This application claims priority to U.S. Provisional Application Serial No, 62/835651 filed April 18, 2019, incorporated by reference herein in its entirety.

Reference to Sequence Listing

Tins application contains a Sequence Listing submitted as an electronic text file named ⁱ 18-l:784-FGT_Seqoesce-I isti3ig_ST25.txt ^!, _; having a size in bytes of 205 kb, nd created on April 19, 2020. The information contained in this electronic file is hereby incorporated by reference in its entirety pursuant to 37 CFR § 1 52(e)(5).

Background

The ability of naturally occurring proteins to change conformation in response to environmental changes is critical to biological function. White there have been advances in the de novo design of ex tremely stable proteins, the design of conformational switches remains a major challenge.

Summary

In one aspect, the disclosure provides non-naturally occurring polypeptides or polypeptide oligomers, comprising a buried hydrogen bond network that comprises at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 pH sensiti ve amino acids located (i) at an intra-chain interface between different structural elements in one polypeptide, or (ii) at an inter-chain interface between structural elements present in different chains of a polypeptide oligomer, wherein the polypeptide or polypeptide oligomer is stable above a given pH, an wherein the polypeptide or polypeptide Oligomer undergoes a coofommtiona! transition when subjected to a pH at or below the given pH In one embodiment the pH sensitive amino acids are selected from the group consisting of histidine, aspartate, and glutamate residues. In another embodiment, the different structural elements are selected from the group consisting of loops, beta sheets, alpha helices, or combinations thereof In another embodiment, the at least one pH sensitive amino acid located is at an intra-chain interface between different structural elements in the polypeptide. In a further embodiment, the at least one pH sensitive ammo acid located Is at an inter-chain interface between structural elements present in different chains of the polypeptide oligomer. In one embodiment, the pH sensitive amino acids comprise histidine residues.

In another embodiment the disclosure provides non-naiuraiiy occurring pH- responsi ve polypeptides, comprising an oligomeric helical bundle comprising at least fouralpha-helical subunits, wherein the oligomeric helical bundle comprises:

one or mote interfaces; and

one or more histidine-containing layers that participate in buried hydrogen bond networks wherein each histidine N« and Ns atoms are hydrogen-bonded across the one or more interfaces;

wherein the polypeptide is stable above a given pH, and wherein oligomers (including but not limited to dimers or trimers} of the polypeptide undergo a confcrmationaS transi tion when subjected to a pH at or below the gives pH.

In a further embodiment, the disclosure provide non-natnrally occurring pH- responsive polypeptides or polypeptide oligomers, comprising a helical bundle comprising at least four alpha-helical subunits, wherein the helical bundle comprises:

one or more interfaces; and

one or more histidine-containing layers that participate in buried hydrogen bond networks, wherein each histidine N« and Ng atoms are hydrogen-bonded across the one or more interfaces;

wherein the polypeptide or polypeptide oligomer is stable above a given pH, and wherein the polypeptid or pol peptide oligomer undergoes a conformational transition when subjected to a pH at or below the given pH.

In various embodiments, the polypeptides comprise a polypeptide of general formula 1 , 2, 3, or 4, as disclosed herein. In one embodiment, the polypeptide or polypeptide oligomers of any embodiment or combination of embodiments further comprises a functional subunit. In some embodiments, the functional subunit comprises a detectable protein or functional fragment thereof including hut not limited to a fluorescent protein or functional fragment thereo In another embodiment, the polypeptides of the disclosure comprise the ammo acid sequence at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide of any one of SEQ ID NOS; i -40, 45-46, 60-66, 69-76, and 81-86.

In another aspect, the disclosure provides non-naturally occurring polypeptides, comprising the amino acid sequence at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 961., 97%, 98%, 991., or 100% identical to the amino acid sequence of one of SEQ ID NOS:! -77 and 81 -86. In another embodiment, the disclosure provides oligomeric polypep tides comprising two or more polypeptides of any embodiment or combination of embodiments disclosed herein. In one embodiment, the oligomeric polypeptides comprise hetero-oligomers, including but not limited to a

heterodlmer of two different polypeptides in another embodiment, the oligomeric

polypeptides comprise homo-oligomers, including but not limited to a homotimer.

The disclosure further comprises nucleic acids encoding the polypeptide of any embodiment or combination of embodimen ts disclosed herein, recombinant expression vectors comprising the nucleic acids operatively linked to a control sequence, ceils comprising the nucleic acid and/or the recombinant expression vector of the disclosure, uses of the polypeptides or the oligomeric polypeptides for any methods as disclosed herein, and methods for designing the polypeptides or the oligomeric polypeptides disclosed herein.

Description of the Figures

Fig. 1A-G. Design of pH-responsive oligomers (p s). Design models indicate cross-sections that contain the histidine hydrogen bond networks. (A) Design strategy·': pre- orgahked histidine residues destabilize mtermoleeular interfaces upon protohation at low pH. (B) The histidine-containing hydrogen bond networks of design pRO~2 (top) are replaced in pRO-2 -noliis with networks with no histidines, but all buried polar atoms satisfied by hydrogen bonds (blue box, bottom). (C) pRO-2 (topk but act pRO-2-noHis (bottom) undergoes cooperative pH-dependent quaternary structure disassociation when the pH is dropped below 5.5. Native mass spectrometry was carried out at indicated pH values at SpM iriiner, (D) The stability of pRO-2 (top) but not pRO-2-aoHi$ (bottom) is strongl pH dependent, as indicated by chemical denatoration with GdmCS monitored by circular dichrois (CD) mean residue elhpiicity (MRE) at 222nm. (E) pRO-2 CD wavelength scan and temperature melt monitoring 222 nm met) for pRO-2 in NasHPCk-Citrate buffer pH 7.0 (black), PBS pH 7,4 (dark), and PBS pH 7 4 with iOniM EDTA (light). (F) Designed homotri er pRO-3 and heterodimers pRO-4 and pRO-5. (G) pH- induced disassembly of designs In (F) monitored by native mass spectrometry, L23A / V130A mutations designed to weaken the interface of pRO-4 increase pH-sensilivit (dashed lines) compared to the parent design (solid lines). In (C) an (G), % oligomer is plotted as the percentage of that species relative to all oligomeric species observed at each pH value; for clarity, not all species are shown _» and in several eases, other oligomeric species were observed at intermediate pH values during the transition to monomer (fig, 20}.

Fig _* 2 A-B, High resolution X-ray crystal structures are very close to design models, (A) Design models of pRO-2.3 and pRO-2.5 are in close agreement with (B) X-ray crystal structures (white); electron density (mesh) shown at a level of LOA; RMSD values between crystal structure and design model are gi ven for heavy-atom superposi tion of the side chains shown in the boxes, and for all backbone atoms (right). Cross-section (layer) labels m, n, and ί correspond to Eq. I and Figure 3. Protein Data Bank (PDB) accession codes are 6MSQ (pR.O-2.3) and 6MSR (pRG~2,5).

Fig. 3 A-E. High Systematic timing of pH transition point and cooperativity. (A) Schematics of designs with different combinations of hydrophobic layers (n, black), histidine network layers (?«}, and polar network layers lacking histidine {/); fee number of each type of layer is given in parenthesis as (n, m, 1). (B) Chemical denatoraiion by guanidinium chloride (Gd Cl) at pH 7,4 measured by circular dichroism (CD) mean residue elliptielfy (MRE) monitoring hell city at 222 nm (C) Theoretical pH-dependence of tri er abundance according to Eq. 1; each curve corresponds to the values ofm, and / for a design in (A) and are colored accordingly. AG *oph«j ic , AG _} * _>i *r , and AG _P< .sa· j were estimated from chemical denaturatioh experiments (B and rig. 11). (D) Native mass spectrometry monitoring pH- induced quaternary structure disruption of the designs in (A) at 1.67 mM or 5 mM wife respect to the irimeric species; curves were fit to the experimental data using Eq. 2. (E) The higher the ratio of m to « (x-axis), the higher the pH transition point pHo (y-axis).

Fig. 4A-E. pH-depeudent membrane disruption. Proteins were added to synthetic liposomes encapsulating quenched suHorhodamine B (5RB) fluorescent dye; activity is measured by normalized dequenching of dye that leaks out fro disrupted membranes, (A) Design pRQ-2 disrupts liposomes in a pH-dependent manner; colors correspond to different pH value (shown on right). (B) pRO-2-naHis, which i not pH-responsive (Figure IC-D), shows no detectable liposome activity at pH 5. (C) Design pRO-3 shows liposome disruption activity at pH 4.75, whereas pRO-3.1 does not despite pRO-3.1 being more pH-responsive (Figure 3D). (B) Comparison between pRO-2, pRO-3, pRO-3.1 suggests that the membrane interacting region is the contiguous hydrophobic stretch at the termini. Top to bottom· SEQ ID NOS:?8, 79, and 80, (E) pRO-2 170N mutation attenuates liposome activity. All liposome experiments used a final protein concentration of 2.5 pM with respect to monomer. All data shown on same plot was collected using the same batch of liposomes. Fig, 5A-G. Imaging of pH-induced membrane permeabi!teation. (A) Tuning

AGi irtpkobs by mutagenesis to increase the pH~sensitivI†y of pRO~2; (left) theoretical curves (Eq, 1) for pRO~2 compared to I56V and A54M mutants; {right) native mass spectrometry of pRG-2 compared to I56V and A54M mutants. Tire pH set point is shifted as predicted without affecting cooperati vity; data are fit to Eq. 2 as in Figure 3, (B) pRG-2 156V has increased membrane permeabilization activity (assay as in Figure 4). (C) Cryo-electron microscopy using purified proteins conjugated to gold-nanopartieies: desi gn pRO-2 I56V interacts directly with liposomes at pH 5 but not pH 8, whereas pRO-2-noHis does not interact with liposomes at _. either pH, At low pH, design pRO~2 156V deforms liposomes and induces the formation of tight extended interfaces between liposomes (white arrow in top middie panel: density between membranes is likely pRO-2 I56V). In all control conditions, liposomes were unperturbed and free protein conjugated gold-nanoparticles were well dispersed. All scale bars are equal to lOff m. (B) Electron tomography of ÷36GFP fusions to pRO-2 and pRG-2 -noHis at pH 5 or 8 , (E) Fluorescence imaging of +36GFP fusions to designs pRO-2, pRO-2 156V, and pRO-2-noHis and composite correlation with lysosorae membrane staining in U2-OS cells. pRO-2 156V but not pRO-2-noHis is clearly localize within lysosomes; the pRO-2-noHis stainin is likely from protease resistant aggregates. (F) Manders' colocalication coefficients representing the fraction ÷36GFP fusion proteins coloeahzing with lysosomal membrane. (G) Ratios of yellow emission and blue emission on U2-03 loaded with LysoSen$or ^5M Yeflow/Blae DND-160 after I hr incubation of pRO-2 (5 mM), pRO-2 156 V (5 mM), pRO-2-noHis (5 mM), BafUomycm A (1 mM, Baf A), Chioroqume (50 mM), and medium (normal). The lower the ratio, the higherthe !ysosome pH; pRO-2 156V increases the lysosomal pH more than the small molecule drags.

Fig. 6Ά-B. (A) Homotrimer design pRO-1 was shown to be primarily dimeric at 7.5 mM dimer concentration by (B) native mass spectrometry. The mass spectrum was acquired on an Exactive Pins EME Orb trap ^{! M} mass spectrometer (Thermo Scientific) modifie with a quadrupole mass filter and an SID device(5b). Unlike successful designs pRO-2 to 5, which have contiguous, extensive histidine networks at each cross section, pRO-1 consists of three separate disjoint networks at each cross section, each with only a single histidine.

Fig. 7A-B. Designed homotrimer 2L6HC3J 3 has no histidine networks and is not pM-sensitive. (A) Native mass spectrometry was carried out at indicated pH values at 5 mM tri er concentration as in Figure 1 , (B) GdmCl denaturation experiment by CD monitoring the helical signal at 222 nM; compared to phosphate buffered saline (PBS) at pH 7.4 (gray), the same experiment in NaaPOt-Ciirate at lower pH showed no destabilization, and in fact, lower pH seems to have a modest stabilizing effect for this particular design.

Fig. 8, Design pRO-2 is pH-resptmsive by size-exclusion chromatography (SEC), whereas design pRO 2 ft«Hh is ant: SEC chromatograms using a Superdex™ 75 column art 25mM Tils pH 8 0 at room temperature (black) or NasPO^-Citrate buffer at pH 4 (ted). Design pRO-2 is a soluble aggregat at pH 4 under thes conditions, whereas by native mass spectrometry, pRO-2 is predominantly monomeric at pH 4 (Figure 1C): differences could be explained by different buffer systems or the vacuum conditions of the native mas

spectrometry.

