Serlδ7Asp +

Ser203Glu + Ser215Asp Gly 62Asp + Asn 96Gln + Serl04Glu + Tyr205Gln +

Thr212Asp + Tyr213Val Val 94Gly + Asn 96Asp + AlalOSGlu + Asnl62Asp +

Asn211Gln + Thr212Gln Ser 97Glu + Serl37Asp + Ser203Glu + Tyr213Gln 4

LeullβGlu + Asn217Gln Gly 64Pro + Serl04Glu + Glyl2βGlu + TyrlββSer 4

TyrlOSGlu 4 Ser215Glu 31yl31Asn Thri32Asp ⁴ Serl87Glu + Pro209Gln +

Tnr214Glu 4 Ser215Glu liyllβGiu 4 SerlβOAsp + TyriββMet + Val202Thr

^" Thr214Asp * Ser215Glu Ser 98Asp GlylOlSer + Glyl30Asp + Serl58Glu 4

Glyl59Asp 4 Leu216Ala Thr 5δGlu + Tyrl03Pro + Glyl30Glu + Serl31Asp +

SerlδδGlu 4 Alal99Pro Gly βOAsp 4 Asn βlAsp Serl31Asp + Glyl53Ser 4

Thrl63Glu + Val202Cys Gly 99Asp + Prol28Asn 4 Asnl62Giu 4 Thr212Ser + Gly βOSer 4 Ser 98Asp + Asnl54Asp + Serl55Glu +

Glyl59Pro 4 Asn211Glu Tyrl03Prc 4 Serl04Glu 4 Leul25Cys + Val202Glu +

Ser203Glu 4 Pro209Glu GlylOlGln 4 Serl04Asp + Leui25Val + Val202Asp +

Ser203Asp + Thr212Asp Thr 65Asn + Ser 9δAsp 4 Glyl59Asn + Glyl65Asp +

Ser215Asp + Leu216Asp Glyl27Glu + GlylβδPro + Tyr205Cys + Ser210Asp +

Ser215Glu + Leu216Glu Val 94Ser 4 ser 98Glu 4 Gly 99Asp GlylδβAsp +

PhelδδLeu 4 Ser203Glu Giy 62Asp + GlylOlGlu + SerlβOGlu GlnlβlAsp 4

Tyr205Met + Thr212Pro Ser 97Asp + Ser 9δGlu 4 Alal05Hιs + Serl57Glu +

Thr212Ser + Ser215Asp Gly 62Gln + Ser 97Glu 4 Ser 9δAsp + Glyl26Glu +

Glyl30Pro + Thr212Glu Val 94Glu + Ser 97Asp 4 Leul25Asn + Thrl63Ser +

Ser210Asp + Leu216Ile Leu 95Glu + Asn 96Gln 4 Glyl59Glu + Tyr205Glu +

Pro209Ser 4 Ser215Glu Serl29Glu + Asnl54Ser + Thr212Glu + Ser215Asp

Gly21δAsp + Thr219Ser Asp 59Glu + Glyl27Gln + Val202Glu + Asn211Glu +

Thr212Ser + Ser215Asp Serl04Asp + Alal05Pro + Serlδ7Glu + Ser203Glu +

Tyr205Asp + Tyr213Pro Thr 5δPro + Asn 96Asp + Glyl27Glu + Serl55Glu +

Asnl62Ser 4- Thr212Ser Thr 5δGly 4 Leul25Asn + GlylSβAsp + GlnlβlGlu

Tyr205Gly + Ser215Asp Gly βOSer 4 Glyl56Asp + Pro209Ser + Thr214Asp

Leu216Thr + Thr219Asp Leul25His + Glyl27Gln Serl31Glu + Ser203Asp

Asn211Asp + Ser215Glu

Thr 58G!u + PrcllδGlu + Glyl65Gln - AialδβHis 4

Ser203Glu + Ser215Glu Serl31Asp + Serl58Glu + Ser203Asp - Tyr205His 4

SerllΞAsp + leullβGin Thr βSA≤r. - Giyl30Asp + SerlSδAsp + Ser2C3Asp 4

Pro209Ser 4 ≤er215Glu Giy όOGln + Thrl02Glu + Serl57Asp + Ser203Asp +

Ser215Asp + Thr219Gln Gl SerlβOGlu + Ser203Asp + Ii Thrl63Asp + Ser203Glu 4 GlyllβSer SeriβOAsp 4 Thr212Glu 4 Ser215Glu 4