Fig, 9. Reversibility of disassembly as deter mined by native MS, 5 pM pRO-2 and pRO-3 1 tamer were measured i 200 i¾M NFUAe (pH 6 8). Acetic acid was added to lower the pH and cause dissociation into monomers (pH 6.8 2.4) Subsequent addition of ammonia (pH 2,4 ® 9.1 ) results in re-association of monomer into trimer. 6.67 mM PRO- 2, 3, pRO-2.4 and pRQ-2 5 trimer were measured in 200 mM NiffAc / 50 usM TEAA (pH 7 0). Acetic acid was added to decrease the pH an cause dissociation into monomers (pH 7.0 3 0). Re-association was induced via buffer-exchange to 200 mM NBrAe / 50 mM TEAA (pH 7.0) by ultrafiltfatioft (Amicon Ultra, M WCO 3 kDa).

Fig, 10. 1.28 A X-ray crystal structure of design pRO-2 (PDB ID 6MSQ): {left) during; refinement, positive (green) density was observed from the difference map where the proton is supposed to be in the designed hydrogen bond network (right) The non-histidine polar network, layer /, extends to make additional hydrogen bonds wife resolved water molecules as part of a very extensive hydrogen bond network.

Fig, 11. AG estimates (top) from GdmCl denaturation experiments (bottom); fro this data, DO for each individual layer type (n. m, l) were estimated by solving a set of linear equations given the &G of folding for each design and its corresponding number of la ers of each type; these values were used for the AG values in the theoretical model (Eq. 1) used to generate the theoretical dissociation curves in Figure 3

Fig. 12A-IX Small-angle X-ray scattering (SAXS) to assess flexibility. SAXS profiles of (A) designs pRO-2, pRD-2.1 , pRO-2.3. pRO-2.4, pRO-2.5, and pRO-2-noHis: (B) experimental scattering data (black) at pH 8,0 is in close agreement with theoretical profiles computed from design models (red) usin FoXS( / 42); radius of gyration (Rg), maximum distance ( ax), and other metrics are also largely in agreement to (he design models (Table 5). However, there are differences noticeable differences between designs that have a histidine network close to the termini (pRO-2 and pRO-2.4) compared to those that do not (pRO-2.1, pRO-2.3, pRO-2 5, and pRO-2-noHis): (C) Scaled Log 10 intensity plots (left) and Kratky plots (right) show that pRO-2 and pRO 2.4 are very similar, with spectra consistent with increased flexibility as compared to pRO-2.3 and pRO~2.5, (0) pRO-2 -noHis at pH 4.0 shows subtle differences in the high q region, but is still in dose agreement in the low q, Gunter region, and consistent with a irinieric species. Plots in (€) made using ScAtter ^iM software.

Fig. 13. Other factors that affect cooperativity; the role of the helical hairpin loop. Replacing the structured hairpin loop connecting the helices of the monomer with a flexible GS linker results in less cooperativity, as assessed by native mass spectrometry at different pH values (left) Design pRO-2-GS loses its homogenous trimeric assembly at neutral pH when the flexible loop is introduced (right) Design pRO-2 3 -GS retains its trimeric assembly at neutral pH, but disassembles with less cooperativity (steepness of transition) in response to lower pH than its parent design (Figure 3D),

Fig. 14, Liposome disruption assay (as in Figure 4) for design pRO-2 at pH 5,0 using liposomes with more native-like lipid compositions.

Fig, 15,4-C.€!> data for pRO-2 mutants 156V and A54M. (A-B) GdmCI denaturation experiments performed at pH 5.89 in NarPCH-Cilrite buffer. (A) Letting the samples sit at low pH for different amounts of time before starting experiments affected results; for this reason, all native MS and CD data at varying pffs in this study were incubated for the same short amount of time before starting each experiment to ensure consistency. (B) 156V and A54M show subtle, but reproducible, changes in stability (data shown is representative from three independent experiments) (C) Free energy of folding calculations Rom denaturation experiments as in Fig. 1 1.

Fig. 16A-B. (A) Representative electron micrographs ofDOFC liposomes and purified designed proteins pRO-2 I56V and pRO-2-MnHis conjugated to 10am goM aaaopartides at pH 5, Free an gold conjugated pRO-2 1S6V are membrane active and associate wi th liposomes at pH 5. Two primary modes Of interaction are observed (Indicated by white arrows): liposome disruption, where foe lipi bilayer appears ruptured and discontinuous, and bi layer bridging, where a tight and extended interlace is formed between two liposomes. Density that likely corresponds to pRO-2 156V can be seen at the interface. Design pRO-2 T56V does not perturb liposomes at pH 8 and foe protein conjugated gold nanoparticles are well dispersed and not associated with liposomes. Design pRO-2-NoHis was similarly membrane inactive at pH 5 and 8. (B) Reconstructed cryo-electron tomograms of 0OPC liposomes with designs pRO-2 I56V (left) or pRO-2-NoHis (right) at pH 5. At pH 5, pRG-2 156V helps create extended interfaces between adjacent liposomes. Design pRO-2-NoHis does not exhibit any membrane activity at pH 5. All scale bars are l OOnm.

Fig. 17. Images of 112-OS cells loaded with LysoSeasor Yellow/Blue D D-160 that ar incubated with pRQ-2 (5 mM, top left), pRO-2 I56V (5 mM, middle left) _» Untreated {bottom left). pRO-2 -No His {5 mM. top right), Chloroquine (50 mM, middl right),

Bafiloraycit A (I mM, bottom right) for 1 hr. Blue images represent intensities of emission acquired in the region of 410-499 a upon 405 nra excitation. Yellow images represent intensities of emission acquired in the region of 500-600 nm upon 405 nm excitation intensity of excitation laser was same for all images and images are sealed to the same maximum intensity _' value.

Fig. 18, Normalised .fluorescence measurements plotted verses pH of bulfer from a fluorescent plate reader. The increase in fluorescence between pH 8 0 and 5.3 is shifted towards lower pH for the 163.2(2+ i )-cpmoxCerulean3 _,, v2 construct (cyan) compared with the (i56V)163.2(2+ l)-cpmoxCert!lean3 v2 construct (blue), which supports the theoretical model that .reduced in terface energy of hydrophobic layers (AGfeyd _fop sioiw) in the helical bundle due to the isoleucine-to-valine mutations increases the pH at which the helical hundld unfolding transition occurs. Proteins are at 5 pg/mL concentration in phosphate-citrate buffer of varying pH with 148.75 niM NaCl and 0.975 mM dithioihreitol (DTT) Data is background-subtracted from blank buffet wells. Error bars represent the standard deviation of 3 technical replicates with propagated error through analysis.

Fig. 19. Topology of# wvo cueidarfy-permisted fluorescent protein {cpFP) ^» based fluorescent pH biosensor construct 163.2(2+1 )-epmGxCerutean3 c2-cfSGFP2 depicted at high pH, At high pH, the helical bundle trimer (grey) is associated, and foe

cpmoxCemlean3. _.. v2 (cyan) acts as a FRET donor to the C-terminal cfSGFP2 (green), which acts as a FRET acceptor* producing a quantifiable FRET signal. At low pH, foe helical bundle trimer dissociates due to histidine residues at the trimer interface becoming protonated, the conformational change of which is couple to the cpmoxCerulean3 jv2 FRET donor increasing in fluorescence brightness. The cpmoxCemlea»3 _ v2 has a low pR» of unfolding, while the cfSGFPS has a high pK» of unfolding, so at low pH the cpmoxCemlean3_v2 remains folded and the cfSGFP2 unfolds reducing its ability to act as a FRET acceptor. Thus, at low pH, because the FRET donor increases in fluorescence brightness while the FRET accep tor decreases in fluorescence brightness, the overall FRET signal is reduced at low pH. The described mechanism: allows the designed conformational change of the helical bundle upon pH change ip be coupled to measnreahie fluorescence readouts.

Fig, MA-Y, pH-induced changes in oligomeric state as determined by native MS: Mass spectra are shown at the indicated pH to illustrate differences in dissociation pathways for ihe designs; the number of suburb i in each observed oligomeric complex is denoted by n (eg. n~3 indicates trimer, and n ^::: i indicates monomer). Trrmers 2L6RC3 13 (A), pRO-2- noHis (B), and pRO-2,2 (E, G) show no significant pH response within pH -7,0 to -3,0 Trimers pRO-2 (€, M\ pRO-2.1 , N), pRO-2.4 (G, Q), pRO-3 (I), pRG-3.1 (0), pRO-2 I56V (S) and pRO~2 AS4M (T) disassemble via tetramer as intermediate, whereas pRO-2,5 (H, R) seems to directly dissociate into monomer at low pH, pRO-2,3 (F, P) forms multiple higher-order oligomers besides tetramer at low pH prior to dissociation into monomer.

Dimers pRO-4 (K) and pRQ-5 (L ) predominantly directly dissociate into monome at low pH. The occurrence of characteristic intermediates in pH-dependent dissociation of the designs was observed to be independent of concentra tion, although concentration does somewhat affect the relative percentages of the different intermediate states observed;

concentrations are with respect to the initial oligomeric state at neutral pH (e.g. 5 pM pRO~2 indicates 5 mM of trimer species in the sample).

Detailed Description

As used herein, the singular forms "a",’’an” and’’the" include plural referents unless the context clearly dictates otherwise.

As used herein, the amino acid residues ar abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (lie; 1), leucine (Leu; L), lysine (L s; R), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; F), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

All embodiments of any aspect of the disclosure can he used in combina tion, unless the context clearly dictates otherwise.

Unless the context clearly requires otherwise, throughout the description and the claims, (he words‘comprise’ _» ‘comprising ^* and the like are to be construed in an inclusive sense as oppose to an exclusive or exhaustive sense; that is to say, in the sense of including, but not limited to”. Words using the singular or plural number also include the plural and singular number _» respectively. Additionally _» the words“herein,”“above,” and “below” and words of similar import, _, when used in this application, shall refer to this application as a whole an not to any particular portions of the application.

The description of embodiments of the disclosure i not intended to fe exhausti ve or to limit the disclosure to the precise form disclosed. While the specific embodiments of and examples for, the di closure are described herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as those skilled in the relevant art will recogntee.

In a first aspect, the disclosure provides non-naturally occurring polypeptides or polypeptide oligomers, comprising a buried hydrogen bond network that comprises at least one pH sensitive amino acid located (i) at an intra-chain interface between different structural elements in one polypeptide, or (ii) at an inter-chain interface between structural elements present in different chains of a polypeptide oligomer, wherein tire polypeptide or polypeptide oligomer is stable above a given pH, and wherein the polypeptide or polypeptide oligomer undergoes a conformational transition when subjected to a pH at or below the given pH

As disclosed the examples, the inventors present _: a general strategy to design pH- polypeptides or polypeptide oligomers by precisely pre-organhdag histidine residues in buried hydrogen bond networks that span across the polypeptide interface or oligomeric interface. The pH range at: which disassembly occurs, as well as the coeperativity of the transition, can be programmed by balancing the number of histidine-containing networks and the strength of the surrounding hydrophobic interactions. In non-limiting embodiments, the polypeptides or polypeptide oligomers (including but not limited to bomotrimers and heterodimers) are stable above pH 6 5, but undergo cooperative, large-scale conformational transitions wire» the pH is lowered and electrostatic and steric repulsion builds up as the network histidines involved in foe buried hydrogen bond network become protonafed The repeating geometric cross-sections allow hydrogen bond networks to be added or subtracted in a modular fashion.

In one embodiment, foe pH sensitive amino acids are selected fro foe group consisting of histidine, aspartate, and glutamate residues. In a specific embodiment, the pH sensitive amino acids comprise histidine residues

hi other embodiments, the buried hydrogen bond network comprises at least 2, 3, 4, 5, 6, or more pH sensitive amino acids.

The polypeptides or polypeptide oligomers may include any suitable“structural element”. In non-limiting embodiments, the different structure! elements are selected from the group consisting of loops, beta sheets, alpha helices, or combinations thereof. In a specific embodiment the structural elements comprise alpha helices.

In another embodiment, the polypeptides or polypeptide oligomers may include at least 2, 3, 4, 5, 6, ?, 8. 9, or more structural elements. The different structural elements in a gi ven polypeptide or polypeptide oligomer may comprise different structural elements linked via an amino acid Sinker, or different structural elements present on separate polypeptides present: i a polypeptide oligomer.

In one embodiment, the at least one pH sensitive amino acid located is at an intra- chain interface between different structural elements in the polypeptide in another embodiment, the at least one pH sensitive amino acid located is at an inter-chain interface between structural elements prese t in different chains of the polypeptide oligomer.

oire embodiment, the buried hydrogen-bond network comprises one or more histidine-containing layers, wherein each histidin N* and Ns atoms ar hydrogen-bonded across the one or more interfaces.

As used herein,‘layers” refer to an interaction between different structural elements in the polypeptide or polypeptide oligomer. The interactions) ma comprise hydrogen- bonding between different structural elements, hydrophobic interactions between different structural elements, or combinations thereof

In some embodiments, the polypeptide or polypeptide oligomer comprises a

polypeptide monomer, as described herein (t .e.: the buried hydrogen bond network comprises at least one pH sensitive amino acid is located at an intra-chain interface between different structural elements in one polypeptide). In another embodiment, the polypeptide or polypeptide oligomer comprises a homo-oligomer, including but not limited to honio-trimers, or a hetero-oligomer, including but not limited to hetero-dimers as describe herein (f e : the buried hydrogen bond network comprises at least one pH sensitive amino acid located at an inter-chain interface between structural elements present in different chains of the

polypeptide oligomer).

In another embodiment, die disclosure provides non-naturally occurring pH- msponsive polypeptides, comprising an oligomeric helical bundle comprising at least four alpha-helical subunits, wherein the oligomeric helical bundle comprises

one or more interfaces; and

one or more histidine-containing layers that participate in buried hydrogen bondnetworks, wherein each histidine N« and s atoms are hydrogen-bonded across the one or more interfaces; wherein the polypeptide is stabl e above a given pH, and wherein Oligomers {including but not limited to dimers or trimers) of the polypeptide .undergo a conformational transition when subjected to a pH at or below the given pH.

As will be understood by those of skill in the art, the helical bundle will include the alpha-helical subunits and a single hairpin loop per subunit; as used herein, a“helical bundle subunit” includes the alpha-helix and the hairpin loop.

In one embodiment, each alpha helix is connected to the next helix along the primary amino acid sequence vi an amino acid Sinker. The linker may be any suitable amino add length and composition. In various embodiments, the amino acid Sinker is between 4-8, 4-7, 5-8, $-7, or 5-6 amino acids in length. Each inner helix can connect to an outer helix through a short designed loop to produce helix-turn-heiix monomer subunits. The short designed loop may be any polypeptide equence or domain that permits formation of the alpha-helical hairpin, Including any functions! domain insertions of interest.

In one embodiment, the polypeptide comprises two or more (ie.; 2, 3, 4, 5, 6, or more) histidine-containing layers.

In one embodiment the given pH is between about pH 4.5 to about pH 6.5. As descri bed below, modification of hydrophobic layers shift the "given pH" transition point lower. As the number of hydrophobic layers increases, therefore the number of hy drophobic layers modulates the pH-responsiveuess Thus; the number of hydrophobic layers can be modified to tune pH responsiveness as deemed appropriate for an intended use.

in one embodiment, polypeptide comprises a polypeptide of formula I ;

Xi -X2-7i3-X4-X5-X6-X7-Xg-X9-XI0-Xl 1-X12-XS.3-XI4-XI5- I 6-X17, wherein: I and XI7 arc independently absent or comprise peptides;

X2, X4, X6, X8, XI 0, XI 2, XI 4, and XI 6 are each 1-2 amino acids that may be comprised of either hydrophobic residues or polar residues, forming a helical secondarystructure, wherein at least 1 2/3, 4, S, 6, 7, or al! 8 ofX2, X4, X6, X8. XI 0, XI 2, XI 4, and X I 6 include a histidine residue;

X3, X5, X7, XI 1 , XI 3, and Xi 5 are 5-6 residue variable amino acid linkers forming a helical secondary structure; ari

X9 comprises a loop, including but not limited to a hairpin loop, of variable amino acids.

The polypeptides are thus composed of a heiix-ioop-heiix secondary structure and hairpin -shaped tertiary structure. ia this embodiment, X2, X4, X6, and X8, X 10, X 12, X14, and X 16 are always buried in the oligomeric interlace upon homo-irimerfeation of the polypeptide. Since a canonicalalpha-helix has ~3.6 residues per 360 degree turn, the .residues in X2 X4, X6, and X8, as well as XI 0, XI 2, 14, and XI 6 are defined every two complete turns of the alpha-helix (ie. since they are each 1-2 amino acids in length and domains X3, X5, X7, XU , XI 3, and XI 5 segments contain the 5-6 intervening residues. In this embodiment, the buried hydrogen bond network comprises at least one pH sensitive His residue. The polypeptides of this embodiment form homoiriraers _: as described in the examples that follow. In this

embodiment, domains X8 and XI 0, X6 and Ί2, X4 and XI 4, and X2 and XI 6 segment pairs interact in the homo-primer to for part of a single“layer" ^: (i.e.: the interaetipp between domains X8 d XI 0 constitutes one layer; the interaction between domains X6 and XI 2 constitutes a second layer, the interaction between domains X4 and X14 constitutes a third layer, and the interaction between domains X2 and XI 6 constitutes a fourth layer). The interactions in each layer may comprise purely hydrophobic interactions, a mix of hydrophobic and polar interactions, and/or a mix of hydrophobic and His interactions. The interactions may occur at an inter-chain interface between domains present in different subunits of the polypeptide oligomer, at an intra-chain interface between different domains in one polypeptide subunit or both. In one embodiment, the interactions primarily may occur at an inter -chain interface between domains present in different subunits of the polypeptide oligomer.

As will be understood by those of skill in the art based on the teachings herein, other embodiments are possible and described below. For example, other polypeptides or polypeptide oligomers (including hotno-iriniers) may comprise 1 , 2, 3 or 4 such layers.

Increased numbers of such layers are also possible.

lit another embodiment, the polypeptide comprises a polypeptide of formula 2;

X6-X7-X8-X9-X10-X1 l-Xl2, wherein;

X6-X8 form a first helical secondary structure;

XiO-X 12 form a second helical structure;

X9 comprises a loop of vari able amino acid length and sequence; and

wherein at least 1 , 2, 3, 4, 5, or all 6 of X6, XT, X8, XKf XI L and XI 2 include a pH sensitive amino acid residue:

wherein the polypeptide or an oligomer comprising the polypeptide undergoes conformational transition when subjected to a pH at or below a given pH in a further embodiment, the polypeptide comprises a polypeptide of formula 3:

X4-X5-X6~X7~XS-.X9-Xl 0-X 1 1 -XI 2- 1 -XI 4, wherein;

X4-X8 form a first helical secondary structure;

XI 0-X 14 form a second helical structure;

X9 comprises a loop of variable amis® acid length and sequence; asid

wherein at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or all lO of X4, X5, X6„X7, X8, X.H), X.1 I, X12, X I 3, and X I4 include a pH sensitive amino acid residue;

wherein the polypeptide or a oligomer comprising the polypeptide undergoes a conformational transition when subjected to a pH at or below a given pH.

In another embodiment, the polypeptide comprises a polypeptide of formula 4;

X2-X3-X4-X5-X6-X7-X8-X9-X! 0-X11 -XI 2-X 13- 14- 15~X ! 6, wherein;

X2-X8 form a first helical secondary structure;

X 10 -X 16 form a second helical structure;

X9 comprises a loop of variable amino acid length and sequence; and

wherein at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, ! 1 , 12. 13, or all 14 ©fX2, X3, X4, X5, X6, X7, X8, X!fo XI 1 , XI 2, X.13, XI 4, XI5, and XI6 include a pH sensitive amino acid residue; wherein the polypeptide or an oligomer comprising the polypeptide undergoes a conformational transition when subjected to a pH at or below a given pH.

In each of these embodiments, the polypeptide, or polypeptide oligomers comprising the polypeptide comprise a buried hydrogen bon network that comprises at least one pH sensitive amino acid located (i) at an intra-chain interface between different domains in one polypeptide, or (ii) at an in tor-chain interlace between domains present in different chains of a polypeptide oligomer, wherein the polypeptide or polypeptide oligomer is stable above a given pH, and wherein the polypeptide or polypeptide oligomer undergoes a conformational transition when subjected to a pH at or belo the gi ven pH

In one embodiment, the pH sensitive amino acids are selected from the group consisting of histidine, aspartate, and glutamate residues. In a specific embodiment, the pH sensiti ve amino acids comprise histidine residues.

In other embodiments, the buried hydrogen bond network comprises at least 2, 3, 4, 5, 6, or more pH sensitive amino acids.

The various X domains in these embodiments may comprise any length or content of amino acids so long as the recited limitations are met. in one embodiment of any of these embodiments, 1 , 2, 3, 4, 5, 6, 7, or all % of X2, X4, X6, X8, XiO, XI 2, X14, and XI 6 (when present) are 1-2 amino acids that may be comprised ofhydrophobic residues, polar residues or both, wherein at least 1, 2, 3, 4, 5, 6, 7, or all 8 of X2, X4, X6, C8 _» Xlff X12, XI 4, and I 6 (when presen t) bid tide a p H sensitive amino acid.

In another embodiment: that can be combined with any of these embodiments, 1, 2 3, 4, 5, or all 6 of X3, X5, X7, XI 1, XI 3, and XI 5 (when present) are 5-6 residue variable amino acid linkers.

In a further embodiment: of any of these embodiments, X9 may comprise a hairpin loop, or may comprise a flexible? linker including but not limited to a flexible OS-based linker.

In a further embodiment of any of these embodiments, additional amino acid residues or functional domains may be present, such as at the N- or C-terminus, as deemed appropriate for an intended use.

As used herein, amino acid residues in a polar layer could be any of the following: C, D, E, G, K, N, Q, R, S, T, Y, W, and H, Amino acid residnes in a hydrophobic layer could be any of the following: A, F, G, I, L, M, P, V, W and norleucme.

Hydrophobic layers shift: the "given pH" transition point lower as the number of hydrophobic layers increases, therefore the number of hydrophobic layers does modulate the pH-resppnsiveness. Thus, the number ofhydrophobic layers can be modified to tune pH responsiveness as deemed appropriate for an intended use.

In one embodiment, 1 , 2, 3, 4, 5, 6, or 7 of X2, X4, X6, X8, XI 0, XI 2, XI 4, and XU are comprised of hydrophobic residues, as deemed suitabl e for an intended use. For example, to shift the“given pH” lower, the number of hydrophobic domains is increased and the number of polar domains is decreased; to shift the“given pH” higher, th number of

hydrophobic domains is decreased and the number of polar domains is increased.

In another embodiment X9 is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, Hi 19, 20, 21, 22, 23, 24, 25 or more amino acids in length.

In a further embodiment, each of XI and XI 7, when present, are the same length.

In one embodiment, one or more of XI, X9 and XI 7 comprise a functional subunit, or the polypeptide further comprises a functional domain at the N-terminns or C-terminus. A “functional subunit” is any domain that can be ad functionality to the polypeptide. Any functional domain may be used as suitable for an intended purpose. In one embodiment, the functional subunit comprises a detectable protein or functional fragment thereof, including but not limited to a fluorescent protein or functional fragment thereof. For example, a functional snbunilcomprising a fluorescent pro tein or functional fragment thereof permi ts coupling of tiie confe iaitonal change due to protanatioh of the buried histidines in the hydrogen bond networks at the interlace of the helical bundle to conformational changes in the ehromophore environment of the fused fluorescent protein. This provides fluorescent readout of the conformation change. As will be understood by those of skill in the art, other functional subunits could he used in a similar «tanner to link the pH-based conformational change with a .readout based on the function of the functional subunit.

In another embodiment, the polypeptide comprises the amino acid sequence at least 23%, 30%, 35%, 40%, 43%, 50%, 55%, 60% _* , 65%, f0%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide selected from the group consisting of SEQ ID NGs: l-40, 45-46, 60-66, 69-76, and 81-86.

Table 1

In this table, the bold residues shew the differences between the modular designs of Figure 3 in the manuscript, which allows mapping of how the layers can be swapped.

Underlined egion is not part of the desi n but hoxah.lstidine tag and T'SV cleavage site for purification ((i.e.: the residues are optional)

Table 2

In this tablet

Hi3tidine-containing hydrogen bond network residues are bolded.

Hon-histidine hydrogen bond network residues are highlighted and underlined Longer underlined region is not part of the esign but hexahietidind tag and TEV cleavage site for purification it is optional),

Table 3 Amin© acid sequences of all designs tested. All constructs were cloned into pS S ^' l-MSSG plasmid except for design pRO-l, which was cloned in p ^' £T28b . Heterodimars pRO-i and pRO~5 were ordered as b cis onic constructs; DNA sequence containing stop codon, additional ribosome binding sequence, and second start codon is shown by the lower case letters in parenthesis (this sequence is not included in the amino acid sequence or associate SEQ ID NO) . Underlined regions are removed after hexahistidine tag cleavage (i.e.: they are optional) . Bold positions indicate

mutations/differences between a design variant and its parent design.

The polypeptides of SEQ ID NOS:l-26 and 33-36 ail form homoirimers and the polypeptides of SEQ ID NOS;2?-32 and 81 86 form heterodiiners. In these embodiments, the buried hydrogen bond network comprises at least; one pH sensitive amino acid located at an inter-chain Interface between structural elements present in different chains of the polypeptide oligomer.