LeullβVal 4 Thr219Ser Giy βOPro 4 GlylOlAsn 4 SerlSδGlu + Tyr205Ile +

Thr212Asp ⁴ 5er215Glu Gly 62Glu Gly 99Ser 4 VaI202Ser 4 Tyr205Asp 4

Thr212Asn + ^' τhr214Ser Val 94Hιs + Thri32Glu 4 Glyl65Asp + TyrlββCys +

Ser203Asp ⁴ Leu216Glu Gly 62Asn + Serl31Asp + Serl57Glu + GlnlβlAsn 4

Ser203Glu + Leu216Glu Giy βOPro + Val 94Asp + Alal05Glu 4 Glyl27Pro +

GlnlβlAsp + Ser210Asp Gly β2Gln Ser 97Asp 4 Asnl54Asp + Serl90Asp +

Ser203Glu 4 Thr212Ser Ser 97Glu + Asnl54Glu + Glyl59Asn 4 Serl90Asp +

Tyr205Thr + Asn211Glu Thr 65Glu + Ser 9δAsp + Thrl02Pro + Glyl26Asp +

Glyl65Asp + Gly21δGln Asn 96Glu + Tyrl03Asp + Glyl59Asp + Asnl62Asp +

Tyr20δLeu + Thr212Pro Gly β2Asp + Serl04Glu 4 IlelOβAsp + Serl57Asp +

Gly21δ ^" ser + Thr219Pro Asn 9 ^* 6Glu 4 Gly 99Asp + SerlβOAsp + Gly201Ser 4

Asn211Gln 4 ^" ser215Glu IlelOβPro 4 Glyl53Asp 4 Glyl65Glu + TyrlβόSer 4

Ser203Asp + Thr212Asp Se 97Asp + Gly 99Asn + Serl5δAsp + Ser210Glu +

Thr212Glu + Leu216Thr Asp 59Glu + Ser 9δAsp + SerlβOGlu + Gly201Ser +

Thr212Gly + Tyr213Asn Val 94Ser + Ser 97Asp + Serl87Glu + Serl90Asp +

Pro209Asp + Leu216Asn GlylOlGln + Glyl26Asp + Serl31Asp 4 Asnl54Glu +

Glyl56Asn + Ser203Asp Gly 6~4Ser + Ser 98Glu + SerlOOGlu + AlalδβGlu +

Thr212Glu + Leu216Pro Ser 97Asp + Gly 99Asp + Val202Asp + Thr207Asn +

Asn211Glu + Leu216Met Thr 65Pro + Thrl32Gly + AlalδβAsp + Val202Ala +

Ser203Glu + Thr212Asp IlelOβAsp + Glyl27Ser + Asnl54Glu + Glyl56Glu +

Thr214Pro + Ser215Glu Tr.r 5-sAsp - 3erl29Glu * ThrlllAsn ⁴ Glyl65Asp - e ΣlβVal 4 Gly216Asp Tr.r ISPro 4 Gly βOGlu 4 Gly βlGlu ⁴ Thrl 2A.sp -