The following embodiments of the polypeptides of the disclosure (SEQ ID NOS: 37- 40, 45-46, 60-66, and 69-76) are single chain monomers, an the buried hydrogen bond network comprises at least one pH sensitive amino acid is located at an intra-chain interface between different structural elements in the polypeptide. The underlined regions of the following sequences are not part of the design but hexaMstidine tag and thrombin or TEV cleavage site for purification (i.e.: the underlined regions are optional). In many of these sequences he monomeric subunits of the homotrimer are fused by linkers/ loops and function domains Into a single polypeptide sequence

pR.02.3, single-chain, with GS linkers on all the loops, asymmetrized, and a TEV site opposite to the termini direction. This allows the pH responsive aimer to be fused at its n- terminus to other proteins, such as a nanoparticle, and confer membrane disruption, Based on the liposome assay described below, the kinetics of dissociation of linked-pH trimer is slower but achieves the same membrane disruption levels as measured by dye leakage over time (on the order of minutes). This performs as well as pR02.3 as measured by the liposome disruption assa in the context of a nanoparticle (ie. fused at Us n-termmus to a nanoparticle).

GSSKSIKRLLEEERKSSSM _^ RRIIEEDDDESKSLnVGGSGSGSErmVEinniLISKRNRTIVEilN IVSILSAIA RVGGSGSGSVEVERILDELRKSSEELDRVTKELKKLTEELDVGGSENLYFQG3G3VEALV RKNVLITRHNDI IVK NNDIINKILKLIAEAVGGSGSGSELERILRELEESTKELRKATESLRRLSEELKVGGSGS GSVEALVRHNEAIVE

:HNK11VENNp11VK.TLET..ITERI (SEQ ID NO: 37)

The next polypeptide is similar to pR02.3, with the TEV site parallel to the termini such that a monomer is released upon cleavage. This monomer Is modified to have aromatic residues (phenylalanine and tryptophan) on the «-terminal helix to enhance membrane disruption. This performs slightly (5-10%) better than the pR02.3 homotrimer in the liposome disruption assay.

GSEEEIKRLLEELRKSSEELRRITKELDDLSKELRVGGSGSGSEMLVEHNKLISEHN RIIVENKRI IVEILEAIA RVGGSGSGSVEVERILDELRKSSEELDRVTKELKKLTEELDVGGSGSGSVEALVRHKVLI TRHNDI IVKNNDIIN KILKLIGEAyGGSEIGLYFQGSGSEFERWLRQLEESTKELRKFTEELRRFSEELKVGGSG SGSVEALniHNEAIVE HEKAIVKSnDirVKILELVEEEI (SSQ ID NO: 38)

Similar to pR02.3, with Thrombin cleavage sites on each loop opposite to the termini. Also has the destabilizing I56V mutation to shift the pH disassembly to a higher pH, This performs close as well as pR02.3 as measured by the liposome disruption assay in the context of a nanoparticle (ie. , fused at its n-temrious to a nanopartide) but with slower kinetics.

GSEEEIKRLLEELRKSSEELRRITKELDDLSKELRVGGSGSGSLVPRGSGSGSGSHA LVEHNKLISEHNRIWEN

MRI IVEILEAIARVGGSGSG3VEVERILDELRKSSEELDRVTKELKKLTEELDVGGSGSGSLV PRGSGSG3GSVE ALVRHNVLITRHNDI'/VKNNDI IMKILKLIA.EA.VGGSGSGSELERILRELEESTKELRKATEELRRLSEELKVGG SG3GSLVFRGSGSGSGSHEALVRHNEAIVEHNKIVVKNKDIIVKILELITERI (SEQ ID NO: 39)

Same as above, but with the third asparagine network mutated such that it is all hydrophobics to destabilize the linked-trimer and increase hydrophobic content for better membrane interaction. This performs 5-10% better than pR02 3 as measured by tire liposome disruption assay in the context of a nanoparticle (ie., fused at its n-terminns to a nanoparticle) but with slower kinetics. GSEEEIKRLLESLRKALEELRRITKELDDLSKELRVGGSGSGSLVPRGSGSGSGSHALVE HNKLISEHNRIWEV LRI IAEILEAIARVGGSGSGSVEVERILDELRKALEELDRVTKELKKLTEELDVGGSGSGSLV PRGSGSGSGSVE ALVRHKVLITRHNDIVVKVLDI IA.KI LKLIAEAVGGSGSGSELERILRELEEALKELRKATEELRRLSEELKVGG SGSGSLVRRGSGSGSOSBSALVRBNSAIVSHNRIVVKVLDITSKXLS ITERI (SEQ ID iJ¾t40>

Additional polypeptides of the disclosure and inactive controls (i.e.: not pH

responsive) are show below. Underlined residues and/or residues in parentheses are optional. s in 1 e a in _^ n *H i s yss_I 63

(MGSSHHHHHHSSGIWPRGS) E SSDSLKYELJKKSTRELQKSTDELEKSTEELERNPSKDVLVENNELIVRNNKI IVKNNIIIVRTKKKGSGGSGDBLESSLKKSTEELDKSTKKLERSTEELKRNPSKDALVEN NKLIVENNTIIVRNN

DirVRTRKKGSGOBGDELKESLEKSTBBLKKBTRELQKSTEELERNPSKDALVKNNK LIADNNRIIVRNNTIIVR DIKAS (SEQ ID NO: 41) Inactive control

s i gle_chain_noHis_ sym_l63

HMGSDELKYELEKSTRELQKSTDELEKSTEELERNPSKDVLVENNELIVRNNKI IVKMMI I IVRTEKKGSGGSGD ELKEELEKSTRELDKSTKKLERSTEELKRNPSKDAL\'ENNKLIVENNTI IVRNNDI IVRTRKKGSGGSGDELKEE LEK3TRELKKSTKELQKSTEELERNPSKDALVKMNKLIADNNRI I\'RNMTI IVRDIKAS { SEQ ID NO : 42 ) Inactive control single chain noHis ssym 162

(MGSSHHnHHHSiGLVPRGSi HMSSDDEDIDEVLSSLRRSTEELDRSTKDLERSTQELRRNPSVDALVKNNNAIV BSNSTIVESSRI LE LSLLLRSIRGSGGSGDRESIKKVLDELRESTERLER3TEELRRSTEELKKNPAVEVLVR NNTIIVKNNKIIVDNNRIIVRVLELLEKTIKGSGGSGDKYEIRKVLKELKDSTEELRNST KNLTDSTEELKRNPS VEILVKNNILIVENNKIIVENNRI IVDVLELIRKAIAS (SEQ ID NO:43) Inactive control single_chain_noHis_ sym_162

HMG3DDEDIDRVLEELRRSTEELDRSTKDLERSTQELRRNPSVDALVKMMNAIVRNNEII VENNRIILEVLELLL RSIKGSGGSGDREEIKKVLDELRESTERLERSTEELRRSTEELKKNPAVEVLVRNNTIIV KNNKIIVDNNRIIVR VLELLEKTIKGSGGSGDKYEIRKVLKELKDSTEELRNSTKNLTDSTEELKRNPSVEILVK NNILIVENNKIIVEN NRIIVDVLELIRKAIAS (SEQ ID NO:44) Inactive control single chain asyit 162

{MOSSEHHHHHSSGLVPKGS) HMGSDDEDIDEVLSSLRRITEELDRITKDLERLTQELRRNPSVDALVKHNNAIV

RHNSIIVEHN ILEVLELLLRSYKGSGGSGDRESIKKVLDELREATERLERATEELRRLTEELKKNPAVEV LVR

HMYIIVRHilKIIVDRNRXXVRVLELLSKTIKGSGGSGDKYEIRKVLKELKDITEEL RNMTKNLTDLTEELKRNPS VEILVKHNILIVEHNKIIVEHNRIIVDVLELIRKAIAS (SEQ ID NO: 45)

single_chain_asyrr _; _l 62

HMGSDDEDIDRVLEELRRITEELDRITKDLERLTQELRRNPSVDALVKHNNAIVRHNEII VEHNRIILEVLELLL RSIKGSGGSGDREEIKKVLDELREATERLERATEELRRLTEELKKNPAVEVLVRHNTIIV KHNKIIVDHNRIIVR VLELLEKTIKGSGGSGDKYEIRKVLKELKDITEELRNMTKNLTDLTEELKRNPS EILVKHNILIVEHNKIIVEH NRIIVDVLELIRKAIAS (SEQ ID NO : 46 )

T ag'GRFS TEV · TagB FP ^¨ Two fiuorescerd proteins TagGFP2 and TagBFP fused together by a TEV protease site linker.

(MGSSHHHHHH33GLVPRGS ) HMSGGEELFAGIVPVLIELDGDVKGHKFSVRGEGEGDADYGKLEIKFICTTGKL PVPWPTLVTTLCY ^' GIQCFARYPEHMKMNDFFKSA PEGYIQERTI FQDDGKYKTRGEVKFEGDTLVNRIELKGK DFKEDGNILGHKLEYSFNSHNVYIRPDKANNGLEANFKTRHNIEGGGVQLADHYQTNVPL GDGPVLIPINHYLST

QTKISKDRNEARDHMVLLESFSACCHTGGSGGSENLYFQGASGGSGSELIKENMHMK LYMEGTVDNHHFKCTSEG

EGKPYEGTQTMRIKWEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQ3FPEGF TWERVTTYEDGGVLTAT QDT3LQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLV GGSHLIANIKTTYRS KKPAKNLKMPGVYYVDYRLSRIKEANNETYVEQHEVAVARY (SEQ ID NO: 47)

HMSGGEELFAGIVPVLIELDGDVHGHKFSVRGEGEGDADYGKLEIKFICTTGKLPVP PTLVTTLCYGIQCFARY PEHMKMNDFFK3AMPEGYIQERTIQFQDDGKYKTRGEVKFEGDTLVNRIELKGKDFKEDG NILGHKLEY3FNSHN VYIRPDKANNGLEANFKTRHNIEGGGVQLADHYQTNVPLGDGPVLIPIMHYL3TQTKISK DRNEARDHMVLLESF SACCHTGGSGG3ENLYFQGASGGSGSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYE GTQTMRIKWEGGPL PFAFDILATSFLYGSKTFINHTQGIPDFFKOSFPEGFTWERVTTYEDGGVLTATQDTSLQ DGCLIYMVKIRGVNF ISNGPVMQKKinNIGEAFTSTIiYPADGGHEGRNDKAX _^ KLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLER CKBMίMETYvgQEE¾VARY (SEQ; ID NO: 48}

TagGFP2-~.s ingle chain noHia &s s& 1 SS-TagBFP

(MGSSHHHHHHSSGLVPRGS) HMSGGSBLFAGIVpyLIELDGDyHGHKFSVRGEGEGDADYGKLEIKFICTTGKL PVPWPTLVTTLCYGIQCFARYPEHMKMNDFFKSAMPEGYIQERTIQFQDDGKYKTRGEVK FEGDTLVNRIELKGK DFKEDGNILGHKLEYSFNSHNVYIRPDKANNGLEANFKTRHNIEGGGVQLADHYQTNVPL GDGPVLIPINHYLST QTKISKDRNEARDHMVLLESFSACCHTGGSGGSDELKYELEKSTRELQKSTDELEKSTEE LERKPSKDVLVENNE LIVRKKKI IVKNNI IIVRTEKKGSGGSGDELKEELEK3TRELDKSTKKLERSTEELKRNPSKDALVENNKLIVE N NTI IVRNMDI IVRTRKKGSGGSGDELKEELEKSTRELKKSTKELQKSTEELERNPSKDALVKNKKLIADN NRIIV RNNTIIVRDIKASGGSGSELIKENMHKKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK WEGGPLPFAFDILA TSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNV KIRGVNFTSNGPVMQ KHIII;WEAFTFTI.YPADGGHFGKNDNAI.KI,VSG3BT,IANIKTTYRSKKPAKNLKM PGVYYVDYRLERIKEANNET YVKQBEVAVARY < 3EC ID NO: 48) inactive control

xa¾GF 2-slngi«_;chain_noHis_asy®_X€3~TagBFP

H SGGSELFAGiVFPLISLDGDVHGHRFSPRGEGSGDADYGKLEIKFICTTGKLPVPWPTLV TTLCYGIQCFARY PEHMKMNDFFKSAMPEGYIQERTIQFQDDGKYKTRGEVKFEGDTLVNRIELKGKDFKEDG NILGHKLEYSFNSHN VYIRPDKANNGLEANFKTRHNIEGGGVQLADHYQTNVPLGDGFVLIPINHYLSTQTKISK DRNEARDHMVLLESF SACCHTGGSGGSDELKYELEKSTRELQKSTDELEKSTEELERNPSKDVLVENNELIVRNN KI IVKMMI IIVRTEK KGSGGSGDELKEELEKSTRELDKSTKKLERSTEELKRNPSKDALVENNKLIVENNTI IVRNNDI IVRTRKKGSGG SGDELKEELSKSTRELKKSTRELQKSISSLERNPSKDALVKNNKLIADMMRIIVRKKTII DIKASGGSGSELI KE^KhSRLYPiSGTYiWHHFKCTSEGSGKPYEGTQTRRIKVVEGGPLPFAFDILATSFLY GSKTFINHTQGIPDFF K¾3FPSGFT??ERVTTYEDGGVLTATQ;DTSLQDGCLXYNVKIRGVNFTSNGPVMQKKT LGJEAFTETLYPADGGLE GRHOMALKLVGGSN _B lANrNTXYRSKKPAKN _B RMPGVYYVDYRLERIKEANNETYVEQHEVAVARY { SEQ ID DO: DO) Inactive control