Serl58Glu 4 Leu216Ala ProllδAsp 4 Asnl54Glu PhelδδVai ⁴ Val202Cys 4

Ser203Asp + Tyr205Asn GlylOlGln 4 Alal05Hιs + Serl55Asp + TyrlββVal 4

AlalδβAsp + Pro209Glu Leu 95Val + Serl55Glu + TyrlββAla + AlalδβGlu

Ser210Asp 4 Leu216Met Asn 9βAsp + GlylOlGlu + Asnl62Glu + Val202Thr 4

Ser210Asp 4 Leu216Asn Serl29Glu 4 Serl57Asp + PhelδδAla 4 Serl90Glu 4

Val202Ser + Tyr205Glu Serl31Asp + SerlδδAsp + SerlβOAsp + Tyr205Ile

Ser215Asp + Leu216Met Gly β2Glu + Leu 95Glu + Tyrl03Pro + AlalδβHis 4

Ser203Glu + Thr207Gly SerlOOGlu + Leul25Ala Serl57Glu + Ser203Asp 4

Tyr205Asp + Leu216Cys ThrlClGin 4 Serl04Asp + Glyl53Pro + Serl58Glu 4

Ser203Glu 4 Tyr205Asp Asn βlGlu 4 Asn 96Gln + Ser 98Asp ⁴ Serl58Asp +

Thr212Asn ⁴ Thr219Asp Val 94Thr 4 Thrl32Asp 4 SerlSSAsp + Serl57Glu 4

Thr207Gly + Ser215Asp Prol26Gly + Glyl30Pro + SerlδδGlu + Serlδ7Glu 4

PhelδδCys 4 Ser203Asp Thr 65Glu 4 Glyl30Pro + Glyl59Glu + GlylβδAsp +

PhelδδMet + Pro209Asp Thrl63Glu + Val202Asp + Tyr205Cys + Thr212Asp 4

Leu216Pro + Gly21δGiu Ser 97Glu + GlylOlAsn + Glyl30Glu + Asnl54Asp *

Thrl63Gly + Gly216Glu Thr 65Gly + Alal05Glu + Serl55Asp + GlnlβlGlu 4

PhelδδVal + Asn217Gln Thr β5Gln + Tyrl03Asp + Prol26Glu + Serl55Asp +

Ser215Asp + Leu216Ser Thrl-32Glu + AlalδβGln + Serlδ7Glu + Val202Pro +

Thr207Gly + Asn217Asp Gly βOGlu + Serl29Glu + Serl31Glu + GlylβδAsn +

Ser203Glu + Pro209Gln GlylOlPro + Prol23Asp + Glyl30Asp + Ser203Glu 4

Tyr205lle + Thr212Asp Val 94Glu + Asn 96Glu + Asnl54Asp + Tyr205Ala +

Thr212Glu + Leu216Cys Ser 97Glu + Thrl02Ser 4 Serl57Asp + Ser215Asp +

Leu216Ser Asn217Glu Ser 9δAsp + GlylδβGlu + SerlδδGlu + GlnlβlSer +

Ser203Asp + Gly218Asn Glyl30Ser + Glyl56Asp + Serl56Glu + Ser210Asp

Thr212Gln + Ser215Asp

SerlOOGlu + ThrlOlAsp + Glyl2δPro + Glyl27Ser +

Serl87Glu + Ser215Giu Asn.96Glu + Glyl27Ser + Prol28Gln 4 Serl90Asp 4

Tyr213Asp + Ser215Glu Prcl23Glu ⁴ Glyi53Ser + Serl5δGlu + GlylβδSer 4

AialSβGly + SerlS^Glu Thr 65Glu 4 Giyl53Asp + Thrl63Asp + Glyl65Asn 4

Metl98Hιs 4 Ser203Glu LeullSGly - Serl29Glu + Thrlβ3Glu + AlalδβHis 4

Ser203Glu 4 Tyr205Leu Thr 5SGlu ⁴ Asn βlSer + Thr β5Gly 4 Serl58Asp 4

Asnl62Asp ⁴ Ser203Glu Thr 65Asn 4 Val 94Thr + Glyl2βGlu + Serl57Asp +

Asnl52Glu + Leu216Asp Asp 59Glu 4 ser 97Glu + Prol2δAsn 4 Thrl32Glu +

Serl57Asp Tyr205Gly Thr 58Glu 4 Giy βOGln 4 Leu 95Glu + Val202Gln +

Tyr205Gln ^{4 "} ser215Asp Asp 59Giu - Serl57Aεp 4 Asnlβ2Asp - TyrlββAsn 4

Leu216Ile - Asn217Glu Serl57Glu Asnl62Glu Phel88Gly 4 Ser203Asp +

Asn211Asp ⁴ Thr212Ser Serl57Glu 4 Asnl62Asp + Ser210Asp + Thr212Ser 4

Tyr213Leu - Leu216Asp Giy βOPro + Ser 97Glu + SerlOOAsp + Alal05Hιs +

Ser203Glu 4 Asn211Asp Asn βlAsp + Leul25His + GlylδβAsn + Pro200Gln 4

Thr212Glu 4 Leu216Asp Thr 65Asp + Ser 97Asp + Leul25Asn + Serl57Glu +

Thrl63Gly - Thr212Asp Gly 52Glu 4 Thr 65Asn 4 Asnl62Glu + Thrl63Ser 4

Asn2IlAsp 4 Ser215Glu Val 94Glu + Asn 96Ser 4 GlylOlPro + Thrl02Asp 4

Serl55Glu + Thr212Asp Gly βOGlu + Ser 98Glu + Gly 99Gln + Serl04Glu +

Alal05Thr 4 Thr214Asp Serl55Asp + Serlδ7Asp + Alal99Gln + Ser203Asp +

Tyr205Val + Thr212Glu Se l04Glu + IlelOβCys + Thrl63Ser + Glyl65Glu +

Serl90Glu + Thr212Asp Val 94Thr 4 IlelOβAsp + Glyl30Asn + Serl31Asp +

Ser203Glu + Tyr205Pro Ser 96Glu + Prol26Glu + Serl55Asp + SerlβOGlu +

AlalδβSer + Pro209Asn Thr 58Glu + Glyl59Ser + TyrlββGlu + Serl90Glu +

Thr212Pro + Leu216Ser Leu 95Glu Leul25His + Glyl2βAsp + Tyr205Gln +

Ser215Asp + Thr219Asn Asp 59Glu + Gly 99Asp + Alal05Glu + AlalδβPro +

Serlδ7Asp 4 Thr212Pro Asn 96Gln + Ser 9δGlu + GlylOlPro + Thrl32Glu +

Val202Asp + Ser215Asp Giy 99Asp + Prol2δAsp + Thrl32Asn 4 Val202Asp +

SerlllAsp ⁴ Leu21βHιs Val 94Gin + ^" GlylOlAsp + Serl04Asp + Thrl63Pro 4

Serl37Asp 4 Ser215Asp Leu ^' 95Met 4 GlylOlAsp 5erl04Glu + Glyl53Glu +

4 ^" ser215Glu Serl04Glu 4 Ser203Asp + Tyr205Ile +

SerllOGlu ⁴ Thr212Prc Val 94Glu 4 SerlDOGlu 4 IlelOδMet + Glyl30Glu +

Vai202Glu 4 Thr214Asn Ser 98Asp + IlelOβAsp + Asnl54Asp + GlylδδAsp 4

Val202Met + Leu216Gln Leu 95Met + Thrl02Ser + Glyl27Asn + Serl90Glu +

Val202Gly + Asn217Asp Gly βlPro IlelOβGln 4 Glyl26Asp + Serl29Glu +

Serl60Asp 4 Ser215Asp Sly lA.sp 4 Asnl54Glu 4 SerlβOGlu + AlalδβThr +

Se 1S ^~ Asp 4 Leu216Thr Sly 1Ser * Thr 65Ser 4 Serl55A.SO ⁴ Giyl59Glu + ^" =er203Glu 4 Thr219GIn

π. Cleaning Compositions

In another embodiment of the present invention, an effective amount of one or more of the enzyme variants are included in compositions useful for cleaning a variety of surfaces in need of proteinaceous stain removal. Such cleaning compositions include detergent compositions for cleaning hard surfaces, unlimited in form (e.g., liquid and granular); detergent compositions for cleaning fabrics, unlimited in form (e.g., granular, liquid and bar formulations); dishwashing compositions (unlimited in form); oral cleaning compositions, unlimited in form (e.g., dentifrice, toothpaste and mouthwash formulations); denture cleaning compositions, unlimited in form (e.g., liquid, tablet); and contact lens cleaning compositions, unlimited in form (e.g., liquid, tablet).