TagGFF2--single__chain_KGHIs_ _j &syss_i 63~TagBFP

¾RCSSRRHRBBGGGIY/PRGS) R SGGSBLFAGIVPVBIELDGDVHGHKFSVRGEGEGDADYGKLEIKFICTTGKL FV PTLVTTECYGlOCFARYPEiMMERNDFFRSAilREGYIQERTIQFQDDGKYKTRGE FEGDTLVMRIELKGK DFKEDGNILGHKLEYSENSKNVYIRPDKANNGLEANFKTRHNIEGGGVQLADKYQTNVPL GDGPVLIPINKYLST QTKISKDRNEARDHMVLLE3FSACCHTGGSGG3DELKYELEKSTRELQKSTDELEKSTEE LERNPSKDVLVEKNE LIVRNNKI IVKNNI I IVRTEKKGSGGSGDELKEELEKSTRELDKSTKKLERSTEELKRNPSKDALVENNKLIVEN NTI IVRNNDI IVRTRKKGSGG,3GDELKEELEKSTRELKKSTKELQKSTEELERNP,3KDALVKNNKLIA DNNRI IV RNNTIIVRDIKA3GGSGSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK WEGGPLPFAFDILA TSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNV KIRGVNFTSNGPVMQ KKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYY VDYRLERIKEANNET

yVEQKSVAVARY {SEQ ID NO: 51} Inactiv control

TagGPP2--singlo chain noHis asys?5 ISS-TagBPP

FMBGGSFGEAGIVFYLXBLDGDFHGsiFSVRGFGEGDADYGSLSIKFICTTGRLPFPiiP TLVTTLCYGIQCFARY FSHMK NDFFKSAMPEGYIQSRTIQFQDDGKYKTRGSVKFEGDTLV EIELRGRDFKEDGNILGHNLSYSFNSHN VYIRPDKANNGLEANFKTRKNIEGGGVQLADHYQTNVPLGDGPVLIPINHYL3TQTKISK DRNEARDHMVLLESF SACCHTGGSGGSDELKYELEKSTRELQKSTDELEKSTEELERNPSKDVLVENNELIVRNN KI IVKNNI IIVRTEK KGSGGSGDELKEELEKSTRELDKSTKKLERSTEELKRNPSKDALVENNKLIVENNTI IVRNNDI IVRTRKKGSGG SGDELKEELEKSTRELKKSTKELQKSTEELERNPSKDALVKNNKLIADNNRIIVRNNTII VRDIKASGGSGSELI KENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKWEGGPLPFAFDILATSFLYGS KTFINHTQGIPDFF KQSFPBGpINSRVVTYEDGGVLTATQDTSnQDGCLIYNVKIRGVNFTSNGPVMQKKTLGW EAFTETLYPADGGLE GKND ALRLVSGSHLIANIKTTYNSRRPAKHLKKPGVYYVDYRLERIKEANNETYVEQHEVAVAR Y { SEQ ID HO: 52) Inactive control cpmoxCer«1ean 2

{MGS3HHHEiHHSSGENAY} FQGS 3GG HGKVYITADKQKNGIKANFGLNSNVEDGSVQLADKYQQNTPIGDGPV LLPDNEYESTQSALSKDRNSKEDRPiVLLSFVTAAGITLGMDELYKGGTGGSMVSKGEEL FTGVVPILVELDGDVN GHKFSVBGEGSGDATNGEAYLKFISTTGKAP SFTLVTALSisGVQSFARYBDHMKQHDFFKSAHPEGYVQSRTT FFKDDGTYRTRAEVKFEGD L RISLKGIDFKEDGNILGHRLSY* (SEQ ID NO; 53 } Inactiv cpmoxCe ruieaxi v2

EQGSGSGGlHGRVYIEAOSiQKNGiKAMFGLNSNVEDSSFQLADHYQQNTRlGDGPFLLP DHHYLSEQSALSRDPN EKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGWPILVELDGDVNGHKFS VRGEGEGDATNGKL TLKFISTTGKLPVPWPTLVTTLSWGVQSFARYPDHMKQHDFFKSAMPEGYVQERTIFFKD DGTYKTRAEVKFEGD TLVNRIELKGIDFKEDGNILGHKLEY* (SEQ ID NO : 54 ) Inactive

SB13 {2+1}“CpmozCsruieariG v2

{MGS3HEHHBBSSGERLY} F GSGSGSTKYELRRALEELSKALRELKKSLDELERSLEELEKNPSEDALVENNRL

HVENNKIIYSVERi SYLKINARSGGSGSGSTEYSLRRALEELEKALRELKKSLDELERSLEELEKNPSSDALV

SNNRANFEMiKIIVEVLMlABVLKINAKSDSSSTHGNVYITADKQKNGIKANFGLNS NVEDGSVQLADHYQQNT

PIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGG SMVSKGEELFTGWPILV

ELDGDVNGHKFSVRGEGEGDATNGKLTLKFISTTGKLPVPWPTLVTTLSKGVQ3FAF .YPDHMKQHDFFK3AMPEG

YVQERTIFFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGMILGHKLEYGGSTK YELRRALEELEKALRELK

KSLDELERSLESLEKNPSEDALVENNRLNVENNKIIVSVLRIIASVLKINAKSD* (SEQ ID NO: 55)

Ina.ctive control

SE13 (2+1) -cpmoxCerulean3_v2

FQGSGSGSTKYELRRALEELEKALRELKKSLDELERSLEELEKNPSEDALVENNRLNVEN NKI IVEVLRI IAEVL KINAKSDGSGSGSTKYELRRALEELEKALRELKKSLDELERSLEELEKNPSEDALVENNR LN\'ENNKI IVEVLRI IAEVLKINAKSDGSGIHGNVYITADKQKNGIKANFGLNSNVEDG3VQLADHYQQNTPIGD GPVLLPDNHYLSTQS AL3KDPKEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDG DVNGHKFSVRGEGEG DATNGKLTLKFI3TTGKLPVPWPTLVTTLSWGVQSFARYPDHMKQHDFFKSAMPEGYVQE RTIFFKDDGTYKTRA EVKESGDIIAN4RXELKGIDFFEDSNILGHKLEYGG3TKYELRRALEELEKALRELKKSL DELERSLEELEKNPSE DAIAfSNNRLNVENNKIJVEVLRITAEVLKISAESO* {SEQ ID NO: 56) Inactive control

SB! 3 {2+1 } --cpi¾oxCertie¾A3_v2~c{;3SF?£

(MGSSHHHHHHSSGENLY) FQGSGSGSTKYELRRALEELEKALRELKKSLDELERSLEELEKNPSEDALVENNRL NVENNKI IVEVLRI IAEVLKINAKSDG3GSGSTKYELRRALEELEKALRELKKSLDELERSLEELEKNPSEDAL V ENNRLNVENNKI IVEVLRI IAEVLKINAKSDGSGIHGNVYITADKQKNGIKANFGLNSNVEDGSVQLADHYQQNT PIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMV SKGEELETGVVPILV ELDGDVNGHKFSVRGEGEGDATNGKLTLKFISTTGKLPVPWPTLVTTLSKGVQ3FAF.YP DHMKQHDFFK3AMPEG YVQERTIFFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGMILGHKLEYGGSTKYEL RRALEELEKALRELK KSLDELERSLEELEKNPSEDALVENNRLNVENNKI IVEVLRIIAEVLKINAKSDMVSKGEELFTGVVPILVELDG DVNGHKFSVSGEGEGDATYGKLTLKFISTTGKLPVPWPTLVTTLTYGVQMFARYPDHMKQ HDFFKSAMPEGYVQE RTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNY SHNVYITADKQKNGIKANFKIRHN IEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGIT LGMDELYK (SEQ ID NO: 77) Inactive control

SB13 {2+1) --epffioxCertlean _* 3_v2--cϊSG??2

FQGSGSGSTKYSARRALSELSKAIRELKKSLDELSRSLEELEKNPSEDALVENNRLNVEN NKI IVEVLRI IAEVL KIRAKSDSSGSGSLEYELHKALSELERAARISLKKBLDELERSLEELEKNPSEDALVENN RLNVENNKI IVEVLRI

lAEVLKIIIAKSDGSGIHGNYYITADR KNGIKANFGLNSNvEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQS ALS ^: KDRMEKRDKisfYLL£FVT.¾¾GITLGMDELYKGGTGGSMV3KGEELFTGV VPILVELDGDVNGHKFSVRGEGEG DATNGKLTLKFISTTGKLPVPWPTLVTTLSNGVQSFARYPDHMKQHDFFKSAMPEGYVQE RTIFFKDDGTYKTRA EVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYGGSTKYELRRALEELEKALRELKKSLD ELERSLEELEKNPSE DALVENNRLNVENNKI IVEVLRI IAEVLKINAKSDMV3KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATY GKLTLKFISTTGKLPVPWPTLVTTLTYGVQMFARYPDHMKQHDFFKSAMPEGYVQERTIF FKDDGNYKTRAEVKF EGDTLVNRIELKGI DFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPI G DGFVLLPDNHYLSYQSKLSKQRNBKEDBMVLLEFVTAAGITLGMDSLYK (SEQ ID NO: 57) Inactive control

SBl 3.2 {2+1 ) ~cp o¾Ceruiean3_v2~cfSGFP2 (MGSSHHHHHHSSGEMLY) FQGSGSGSTKYELRRALEELEKALRELKKSLDELERSLEELEKNPSEDALVENNRL NVENNKIIVEVLRI IAEVLKINAKSDGSGSGSTKYELRRALEELEKALRELKKSLDELERSLEELEKNPSEDAL V ENNRLNVENNKI IVEVLRI IAEVLKINAKSDGSGIHGNVYITADKQKNGIKANFGLNSNVEDGSVQLADHYQQNT PIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMV SKGEELFTGWPILV ELDGDVNGHKFSVRGEGEGDATNGKLTLKFISTTGKLPVPWPTLVTTLSWGVQSFARYPD HMKQHDFFKSAMPEG YVQERTIFFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYGSGSTKYE LRRALEELEKALREL KKSLDELERSLEELEKNPSEDALVENNRLNVENNKI IVEVLRI IAEVLKINAKSDMVSKGEELFTGWPILVELD GDVNGHKFSVSGEGEGDATYGKL!LKFISTTGKLPVPWPTLVTTLTYGVQMFARYPDHMK QHDFFKSAMPEGYVQ EP.TXFFRDDGX5YKGEASYKFSGOILVMRI LEGIQFKEDGNILGHKLEYNYNSHNVYITADKQKNGIRANFKIRH NtSDGGVULADEYQONTRlfiDGIHLLEDN!tYLSTQSKLSKDPNEKRDHMVLLEFVTAA GITLGMDELYK (SEQ ID NO:S8) Inactive control

SE13.2 (2 + 1) - cpffiOxCeruIesri3_v2-cfSGFF2

FQGSGSGSTKYELRRALEELEKALRELKKSLDELERSLEELEKNPSEDALVENNRLNVEN NKI IVEVLRI IAEVL KINAKSDGSGSGSTKYELRRALEELEKALRELKKSLDELERSLEELEKNPSEDALVENNR LNVENNKI IVEVLRI IAEVLKINAKSDGSGIHGNVYITADKQKNGIKANFGLNSNVEDGSVQLADHYQQNTPIGD GPVLLPDNHYLSTQS AL3KDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGWPILVELDGD VNGHKFSVRGEGEG DATNGKLTLKFISTTGKLPVPWPTLVTTLSWGVQSFARYPDHMKQHDFFKSAMPEGYVQE RTIFFKDDGTYKTRA EVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYGSGSTKYELRRALEELEKALRELKKSL DELERSLEELEKNPS EDALVENMRLNVENNKIIVEVLRI IAEVLKINAKSDMVSKGEELFTGWPI LVELDGDVMGHKFSVSGEGEGDAT YGKLTLKFISTTGKLPVPWPTLVTTLTYGVQMFARYEDK FQHDFFKS^iPHGYVQFRTIFFKDEGNYKYSAFVF FEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITARKQKNGIEANFEXEHNIED GGVQIPXDHYQQNTPT

GDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMOELYK (SEQ ID EGrSS) Inactive control This embodiment shows pH-responsive fluorescence intensity modulation due to fused helical bundle pH-responsive conformational switching that is al iosterically coupled to chromophore environment.