' The cleaning compositions also comprise, in addition to the Subtilisin DY variants described hereinbefore, one or more cleaning composition materials compatible with the protease enzyme, the term "cleaning composition material", as used herein, means any liquid, solid or gaseous material selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, granule, bar, spray, stick, paste, gel), which materials are also compatible with the Subtilisin DY variant used in the composition, the specific selection of cleaning composition materials are readily made by considering the surface material to be cleaned, the desired form of the composition for the cleaning condition during use (e.g., through the wash detergent use). The term "compatible", as used herein,

means the cleaning composition materials do not reduce the proteolytic activity of the Subtilisin DY variant to such an extent that the protease is not effective as desired during normal use situations Specific cleaning composition materials are exemplified in detail hereinafter

As used herein, "effective amount of enzyme variant" refers to the quantity of enzyme variant necessary to achieve the enzymatic activity necessary in the specific cleaning composition Such effective amounts are readily ascertained by one of ordinary skill in the art and is based on many factors, such as the particular enzyme variant used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e g , granular, bar) composition is required, and the like Preferably the cleaning compositions comprise from about 0 0001 % to about 10% of one or more enzyme variants of the present invention, more preferably from about 0 001 % to about 1 %, more preferably still from about 0 01 % to about 0 1 % Several examples of various cleaning compositions wherein the enzyme variants may be employed are discussed in further detail below All parts, percentages and ratios used herein are by weight unless otherwise specified

As used herein, "non-fabric cleaning compositions" include hard surface cleaning compositions, dishwashing compositions, oral cleaning compositions, denture cleaning compositions and contact lens cleaning compositions A Cleaning Compositions for Hard Surfaces. Dishes and Fabrics

The enzyme variants of the present invention can be used in a variety of detergent compositions where high sudsing and good insoluble substrate removal are desired Thus the enzyme variants can be used with various conventional ingredients to provide fully-formulated hard-surface cleaners, dishwashing compositions, fabric laundering compositions and the like Such compositions can be in the form of liquids, granules, bars and the like Such compositions can be formulated as modern "concentrated" detergents which contain as much as 30%-60% by weight of surfactants

The cleaning compositions herein can optionally, and preferably, contain various anionic, nonionic, zwitteπonic, etc , surfactants Such surfactants are typically present at levels of from about 5% to about 35% of the compositions.

Non miting examples of surfactants useful herein include the conventional Cι ι-C<|8 alkyl benzene sulfonates and primary and random

alkyl sulfates, the C10- 18 secondary (2,3) alkyl sulfates of the formulas CH ₃ (CH ₂ )x(CHOSθ3)-M ⁺ )CH3 and CH ₃ (CH ₂ )y(CHOS0 ₃ -M ⁺ ) CH ₂ CH ₃ wherein x and (y+1 ) are integers of at least about 7, preferably at least about 9, and M is a water-solubi zing cation, especially sodium, the CIQ- C-|8 alkyl alkoxy sulfates (especially EO 1 -5 ethoxy sulfates), C-| n- i8 alkyl alkoxy carboxylates (especially the EO 1 -5 ethoxycarboxylates), the Ciø- C-|8 alkyl polyglycosides, and their corresponding sulfated polyglycosides, 12-C18 alpha-sulfonated fatty acid esters. C12- 18 alkyl and alkyl phenol alkoxylates (especially ethoxylates and mixed ethoxy/propoxy), C12-C-18 betames and sulfobetames ("sultames"), C10-C18 amine oxides, and the like The alkyl alkoxy sulfates (AES) and alkyl alkoxy carboxylates (AEC) are preferred herein (Use of such surfactants in combination with the aforesaid amine oxide and/or betame or sultame surfactants is also preferred, depending on the desires of the formulator ) Other conventional useful surfactants are listed in standard texts Particularly useful surfactants include the C-| rj-C-| 8 N-methyl glucamides disclosed in US Patent 5, 194,639, Connor et al , issued March 16, 1993, incorporated herein by reference

A wide variety of other ingredients useful in detergent cleaning compositions can be included in the compositions herein, including other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, solvents for liquid formulations, etc If an additional increment of sudsing is desired suds boosters such as the C10- 16 alkolamides can be incorporated into the compositions, typically at about 1 % to about 10% levels The C-|n-C 1 monoethanol and diethanol amides illustrate a typical class of such suds boosters Use of such suds boosters with high sudsing adjunct surfactants such as the amine oxides, betames and sultames noted above is also advantageous If desired, soluble magnesium salts such as MgCI ₂ , MgSO and the like, can be added at levels of, typically, from about 0 1 % to about 2%, to provide additionally sudsing

The liquid detergent compositions herein can contain water and other solvents as carriers Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol, propanol, and isopropanol are suitable Monohydπc alcohols are preferred for solubilizmg surfactants, but polyols such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e g , 1 ,3-propanedιol, ethylene glycol, glycerine, and 1 ,2-propanedιol) can also be used The compositions may

contain from about 5% to about 90%, typically from about 10% to about 50% of such carriers

The detergent compositions herein will preferably be formulated such that during use in aqueous cleaning operations, the wash water will have a pH between about 6 8 and about 11 0 Finished products thus are typically formulated at this range Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc , and are well known to those skilled in the art

When formulating the hard surface cleaning compositions and fabric cleaning compositions of the present invention, the formulator may wish to employ various builders at levels from about 5% to about 50% by weight Typical builders include the 1 -10 micron zeolites, polycarboxylates such as citrate and oxydisuccmates layered silicates, phosphates, and the like Other conventional builders are listed in standard formularies

Likewise, the formulator may wish to employ various additional enzymes, such as cellulases, lipases, amylases and proteases in such compositions, typically at levels of from about 0 001 % to about 1% by weight Various detersive and fabric care enzymes are well-known in the laundry detergent art

Various bleaching compounds, such as the percarbonates, perborates and the like, can be used in such compositions, typically at levels from about 1 % to about 15% by weight If desired, such compositions can also contain bleach activators such as tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate, and the like, which are also known in the art Usage levels typically range from about 1 % to about 10% by weight

Various soil release agents, especially of the anionic o goester type, various chelating agents, especially the ammophosphonates and ethylenediaminedisuccinates, various clay soil removal agents, especially ethoxylated tetraethylene pentamme, various dispersing agents, especially polyacrylates and polyasparatates, various brighteners, especially anionic bπghteners, various suds suppressors, especially si cones and secondary alcohols, various fabric softeners, especially smectite clays, and the like can all be used in such compositions at levels ranging from about 1 % to about 35% by weight Standard formularies and published patents contain multiple, detailed descriptions of such conventional materials

Enzyme stabilizers may also be used in the cleaning compositions Such enzyme stabilizers include propylene glycol (preferably from about 1 %

to about 10%), sodium formate (preferably from about 0 1 % to about 1 %) and calcium formate (preferably from about 0 1% to about 1%) 1 Hard surface cleaning compositions

As used herein "hard surface cleaning composition" refers to liquid and granular detergent compositions for cleaning hard surfaces such as floors, walls, bathroom tile, and the like Hard surface cleaning compositions of the present invention comprise an effective amount of one or more enzyme variants of the present invention, preferably from about 0 001 % to about 10%, more preferably from about 01% to about 5%, more preferably still from about 05% to about 1 % by weight of active enzyme of the composition In addition to comprising one or more of the enzyme variants, such hard surface cleaning compositions typically comprise a surfactant and a water-soluble sequestering builder In certain specialized products such as spray window cleaners, however, the surfactants are sometimes not used since they may produce a filmy/streaky residue on the glass surface

The surfactant component, when present, may comprise as little as 0 1 % of the compositions herein, but typically the compositions will contain from about 0 25% to about 10% more preferably from about 1 % to about 5% of surfactant

Typically the compositions will contain from about 0 5% to about 50% of a detergency builder, preferably from about 1% to about 10%

Preferably the pH should be in the range of about 8 to 12 Conventional pH adjustment agents such as sodium hydroxide, sodium carbonate or hydrochloric acid can be used if adjustment is necessary

Solvents may be included in the compositions Useful solvents include, but are not limited to, glycol ethers such as diethyleneglycol monohexyl ether, diethyleneglycol monobutyl ether, ethyleneglycol monobutyl ether, ethyleneglycol monohexyl ether, propyleneglycol monobutyl ether, dipropyleneglycol monobutyl ether, and diols such as 2,2,4-trιmethyl-1 ,3-pentanedιol and 2-ethyl-1 ,3-hexanedιol When used, such solvents are typically present at levels of from about 0 5% to about 15%, preferably from about 3% to about 11 %

Additionally, highly volatile solvents such as isopropanol or ethanol can be used in the present compositions to facilitate faster evaporation of the composition from surfaces when the surface is not rinsed after "full strength" application of the composition to the surface When used, volatile

solvents are typically present at levels of from about 2% to about 12% in the compositions

The hard surface cleaning composition embodiment of the present invention is illustrated by the following examples

Examples 7-12

^* Dιsodιum N-dιethyleneglycol-N,N-ιmιπodιacetate **Na ethylenediamine diacetic acid ^* **Dιethyleneglycol monohexyl ether ^* ***AII formulas adjusted to pH 7

In Examples 7-10, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Phe188Asp, with substantially similar results

In Examples 11-12, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Phe188Asp and Val202Ala, with substantially similar results

Examples 13-18 Spray Compositions for Cleaning Hard Surfaces and Removing Household Mildew

Product pH is about 7

In Examples 13-16, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Asn 96Asp + Tyr103Hιs, with substantially similar results

In Examples 17-18, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Asn 96Asp + Tyr103Hιs and Pro200Gly + Gly201 Ser + Ser210Asp, with substantially similar results 2 Dishwashing Compositions

In another embodiment of the present invention, dishwashing compositions comprise one or more enzyme variants of the present invention As used herein, "dishwashing composition" refers to all forms for compositions for cleaning dishes, including but not limited to, granular and liquid forms The dishwashing composition embodiment of the present invention is illustrated by the following examples

Examples 19-24 Dishwashing Composition

Example No.