(MGSSHHHHHHSSGEMLY) FQGSGSGSEALYELEKATRELKKATDELERATEELEKNPSEDALVEHNRLIAEHNK I IAEHNRI IAKVLKGSGSGSEALYELEKATRELKKATDELERATEELEKNPSEDALVEHNRLIAEHNK IIAEHNR IIAKVLKGSGIKGNVYITADKQKNGIKANFGLNSNVEDGSVQLADKYQQNTPIGDGPVLL PDNHYLSTQSALSKD PNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGWPILVELDGDVNGHK FSVRGEGEGDATNG KLTLKFISTTGKAPYE^PTI.VTTESSGVQSF&RYPDHMKQHDFFKSAMSEGYVQE RTXFFKDDGTYKTRASVKFE GDTIVNEIELKGXDFKEDGNILGHKLSYGGSEALySLSKATRBLKKATDELSEATS:KLE KMFSEDAIGISHNELIA ZWX1I¾SHSRI I&KVl* (3SQ ID NO: 60)

X63 (2*1} -c fflo{Csruie«n3_v3 This embodiment shows pH-responsrve fluorescence intensity modulation due to fused helical bundle pH-responsive conformational switching that is allosterically coupled to chromophore environment

FQGSGSGSEALYELEKATRELKKATDELERATEELEKEESEDALVEKDRLXABBHRTIAS HDRIIAKYLKGSGSG

SEALYELEKATRELKKATDSLERATEELEKKPSEDA VERnRLXASHNKIIAEimRXIARYLRGaGlHGNVYXXA DKQKNGIKANFGLNSNVEDGSVQLADHYQQNTPIGDGPVLLPDNKYLSTQSALSKDPNEK RDKMVLLEFVTAAGI TLGMDELYKGGTGGSMVSKGEELFTGWPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK FISTTGKLPVP PT LVTTLSWGVQSFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGTYKTRAEVKFEGDTL VNRIELKGIDFKEDG GILGHRLEYGGSEALYFLEKAIRELKKATDEIGSFATEELEKNPSEDALVEHNRLIAEHN KIIAEHNRIIAKVLK

(SSQ ID 1X0:61}

163-2 iE--i -cpKoxCeryi anS vs : This embodiment shows pH-responsive fluorescence intensity modulation due to fused helical bundle pH-responsive conformational switching that is ai!osiencal!y coupled to chromophore environment.

(MGSSHHaHRHStoGEMLYj gOgSGSGSBAEYELmAYRSLKKATDELBRATEELSKDPSSOALVBHlffiLXABHNK X IAEHNRIIAKVLKGSGSGSBALYELSKA.TRELRKATDELERA'-ASELERNFSESALVE BMELIAEHNRXXAEHiiR IIAKVLKGSGIHGNVYITADKQKNGIKANFGLNSNVEDGSVQLADHYQQNTPIGDGPVLL PDNHYLSTQ3ALSKD PNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGWPILVELDGDVNGHK FSVRGEGEGDATNG KLTLKFISTTGKLPVPWPTLVTTLSWGVQSFARYPDHMKOHDFFK3AMPEGYVQERTIFF KDDGTYKTRAEVKFE GDTLVKRIELKGIDFKED'GNILGHKLEYGSGSEALYELEKA-TRELKKATDELERATEE LEKNPSEDALVEHNRLI AEHNKI IAEHNRI IAKVLK (SEQ ID NO: 62} 1 «3.2 {2+1} ~cpisoKee ru1 ¾ n 3_v2 : This embodiment shows pH-responsive fluorescence intensit modulation due to fused helical bundle pH-responsi ve conformational switching that is allosterically coupled to chromophore environment.

FQGSGSGSEALYELEKATRELKKATDELERATEELEKNPSEDALVEHNRLIAEHNKIIAE HNRI IAKVLKGSGSG SEALYELEKATRELKKATDELERATEELEKNPSEDALVEHNRLIAEHNKIIAEHNRIIAK VLKGSGIHGNVYITA DKQKNGIKANFGLNSNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEK RDHMVLLEFVTAAGI TLGMDELYKGGTGGSMVSKGEELFTGWPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK FISTTGKLPVPWPT LVTTLSWGVQSFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGTYKTRAEVKFEGDTL VNRIELKGIDFKEDG NILGHKLEYGSGSEALYELEKATRELKKATDELERATEELEKNPSEDALVEHNRLIAEHN KI IAEHNRIIAKVLK

(SEQ ID MO : 63)

(xsev'i 163.2(2+1) -cpr oxCar«iean3_ 2 : This embodiment shows pH-responsive fluorescence intensity modulation due to fused helical bundle pH-responsive conformational switching that is allosierically coupled to chromophore environment

(RKI3S nmHHSSGENL:Y)FQGSGSGSKALyELEKATFBLKRATDSLER&TFSLSEK?SP0A LVE8NRLXS£HNiv IVAEHNRIIAKVLKGSGSGSEALYELEKATRELKKATDELERATEELEKNPSEDALVEHN RLIAEHNKIVAEHNR IIAKVLKGSGIHGNVYITADKQKNGIKANFGLNSNVEDGSVQLADHYQQNTPIGDGPVLL PDNHYLSTQSALSKD PNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGWPILVELDGDVNGHK FSVRGEGEGDATNG KLTLKFISTTGKLPVPviPTLVTTLSWGVQSFARYPDHMKQHDFFKSAMPEGYVQERTIF FKDDGTYKTRAEVKFE GDTLVNRIELKGIDFKEDGNILGHKLEYGSGSEALYELEKATRELKKATDELERATEELE KNPSEDALVEHNRLI AEHNKIVAEHNRIIAKVLK (SEQ ID NO: 64)

iisev 163.2 -Gpmo¾c _& Niean3__Y2 : This embodiment shows pH-responsive fluorescence intensity modulation due to fused helical bundle pH-responsive conformational switching that is ailostericaily coupled to chromophoie environment.

FQGSGaGSSALYKLKRATKSLKFATDSLERA EElSKNPSEDALVSRNRLXAEHNKXVASHNRIXARVLKGSGSG SEALYELEKATRELKKATDELERATEELSKNPSEDALVEKNRLIAEHNKIVAEHNRIIAK VLKGSGIHGNVYITA DKQKNGIKANFGLNSMVEDG3VQLADHYQQNTPIGDGPVLLPDNHYLSTQSAL3KDPNEK RDHMVLLEFVTAAGI TLGMDELYKGGTGGSMVSKGEELFTGWPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK FISTTGKLPVPWPT LVTTLSWGVQSFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGTYKTRAEVKFEGDTL VNRIELKGIDFKEDG NILGHKLEYGSGSEALYELEKATRELKKATDELERATEELEKNPSEDALVEHNRLIAEHN KIVAEHNRIIAKVLK

iSSQ ID NO : 65)

163,2 (2-ri) -cj¾»osiC¾rui¾an3_v2~cfSGFP2 : This embodiment shows pH-responsive fluorescence intensity modulation due to fused helical bundle pH-responsive conformational switching that is allosterically coupled to chromophore environment.

MGSGSEALYELEKATRELKKATDELERATEELEKNPSEDALVEHNRLI EHNKI IAEHNRIIAKVLKGSGSGSEA LYELEKATRELKKATDELERATEELEK PSEDALVEHNRLI EHNKI IAEHNRI IAKVLKGSGIHGNVYITADKQ KNGIKANFGLN3NVEDGSVQLADHYQQNTPIGDGPVLLPDNHYL3TQS LSKDPNEKRDHMVLLEFVTAAGITLG MDELYKGGTGGSMVSKGEELFTGWPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFIS TTGKLPVPWPTLVT TLSWGVQSFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGTYKTRAEVKFEGDTLVNR IELKGIDFKEDGNIL GHKLEYGSGSEALYELEKATRELKKATDELERATEELEKNPSEDALVEHNRLIAEHNKII AEHNRIIAKVLKMVS KGEELFTGWPILVELDGDVNGHKFSV3GEGEGDATYGKLTLKFI3TTGKLPVPWPTLVTT LTYGVQMFARYPDH MKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNIL GHKLEYNYNSHNVYI TADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPN EKRDHMVLLEFVTAA GITLGMDELYK (SEQ ID NO: 66)

Control tvsior· of cp«orCer¾lean3_v2 (a novel cpFP) an ofSGFPl

(UGSSHHHHHHSSGSNLY) FQGSGSGIKGNVYITADKQKNGIKANFGLNSNVSDGSVQL&DHYQQNTPIGDGPVL LP0RRYLS:TQSSLSKD?NEKRDRiiVLLEFVTAAGITLG DELYKGGTGGSRVSKGRRLFTGVVPILVEIDG0VNG HKF3VEGEGSGDATNGKLTLKFISTTGKLPVPRPTLVTTLSWGVQSFARYPDHMKQHDFF KSA PEGYVQSRTIF FKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYGSGSGMVSKGEELETG WPILVELDGDVNGH KF3VSGEGEGDATYGKLTLKFISTTGKLPVPWPTLVTTLTYGVQMFARYPDHMKQHDFFK SAMPEGYVQERTIFF KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNG IKANFKIRHNIEDGG VQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDE LYK (SEQ ID NO: 67) Inactive control Control fusion of c. tsoxCer«lears3 v2 (a novel cpFP) and cfSGFP2

FQGSGBGIHGNVYITADKQKltolRANFGLNSNVEDGSVQLADHYQQNTPIGDGPVLLPD NHYLSTQSALSKDPNE KRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFiGVVPILVELDGDVNGHKFS VRGEGEGDATNGKLi LKFISTTGKLPVPWPTLVTTLSWGVQSFARYPDHMK HDFFKSAMPEGYVQERTIFFKDDGTYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYGSGSGMVSKGEELFTGWPILVELDGDVNGHKFSVS GEGEGDATYGKLTL KFISTTGKLPVPWPTLVTTLTYGVQMFARYPDHMKQKDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTL VNRIELKGIDFKEDGMILGHKLEYNYNSKNVYITADKQKKGIKANFKIRHNIEDGGVQLA DKYOOMTPIGDGPVL LP&^SYLS QSKGGKDPGSKRDKHVLLSF GAAGITLG SELYK: (3BQ: ID NO: 68i Inactiva coatrsl pH-responsive cpFP pH sensor with optimized linker, with C-ter inai

of SGFP2. This _: embodiment shows pH-responsive fluorescence intensity modulation doe to fused helical bundle pH-responsive conformational switching that is aUosterically coupled to chromophore environment.

(MGS3HH¾HHH3SGENL ) FQGSGSGDQEDIDRVLEELEEITSEGDRITKDLERLTQELRRMPSVDALVKHKGAI GGGiNSIIVEHURIILEVLELLLRSTGSGSGDREElRRVLDERREATSRLERAGSSLRRL rESLKKNPAVSVLVRH NTI IVKHNKI IVDHNRIIVRVLELLEKTIGSGIHGNVYITADKQKNGIKAJFGLNSNVEDGSVQLADHYQ QNTPI GDGPVLLPDNHYLSTQSAL3KDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSK GEELFTGWPILVEL DGDVIASHKFSVRGEGEGDATNGKLTLKFISTTGKLPVPWPTLVTTLSWGVQSFARYPDH MKQHDFFKSAMPEGYV QERTIFFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYGSGDKYEIRK VLKELKDITEELRNM TKNLTDLTEELKRNPSVEILVKHNILIVEHNKIIVEKNRIIVDVLELIRKAIMVSKGEEL FTGWPILVELDGDV MGHKFSVSGEGEGDATYGKLTLKFISTTGKLPVPWPTLVTTLTYGVQMFARYPDHMKQHD FFKSAMPEGYVQERT IFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQ KNGIKANFKIRHNIE

pH-responsive cp F H sensor with optimized linker, with C-terminal of SGFE-2. This embodiment shows pH-responsive fluorescence intensity modulation due to fused helical bundle pH-responsive conformational switching that is aUosterically coupled to chromophore environment.

FQG3GSGDDEDIDRVLEELRRITEELDRITKDLERLTQELRRNPSVDALVKHNNAIVRHN EI IVEHMRIILEVLE LLLRSIGSGSGDREEIKKVLDELREATERLEPA.TEELRRLTEELKKNPAVEVLVRHNTI IVKHNKI IVDHNRIIV RVLELLEKTIGSGIHGMVYITADKQKNGIKANFGLN3NVEDGSVQLADHYQQNTPIGDGP VLLPDMHYL3TQSAL SKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDV NGHKFSVRGEGEGDA TMGKLTLKFISTTGKLPVPWPTLVTTLSWGVQSFARYPDHMKQHDFFKSAMPEGYVQERT IFFKDDGTYKTRA.EV KFKGDTLVNRIELKGIDFKKDGNILGHKLEYGSGDKYKIRKVLKELKDITEELRNMTKNL TDLTEELKRNPSVEI LVKHNILIVEHNKI IVEHNRIIVDVLELIRKAIMVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGK LTLKFISTTGKLPVPWPTLVTTLTYGVQMFARYPDHMKQHDFFKSAMPEGYVQKRTIFFK DDGNYKTRAKVKFEG DTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGGV QLADHYQQNTPIGDG PVLLPDKHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 70) H-responsive cpFP pH sensor with optimized linkor using h te pdimer

SCOR133, _: with e- ort nai cf3G?R2. This embodiment shows pH-resppnstve fluorescence intensity modulation due to fused helical bundle pH-responsive conformational switching that is aUosterically coupled to chromophore environment,

(8toSSHHHB¾H¾3GB¾EY) DQGSGSGSDKSYKLDRIhRRLDELIHQLSRlLSSISRI-VDSLERBPhDDKSVQOVI ERXVBLIDBHEEIE/KEyiKLLEBYIKTTKGSGIHGNYYIEADKQKNGIKAHFGLESEVS DGSVQIcADHYQQNTPI GDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFYTAAGITLGMDELYKGGTGGSMVSK GEELFTGWPILVEL DGDVNGHKFSVRGEGEGDATNGKLTLKFISTTGKLPVPWPTLVTTLSWGVQSFARYPDHM KQHDFFKSAMPEGYV QERTIFFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYGSGSPSKEYQ EKSAERQKELLHEYE KLVRHLRELVEKLQRRELDKEEVLRRLVEILERLKDLHKKIEDAHRKNEEAHKENKMVSK GEELFTGVVPILVEL DGDVKGHKFSV3GEGEGDATYGKLTLKFISTTGKLPVPWPTLVTTLTYGVQMFARYPDHM KQHDFFKSAMPEGYV QERTIFFKDDGNYKTRAEVKFEGDTLVNRIBLKGIDFKEDGNILGHKLEYNYNSHNVYIT ADKQKNGIKANFKIR HNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAG ITLGMDELYK (SEQ ID NO : 71 ) pH- responsive cpFP pH s¾ns<>r with opt true-ad linker using hoterodiiner

HCOH1.33, with c- terminal ofsGFFS . This embodiment shows pH-responsive fluorescence intensity modulation due to fused helical bundle pH-responsive conformations! switching that is allosterically coupled to chromophore environment.