Component 19 20 21 22 23 24

Gly127Asn 0.05

Gly201Asn + Tyr205Gly + Thr207Gln + Tyr213lle 12" ^C 14 N-methyl- glucamide 0.90

C12 ethoxy (1 ) sulfate 12.00

2-methyl undecanoic acid 4.50

C-|2 ethoxy (2) carboxylate 4.50

C-|2 alcohol ethoxylate (4) 3.00

C-J2 amine oxide 3.00

Sodium cumene sulfonate 2.00

Ethanol 4.00

Mg ⁺⁺ (as MgCl2) 0.20

Ca ⁺⁺ (as CaCI ₂ ) 0.40

Water

Product pH is adjusted to 7.

In Examples 19-22, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly127Asn, with substantially similar results.

In Examples 23-24, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly127Asn and Gly201Asn + Tyr205Gly + Thr207Gln + Tyr213lle, with substantially similar results. 3. Fabric cleaning compositions

In another embodiment of the present invention, fabric cleaning compositions comprise one or more enzyme variants of the present invention. As used herein, "fabric cleaning composition" refers to all forms for detergent compositions for cleaning fabrics, including but not limited to, granular, liquid and bar forms. Preferred fabric cleaning compositions are those in the liquid form, a. Granular fabric cleaning compositions

The granular fabric cleaning compositions of the present invention contain an effective amount of one or more enzyme variants of the present

invention, preferably from about 0 001 % to about 10%, more preferably from about 0 005% to about 5%, more preferably from about 0.01 % to about 1 % by weight of active enzyme of the composition. In addition to one or more enzyme variants, the granular fabric cleaning compositions typically comprise at least one surfactant, one or more builders, and, in some cases, a bleaching agent.

The granular fabric cleaning composition embodiment of the present invention is illustrated by the following examples.

Examples 25-28 Granular Fabric Cleaning Composition

In Examples 25-26, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly156Glu + Asn162Gln, with substantially similar results.

In Examples 27-28, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly156Glu + Asn162Gln and Leu 95Glu, with substantially similar results.

Examples 29-32 Granular Fabric Cleaning Composition

In Examples 29-30, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gln16 Glu, with substantially similar results.

In Examples 31-32, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gln161 Glu and Ala186Ser + Phe188Ala + Ser190Glu. with substantially similar results.

Examples 33-36 Granular Fabric Cleaning Composition

In Examples 33-34, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Asn 96Gln + lle106Ala, with substantially similar results

In Examples 35-36, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Asn 96Gln + He106Ala and Gly 60Asn + Asn 61 Glu + Gly153Ser + Pro209Asp + Asn211Asp + Ser215Glu, with substantially similar results

Examples 37-40 Granular Fabric Cleaning Composition

In Examples 37-38, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly60Asn, with substantially similar results

In Examples 39-40, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for GlyβOAsn and Ala186Asn + Ser187Asp + Phe188Gln + Ser190Glu, with substantially similar results

Examples 41-42 Granular Fabric Cleaning Composition

Examples 43-44 Granular Fabric Cleaning Composition

Alkyl Sulphate 8 0

Alkyl Ethoxy Sulphate 2 0

Mixture of C25 and C45 alcohol 3 and 7 times ethoxylated 6 0

Example 47 Granular Fabric Cleaning Composition

Component Weight %

Linear alkyl benzene sulfonate 6.92

Tallow alkyl sulfate 2.05

C 4-15 alcohol 7 times ethoxylated 4.4

C 12-15 alkyl ethoxy sulfate - 3 times ethoxylated 0.16

Zeolite 20.2

Citrate 5.5

Carbonate 15.4

Silicate 3.0

Maleic acid acrylic acid copolymer 4.0

Carboxymethyl cellulase 0.31

Soil release polymer 0.30

Gly 60Asp + Asn 61 Asp + Gly 62Asp + Thr 65Gln 0.2

Lipase 0.36

Cellulase 0.13

Perborate tetrahydrate 11.64

Perborate monohydrate 8.7

Tetraacetylethylene diamine 5.0

Diethylene tramine penta methyl phosphonic acid 0.38

Magnesium sulfate 0.40

Brightener 0.19

Perfume, silicone, suds suppressors 0.85

Minors Up to 100 b. Liquid fabric cleaning compositions

Liquid fabric cleaning compositions of the present invention comprise an effective amount of one or more enzyme variants of the present invention, preferably from about 0.005% to about 5%, more preferably from about 0.01% to about 1%, by weight of active enzyme of the composition. Such liquid fabric cleaning compositions typically additionally comprise an anionic surfactant, a fatty acid, a water-soluble detergency builder and water.

The liquid fabric cleaning composition embodiment of the present invention is illustrated by the following examples.