FQGSGSGSDKKYKLDRILRRLDELIKQLSRILEEIERLVDELERKPLDDKEVQDVIERIV ELIDEHLELLKEYIKLLEEYIKTT&GSGIHGUVYrT&pKQKNGIKANFwLNS NVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSAL S DPNE8R!¾IKV »B VT¾¾OåTX^f0BI»VK<5GiP66SMVSKGEELFTGWPILVELDGDVNGHKFSV RGEGEGDA TNGKLTLKFISTTGKLPVPWPTLVTTL3WGVQSFARYPDHMKQHDFFKSAMPEGYVQERT IFFKDDGTYKTRAEV KFEGDTLVNRIELKGIDFKEDGNILGHKLEYGSGSPSKEYQEKSAERQKELLHEYEKLVR HLRELVEKLQRRELD KEEVLRRLVEILERLKDLHKKIEDAHRKNEEAHKENKMVSKGEELFTGWPILVELDGDVN GHKFSVSGEGEGDA TYGKLTLKFISTTGKLPVPWPTLVTTLTYGVQMFARYPDHMKQHDFFKSAMPEGYVQERT IFFKDDGNYKTRAEV KFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIE DGGVQLADHYQQNTP IGDGPVPLRDHHYLSTQPKGSROPHRKRDH VLLEFVYARGXYLGMOELYK {SEQ ID HO: 72} pH- responsive cpFP pH. sensor with optimised linker using heterodirrer 3CON133 with subunits in reverse order in primary sequenc , with C~termin&i of SOFP2. This embodiment shows pH-responsive fluorescence intensity modulation due to fused helical bundle pH-responsive conformational switching that is ailosterieaily coupled to chromophore environment.

(MGSSHHHHHHSSG5NLY) FQGSGSGSPSKBYQEKSAERQKELLHBYBKLVRHLRELVEKLQRRBLDKEEVLRRL VEILERLKDLHKKIEDAHRKNEEAHKENKGSGIHGNVYITADKQKNGIKANFGLNSNVED G3VQLADHYQQNTPI GDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSK GEELF GVVPILVEL DGDVNGHKFSVRGEGEGDATNGKLTLKFISTTGKLPVPWPTLVTTLSWGVQSFARYPDHM KQHDFFKSAMPEGYV QERTIFFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYGSGSDKEYKL DRILRRLDELIKQLS RILEEIERLVDELEREPLDDKEVQDVIERIVELIDEHLELLKEYIKLLEEYIKTTKMVSK GEELFTGVVPILVEL DGDVNGHKFSV3GEGEGDATYGKLTLKFISTTGKLPVPWPTLVTTLTYGVQMFARYPDHM KQHDFFKSAMPEGYV QERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGKKLEYNYN3HNVYIT ADKQKNGIKANFKIR HNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAG ITLGMDELYK (SEQ ID UO: 73}

pH-responsive cpFF pH sensor with optimized linker heterodimer 2COMI33 with subunits in reverse order in primary sequence, with C-ter in&I cfSGFFr,

This embodiment shows pH-responsive fluorescence intensity modulation due to fuse helical bundle pH-responsive conformational switching that is allosterically coupled to chromophore environment,

FQGSGSGSPSKE?QSitSABRaKELLHS?EKLVRaiKSLVEKLQRRSLOKESytiRRl.V EI SniiKnr,aKKåED^ RNESAKKEKKGSGIBGiWYITADRQRNGIKANFGLNSNVSDSSVQi OHYQQNTPIGDGFVLLPOMHYLSTQSAL SKDPKEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGWPILVELDGDVN GHKFSVRGEGEGDA TNGKLTLKFISTTGKLPVPWPTLVTTLSWGVQSFARYPDHMKQHDFFKSAMPEGYVQERT IFFKDDGTYKTRAEV KFEGDTLVNRIELKGIDFKEDGNILGHKLEYGSGSDKEYKLDRILRRLDELIKQLSRILE EIERLVDELEREPLD DKEVQDVIERIVELIDEHLELLKEYIKLLEEYIKTTKMVSKGEELFTGVVPILVELDGDV NGHKFSVSGEGEGDA TYGKLTLKFISTTGKLPVPWPTLVTTLTYGVQMFARYPDHMKQHDFFKSAMPEGYVQERT IFFKDDGNYKTRAEV KFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIE DGGVQLADHYQQNTP IGDGPVLLPDNHYLSTQSKL3KDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 74)

In one embodiment, the polypeptide includes changes to th highlighted residues (i.e., resi ues involve in hydrogen-bind networks) in T able 1 , 2 _» or 3 of the polypeptides of 1 -36 only to other polar amino acids.

In another embodiment, the polypeptide includes no changes to the highlighted residues of the polypeptides of S.EQ ID NOs: 1 -36. In a further embodiment, all amino acid substitutions relative to the am o acid sequence of SEQ ID NOs; 1 -40, 45-46, 60-66, 69-76, and 81-86 are conservative annuo acid substitutions. In various embodiments, a given amino add can be replaced by a residue having similar physiochemical characteristics _* e.g., substituting one aliphatic residue for another (such as lie, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Gin and Asp; or Gin and As»). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobic! ly characteristics, are known. Polypeptides comprising

conservative amino ac id substitutions can be tested in any one of the assay s described herein to confirm that die desired activity is retained. Amino acids can be grouped according to similari ties in the properties of their side chains (in A. L Lehninger, in Biochemistry, second ed., pp. 73-75 _* Worth Publishers, New York (1975)): (1.) non-polan Ala (A), Val (V), Leu (L), lie (1), Pro (P), Phe (F), Trp (W), Met (M ; (2) uncharged polar: Gly (G), Ser (S), Thr (I), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Gin (E); (4) basic; Lys (K), Arg (R), His il l).

Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Noriencme, Met, Ala, Val, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Gin; (4) basic; His, Lys, Arg:

(5) residues that influence chain orientation. G!y, Pro; (6) aromatic Tip, Tyr, Phe. Non- conservative substitu tions will entail exchanging a member of one of these classe for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into H is; Asp into Gin; Cys into Ser; Gin into Asn; Gin into Asp; Gly into Ala or into Pro; His into Asn or into Gin; lie into Leu or into Val; Leu into lie or into Val; Lys into Arg, into Gin or into GIu; Met into Leu, into Tyr or into ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp Into Tyr; Tyr into Trp; and/or Phe into Val, into lie or Into Leu.

In another aspect, the disclosure provides non-natumily occurring polypeptide, comprising the amino acid sequence at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting ofSEQ ID NOS: 1-77 and 81 -86. In one embodiment, the polypeptide includes changes to the highlighted residues in Table 1 , 2, or 3 of the amino acid sequence selected from the group consisting of SEQ ID NOS ; 1 -36oniy to other polar amino acids. In a further embodiment, tile polypeptide includes no changes to the highlighted residues in Table 1 , 2, or 3 of the amino acid sequence selected from the group consisting of SEQ ID NGS: 1-36 In a further embodiment, all amino acid substitutions relative to the amino acid sequence selected from the group consisting of SEQ ID NOS: 1-77 and 81-86 are conservative amino acid substitutions. In another embodiment, the disclosure comprises oligomeric polypeptide comprising two or more polypeptides of any embodiment or combination of embodiments disclosed herein. In one embodiment, the oligomeric polypeptides comprise a hetero-oligomer. The hetero-oligomer may be any suitable hetero-oligomer, including but not limited to

heterodimers, Exemplary heterodimers provided herein include heterodimers between polypeptides comprises the amino add sequence at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 0%, 95%, 96%, 97%, 98%, 99%, or 100% identical to:

(a) the amino acid sequence ofSJBQ ID NO: 81 and the amino acid sequence of SEQ ID NO: 82 (prod);

(b) the amino acid sequence of SEQ ID NO:81 an the amino acid sequence of SEQ I D NO : 84 (pro4) ;

(c) the amino acid sequence of SEQ ID NO: 83 and the amino acid sequence of SEQ ID NG;82 (pro4);

(d) the amino aci sequence of SEQ ID NO:83 and the amino acid sequence of SEQ ID NO: 84 (pro4); or

(e) the amino acid sequence of SEQ ID O:85 and the amino acid sequence of SEQ ID NO:86 Cpro5).

In another embodiment, the oligomeric polypeptides comprise a homo-oligomer. The homo-oligomer may be any suitable homo-oligomer, including hut not limited to

homotrmrers. Exemplary heterodimers provided herein include homotrimers of the polypeptide comprising the amino acid sequence at least 25%, 30%, 35%, 4014, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 9954, or 100% identical to a pRQ- 1 polypeptide (SEQ ID NQs: 13-34), a pRQ-2 polypeptides (SEQ ID MQs: 3-12, 15- 22, and 33-36), or a pRO-3 polypeptide (SEQ ID NOs:23~26).

The polypeptides of the disclosure may !ucTud additional residues at the N-termlnns, C-terminus, internal to the polypeptide, or a combination thereof; these additional residues are not included in determining the percent identity of the polypeptides of the invention relative to the reference polypeptide. Such residues may be any residues suitable for an intended use, including but not limited to detectable proteins or fragments thereof (also referred to as“tags”). As used herein,“tags” include general detectable moieties (i.e.;

fluorescent proteins, antibody epitope tags, etc.), therapeutic agents, purification tags (Hi tags, etc.), linkers, ligands suitable for purposes of purification, ligands to drive localization of the polypeptide, peptide domains that add functionality to the polypeptides, etc. Examples are provided herein.

For example, by fusing the polypeptide to a fluorescent protein, we are coupling the conformational change due to protonatiou off he buried histidines in the hydrogen bond networks at the interface of the helical bundle to conformational changes in the ehromophore environment of the fused fluorescent protein. This provides a fluorescent readout of the conformation change. As will be understood by those of skill in the art, other functional subunits could he used in a similar manner to link the pR based conformationa change with a readout based on the function of the functional subuni t.

As used throughout the present application, the term "polypeptide”,“peptide ', and “protein” are used interchangeably in their broadest sense to refer to a sequence of subunit amino acids of any length, which cah include genetically code and non-geneticaly coded amino acids, chemically or biochemically modified or derivataed amino acids, and poly peptides having modified peptide backbones. The polypeptides of the in vention may comprise L-amino acids t- glycine, B-amino acids + glycine (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D~ and L-amino acids + glycine. The polypeptides described herein may be chemically synthesized or recorohinanily expressed. The polypeptides may be linked to other compounds to promote an increased hall- life in vivo, such as by PEGyiation, HESylatiou, PASylation, glycosylation, or may be produced as an Fc-fosiou or in deimffiuflized variants. Such linkage can be covalent: or non-Co valent as is understood by those of skill in the art.

In another aspect, the disclosure provides nucleic acids encoding the polypeptide of any embodiment or combination of mbodiments of each aspect disclosed herein. The nucleic acid sequence may comprise single stranded or double stranded RNA or DNA in genomic or oDKA form, or DNA-RNA hybrids, each of which may incl ude chemically or biochemically modified, non-natural, or derivaimed nucleotide bases. Such nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded polypeptide, including but not limited to poly A sequences, modified Kozak

sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on th teachings herein, what nucleic acid sequences will encode the polypeptides of the disclosure.

In a further aspect, the disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable con trol sequence. "Expression vector" includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product “Control sequences’ ^* operably linked to the nucleic acid sequences of the disclosure are nucleic add sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need nqt be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof Thus, fo example, intervening untran lated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered "operably linked" to the coding sequence.