Examples 48-52 Liquid Fabric Cleaning Compositions

In Examples 48-50 the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Val94Gly + Gly99Pro + lle106Val, with substantially similar results

In Examples 51-52, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Val94Gly + Gly99Pro + He106Val and Gly218Pro, with substantially similar results

Examples 53-57 Liquid Fabric Cleaning Compositions

In Examples 53-55 the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly156Ser, with substantially similar results

In Examples 56-57, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly156Ser and Ser187Glu, with substantially similar results

Examples 58-59 Liquid Fabric Cleaning Composition

In each of Examples 58 and 59 herein, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for GlylOl Glu + Ser104Glu + Ser203Asp + Tyr205lle + Ser210Glu + Thr212Pro, with substantially similar results.

Examples 60-62 Liquid Fabric Cleanin Composition

Bar fabric cleaning compositions of the present invention suitable for hand-washing soiled fabrics contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0 001% to about 10%, more preferably from about 0 01 % to about 1 % by weight of the composition

The bar fabric cleaning composition embodiment of the present invention is illustrated by the following examples

Examples 63-66 Bar Fabric Cleaning Compositions

^* Can be selected from convenient materials such as CaCO3, talc, clay, silicates, and the like

In Examples 63-64 the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Val202Ser + Asn211Asp, with substantially similar results

In Examples 65-66, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Val202Ser + Asn211Asp and Gly156Gln + lle164Thr, with substantially similar results

Examples 67-70 Bar Fabric Cleaning Compositions

*Can be selected from convenient materials such as CaCO3, talc, clay, silicates, and the like

In Example 67, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Val204Thr + Pro209Asp + Tyr213Cys, with substantially similar results

In Example 68, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly126Gln + Thr214Gly + Ser215Glu, with substantially similar results

In Examples 69-70, any combination of the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Val204Thr + Pro209Asp + Tyr213Cys and Gly126Gln + Thr214Gly + Ser215Glu, with substantially similar results B Additional Cleaning Compositions

In addition to the hard surface cleaning, dishwashing and fabric cleaning compositions discussed above, one or more enzyme variants of the present invention may be incorporated into a variety of other cleaning

compositions where hydrolysis of an insoluble substrate is desired. Such additional cleaning compositions include but are not limited to, oral cleaning compositions, denture cleaning compositions, and contact lens cleaning compositions.

1. Oral cleaning compositions

In another embodiment of the present invention, a pharmaceutically- acceptable amount of one or more enzyme variants of the present invention are included in compositions useful for removing proteinaceous stains from teeth or dentures. As used herein, "oral cleaning compositions" refers to dentifrices, toothpastes, toothgels. toothpowders, mouthwashes, mouth sprays, mouth gels, chewing gums, lozenges, sachets, tablets, biogels, prophylaxis pastes, dental treatment solutions, and the like. Preferably, the oral cleaning compositions comprise from about 0.0001 % to about 20% of one or more enzyme variants of the present invention, more preferably from about 0.001 % to about 10%, more preferably still from about 0.01 % to about 5%, by weight of the composition, and a pharmaceutically-acceptable carrier. As used herein, "pharmaceutically-acceptable" means that drugs, medicaments or inert ingredients which the term describes are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, incompatibility, instability, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio.

Typically, the pharmaceutically-acceptable oral cleaning carrier components of the oral cleaning components of the oral cleaning compositions will generally comprise from about 50% to about 99.99%, preferably from about 65% to about 99.99%, more preferably from about 65% to about 99%, by weight of the composition.

The pharmaceutically-acceptable carrier components and optional components which may be included in the oral cleaning compositions of the present invention are well known to those skilled in the art. A wide variety of composition types, carrier components and optional components useful in the oral cleaning compositions are disclosed in U.S. Patent 5,096,700, Seibel, issued March 17, 1992; U.S. Patent 5,028,414, Sampathkumar, issued July 2, 1991 ; and U.S. Patent 5,028,415, Benedict, Bush and Sunberg, issued July 2, 1991 ; all of which are incorporated herein by reference.

The oral cleaning composition embodiment of the present invention is illustrated by the following examples.

Examples 71 -74 Dentifrice Composition

^* PEG-6 = Polyethylene glycol having a molecular weight of 600. "Precipitated silica identified as Zeodent 119 offered by J.M. Huber. ^*** Carbopol offered by B.F. Goodrich Chemical Company. ^**** lota Carrageenan offered by Hercules Chemical Company.

In Examples 71 -74 the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Thr214Asp, with substantially similar results.

Examples 75-78 Mouthwash Composition

In Examples 75-78, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Thr132Glu, with substantially similar results

Examples 79-82 Lozenge Composition

Example No.

Component 79 80 81 82

Ser 97Glu + lle106Leu

Sorbitol

Mannitol

Starch

Sweetener

Flavor

Color

Corn Syrup

In Examples 79-82, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Ser 97Glu + He106Leu, with substantially similar results.

Examples 83-86 Chewing Gum Composition

Example No Component 83 84 85 86

^• Supplied by L A Dreyfus Company

In Examples 83-86, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Leu125Ala + Ser129Glu + Gly130Pro + Ser131Asp, with substantially similar results 2 Denture cleaning compositions

In another embodiment of the present invention, denture cleaning compositions for cleaning dentures outside of the oral cavity comprise one or more enzyme variants of the present invention Such denture cleaning compositions comprise an effective amount of one or more of the enzyme variants, preferably from about 0 0001 % to about 50% of one or more of the enzyme variants, more preferably from about 0 001 % to about 35%, more preferably still from about 0 01 % to about 20%, by weight of the composition, and a denture cleansing carrier Various denture cleansing composition formats such as effervescent tablets and the like are well known in the art (see for example U S Patent 5,055,305, Young, incorporated herein by reference), and are generally appropriate for incorporation of one or more of the enzyme variants for removing prote aceous stains from dentures

The denture cleaning composition embodiment of the present invention is illustrated by the following examples

Examples 87-90 Two-layer Effervescent Denture Cleansing Tablet

^* Tetraacetylethylene diamine

In Examples 87-90, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Thr212Pro + Thr219Pro, with substantially similar results. 3. Contact Lens Cleaning Compositions

In another embodiment of the present invention, contact lens cleaning compositions comprise one or more enzyme variants of the present invention. Such contact lens cleaning compositions comprise an effective amount of one or more of the enzyme variants, preferably from about 0.01 % to about 50% of one or more of the enzyme variants, more preferably from

about 0 01 % to about 20%, more preferably still from about 1 % to about 5%, by weight of the composition, and a contact lens cleaning carrier Various contact lens cleaning composition formats such as tablets, liquids and the like are well known in the art (see for example U.S. Patent 4.863,627, Davies. Meaken and Rees, issued September 5, 1989; U S Patent Re. 32.672, Huth, Lam and Kirai, reissued May 24, 1988; U.S Patent 4.609,493. Schafer. issued September 2, 1986; U.S. Patent, 4,690,793, Ogunbiyi and Smith issued September 1 , 1987, U.S. Patent 4,614,549, Ogunbiyi, Riedhammer and Smith, issued September 30, 1986, and U S Patent 4,285,738, Ogata, issued August 25, 1981 ; each of which are incorporated herein by reference), and are generally appropriate for incorporation of one or more enzyme variants of the present invention for removing proteinaceous stains from contact lens.

The contact lens cleaning composition embodiment of the present invention is illustrated by the following examples

Examples 91-94 Enzymatic Contact Lens Cleaning Solution

In Examples 91 -94, the Subtilisin DY variants recited in Tables 2-36, among others, are substituted for Gly156Glu + Thr163Ser + Asn211 Ser + Thr212Asn, with substantially similar results.

While particular embodiments of the subject invention have been described, it will be obvious to those skilled in the art that various changes and modifications of the subject invention can be made without departing

from the spirit and scope of the invention It is intended to cover, in the appended claims, all such modifications that are within the scope of the invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: BRODE, PHILIP F. BARNETT, BOBBY L. RUBINGH, DONN N. (ii) TITLE OF INVENTION: SUBTILISIN DY VARIANTS HAVING DECREASED ADSORPTION AND INCREASED HYDROLYSIS

(iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: THE PROCTER & GAMBLE COMPANY

(B) STREET: 11810 East Miami River Road

(C) CITY: ROSS

(D) STATE: OH (E) COUNTRY: USA

(F) ZIP: 45061

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: HAKE, RICHARD A.

(B) REGISTRATION NUMBER: 37,343

(C) REFERENCE/DOCKET NUMBER: 5606

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (513) 627-0087

(B) TELEFAX: (513) 627-0260

(2) INFORMATION FOR SEQ ID NO: 1

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 274 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :

Ala Gin Thr Val Pro Tyr Gly lie Pro Leu lie Lys Ala Asp Lys Val 1 5 10 15

Gin Ala Gin Gly Tyr Lys Gly Ala Asn Val Lys Val Gly lie lie Asp 20 25 30

Thr Gly lie Ala Ala Ser His Thr Asp Leu Lys Val Val Gly Gly Ala 35 40 45

Ser Phe Val Ser Gly Glu Ser Tyr Asn Thr Asp Gly Asn Gly His Gly 50 55 60 Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val 65 70 75 80

Leu Gly Val Ala Pro Asn Val Ser Leu Tyr Ala lie Lys Val Leu Asn 85 90 95

Ser Ser Gly Ser Gly Thr Tyr Ser Ala lie Val Ser Gly lie Glu Trp 100 105 110

Ala Thr Gin Asn Gly Leu Asp Val lie Asn Met Ser Leu Gly Gly Pro 115 120 125

Ser Gly Ser Thr Ala Leu Lys Gin Ala Val Asp Lys Ala Tyr Ala Ser 130 135 140 Gly lie Val Val Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Ser 145 150 155 160

Gin Asn Thr lie Gly Tyr Pro Ala Lys Tyr Asp Ser Val lie Ala Val 165 170 175

Gly Ala Val Asp Ser Asn Lys Asn Arg Ala Ser Phe Ser Ser Val Gly 180 185 190

Ala Glu Leu Glu Val Met Ala Pro Gly Val Ser Val Tyr Ser Thr Tyr 195 200 205

Pro Ser Asn Thr Tyr Thr Ser Leu Asn Gly Thr Ser Met Ala Ser Pro 210 215 220 His Val Ala Gly Ala Ala Ala Leu lie Leu Ser Lys Tyr Pro Thr Leu

225 230 235 240

Ser Ala Ser Gin Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Asn Leu 245 250 255

Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu lie Asn Val Glu Ala Ala 260 265 270

Ala Gin