Other such control sequences include, but are not limited to, polyadeiiy!ation signals, termination signals, and ribosome binding sites. Such expression vectors can. be of any type, including but not: limited plasmid and viral-based expression vectors. The control sequence used to drive expression of die disclosed nucleic acid sequences in a mammalian system may he constitutive (driven by any of a variety of promoters, including but not limited to, CMV SV40, RSV, actin, EE) or inducible (driven by an of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DMA In various embodiments, the expression vector may comprise a plasmid, viral-based vector, or any other suitable expression vector.

In another aspect, the disclosure provides host cells that comprise the expression vectors disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably engineered to incorporate the expression vector of tire disclosure, using techniques including but not limited to bacteria! transformations, calciu phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationie mediated-, or viral mediated transfection, A method of producing a polypeptide according to the disclosure is an additional part of the disclosure. In one embodiment, the method comprises the steps of (a) culturing a host according to this aspect of di disclosure under conditions conducive to the expression of the polypeptide, and (b) optionally,

recovering the expressed polypeptide. The expressed polypeptide can he recovere from the cell free extractor recovered from the culture medium. In another embodiment, the method comprises chemically synthesizing the polypep tides.

In another aspect, the disclosure provides methods for use of the polypeptides or the oligomeric polypeptides of any embodiment or combina tion of embodiments of the disclosure, for any suitable purpose, includin but not limited to delivery of biologies into the cytoplasm through endosoma! escape. Delivery methods relying on ceil penetrating peptides. supercharged proteins, and fipid-ftising chemical reagents can be toxic because of nonspecific interactions with many types of membranes in & pB-Independent manner. Thus, the disclosed polypeptides and oligomeric polypeptides provide a significant improvement over currently available tools.

In another aspect, the disclosure provide methods for designing the polypeptides or the oligomeric polypeptide of any embodiment: or combination of embodiments of the disclosure, comprising a method as described in the examples that follow.

Examples

Abstract; The abil ity of naturally occurring proteins to change conformation in response to environmental changes is critical to biological function. The design of conformational swi tches remains a major challenge. Here we present a general strategy to design pH-responsive protein conformational switches by precisely pre-organizing histidine residues in buried hydrogen bond networks. We design homotrimers and heterodimers that are stable above pH 6 5, but undergo cooperative, large-scale conformational transitions when the pH is lowered and electrostatic and stone repulsion builds up as the network histidines become protonated. The pH range at which disassembly occurs, as well as the cooperatively of the transition, can be programmed by balancing the number of histidine- containing networks and the strength of the surrounding hydrophobic interactions. Upon disassembly, the designed proteins disrupt: lipid membranes both in vitro and in vivo after being endocytosed in mammalian cells; the extent of disruption and the pH-depeudence of membrane activity ca be tuned such that no membrane activity is observed at pH ? and substantial membrane activity is observed at and below pH 6. Our results are dynamic de novo proteins with switchable, conformation-dependent functions that provide a new route to addressing the endosomaS escape challenge lor intracellular delivery.

We explored the de novo design of protein systems undergoing pH-dependeni

Conformation changes, both because the subtlety of the protoftation state changes makes pH- dependence an excellent model problem and a challenging test of our understanding of protein energetics, and because programmable pH-induce conformational changes could have applications for engineering pH-dependent materials an intracellular delivery agents of biological cargo. We set out to create tunable pH-responsive oligomers (pRO’s) by de novo designing parametric helical bundles with extensive histidine-containing networks in which the histidine N« and N« atoms are each making hydrogen bonds (Figure 1). We hypothesized that designing networks with histidine residues that hydrogen bon across the oligomeric. interface would result in disassembly at low pH because histidine side chain profanation would disrupt the hydrogen bond network, energetically destabilizing the assembled protein because of both the resultant steric and electrostatic repulsion and buried polar atoms that are unable to make hydrogen bonds (Figure 1A). The repeating geometric cross-sections of parametric helical bundles allows hydrogen bond networks to be added or subtracted in a modular fashion, and we hypothesized that the pH range of disassembly, as well as the cooperativity, could be tuned b varying the number of histidine networks relati ve to the surrounding hydrophobic contacts:.

We used a three-step procedure to computationally design helical bundles with extensive histidine-containing hydrogen bond networks that span inter-helical interfeces. First, oligomeric protein backbones with an inner and outer ring of ct-belices were produced by systematically varying helical parameters «slug the Crick generating equations. Each inner helix was connected to an outer helix through a short designed loop to produce helix- turn-helix monomer subunits Second, the HBNet™ method in Rosette ^IM was extended to computationally design networks with buried histidine residues that accept a hydrogen bond across the oligomeric in terface, and then used to select the very small fraction o f backbones that accommodate multiple Msiidirie networks (see Computational Design Methods), Third, the sequence of the rest of the protein (surface resi dues and the hydrophobi c contacts Surrounding the networks) was improved while keeping the histidine networks constrained. Synthetic genes encoding five parent designs (named pRCM to pRO-5) with multiple histidine-containing hydrogen bond networks and tight, complementary hydrophobic packing around the networks, along with variants (named pRO-2.1, pRO-2 2, eto ) were constructed (table 3).

All of the designed proteins were well-expressed, soluble, and readily purified by Ni- NTA affinity chromatography, hexakistidine lag cleavage, an a second Ni-NTA step followed by gel filtration. Oligomeric state was assessed by size-exclusion chromatography (SEC) and native mass spectrometry (24). All parent designs assembled to the intended oligomeric state at pH 7 (Figure 1) except for homotrimer design pEO-l , which appeared to be trimeric at high concentration by SEC but was primarily dimeric by native mass spectrometry at lower concentrations (Figure 6); pRO-1 contains smaller, disjoint networks, each with a single histidine, whereas the successful parent designs all have highly-connected hydrogen bond networks that span across all helices of the bundle cross section. To assess the effectiveness of the design strategy, we used native mass spectrometry to study the effect of pH on oligomerization shne(2l, 2d), evaluating each protein from pH 7 down to pH 3 (see Experimental Methods); designs pRO-2 through pRO-5 all exhibited pH-Indneed loss of the initial oligomeric state (Figure 1). As a control, we subjected a previous design

(2L6HC3_13(7$; FOB ID 5.T0H) with a structure similar to pRO-2 but lacking buried histidines to the same assays: changing buffet pH from 7 to as low as pH 3 resulted In no change in oligomeric stale (Figure 7 A) or stability (Figur 7B;I Design pRO-2 was chosen for further characterization, as it exhibited pH-induced disassembly between pH 5 and 6, which is within the range of endosomal pR(27, 28).

The pH-dependent conformational switching is due to the designed histidine networks To specifically evaluate the role of the histidine networks In the pH-induced transition of pR.O-2, we sought to design a variant that lacked the histidine residues but was otherwise identical In sequence. Mutating all histidine residues to asparagine resulted in poor soluble expression and aggregation, likely because the burled asparagine residues are unable to participate in hydrogen bonds; using HBNei ^M , we rescued the histidine to asparagine mutations by generating netw orks in w nch all buried polar atoms participate in hydrogen bonds (Figure IB, him enm-secimm). This new design {pRO-2-noHis), which differs by only six amino acids in each monomeric subunit, is well-behaved in solution an assemble to the intended dimeric state, but unlike pRO-2, remained trimeric at low pH (Figure 1C and fig. 8). Circular dichroism (CD) experiments showed that both proteins were helical and well-folded, and chemical denatur&tfon by gnanidi ium chloride (GdmCl) showed that pRO- 2 has decreased folding stability at low pH, whereas pR0-2-noHis stabilit was unaffected by change In pH (Figure 1 D). The histidines of pRO-2 do not participate in _; unintended metal Interactions that contribute to assembly/disassembly, as addition of 10 mM EDTA had no effect on the helical fold or thermostability of design pRO-2 (Figure IE), Collectively, these results indicate that the observed pH -response is due to the designed histidine networks.

We set out to structurally characterize these designs, but both pRO-2 and pRO-2- noHis were resistant to crystallization efforts. To both test the modularity of our design strategy, as well as to generate additional constructs for crystallization, designs were made that combined networks fro each of pRO-2 and pRO-2-noHts (Table 3). These variants remained soluble after disassembling and reassembled to their designed oligomeric state upon subsequent increase back to pH 7 (figure 9), Designs pRO-2.3 and pRO-2.5 (Figure 2A) readily crystallized and X-ray crystal structures were determined at 1 ,28 A and t .55 A resolution, respectively (Figure 2B, Figure 10, and Table 4). Design pRO-2.3, which differs from parent design pRQ-2 by only two amino acids in each subunit, contains two histidine networks (red cross-sections) and one non-histidine network (blue cross-section);.design JJRG-2.5 differs from pRO-2 by five amino acids in each subunit and contains one histidine network and two non-histidine networks. In all cases, the hydrogen bond networks were nearly identical between the experimentally determined structures and die design models {Figure 2). The ability to swap different types and placements of hydrogen bond networks at each layer without sacrificing structural accuracy highlights the modularity of our design strategy.

Tuning of pH set point and coopera tivity

We take advantage of this modularity to systematically time the pH response by developing a model of the pH-dependence of the free energy of assembly lor a homofrimer with n pH -independent hydrophobic layers, m pE-dependent hydrogen bond network layers each containing three histidine residues, and l hydrogen bond network layers lacking histidine. We assume that the protonation of individual histidine residues within a network layer is cooperative . this is plausible since the protonation of one histidine residue will likely destabilize its surrounding interface, making the remaining histidine residues more accessible and substantially reducing the free energy cost of protonation. The pH- dependence of homotrimer assembly for such a s stem is then

are the free energies of formation of hydrophobic layers, pH-responsive polar layers, and pH-independent polar layers

respectively; R is the gas constant:, and pKa¾.v (the pKa of solvent-exposed histidine) is taken to he 6,0. Equation 1 requires estimates of DOr«! ; m, and L(¾ _M !*L which we obtained from guanidine denaturation experiments (Figure 3B and fig. 1 1 ). In tins model, increases in n shift the pH of disassembly to lower pH values without affecting cooperativity (Figure 3C fop), and varying m while n and (m 4- ) are kept constant changes the cooperativity (steepness) of the transition without as large of an effect on the midpoint (Fig 3C bottom).

To test the tuning of the pH-dependence of disassembly, we generated additional designs based on pRG-2 with different values of m , n and I by swapping one or two of the histidine networks (red cross-sections) for either hydrophobic-only interactions (black cross- sections) or the equivalent hydrogen bond network tacking histidine (blue cross-sections) in different combinations (Figure 3AJ. These new designs were assessed by native mass spectrometry and found to assemble to the intended trimeric state at pH 7 and disassemble at a range of pH values (Figure 30). Because of the context-dependent effects discussed below, we did not directly fit these data to Eq, 1 ; instead the cooperativity of th transition (¾) and the pH set point ( pHQ ) were assessed by fitting the experimental data to a simple sigmoid model that assumes that the starti ng point is 1130% trimer and fee endpoint is 0% trimer:

Eq. 2 % trimer - -· - ·¾¾·· ·^

We compare the observed dependence of k rndpHO on m, n and l with the predictions of the model (Eq. I ) in the following sections.

Tuning pH set point (Figure 3C-D top)

In Equation 1 , the pH set point (pHQ) is fee pH at which the free energy of assembly (the quantity in square brackets) is zero. Designs wife histidine networks replaced by hydrophobic layers have higher stability as assessed by chemical denaiuration experiments (Figure 3B); thus as expected, D<½ώ _{R >>«} is greater than The free energy of assembly at fee pHa of histidine is given by the stun of the first three terms, and since AC%top«> is greater than this sum can be increased by increasing the number of hydrophobic layers and reducing the number of histidine layers. The larger the sum, fee greater the pH change required for the net free energy of assembly to be zero— hence pHO can be lowered by increasing n (fee number of hydrophobic layers) and/or reducing (th number of histidine networks). Consistent with this prediction, replacing a single histidine network with a hydrophobic network (design pRO-2,1, purple curves) shifts the transition pH from above 5 down to "3 5, and replacing two histidine networks with hydrophobic networks (design pRO-2:2, pink curves) eliminates the pH response altogether (Figure 3D top).

Designs pRO-3 (red curves) and pRO~3.1 (orange curves) have two fewer total layers than pRO-2 an also behave as predicted: replacing a single histidine network layer with hydrophobics in these shorter designs increase the pH set point (Figure 3D top). The Equation 1 model holds over fee full set of designs tested: the larger the ratio of to , the higher the transition pH (Figure 3£).

Tuning eonperatrvity (Figure 3C-D bottom) in Equation I , the transition cooperativity (k) is simply 3 m, and replacing the histidine networks ( m ) with polar networks lacking histidines (!) with roughly equal contribution to stability at the pKa of histidine (AGpotsr » roughly equal to AGp^fer ) allows fortuning of the cooperativity of disassembly with litle effect on stability (Figure 3B and 3C), At 5 mM trimer (Figure 3D, bottom right panel), the cooperativity decrease through the series 1=0) (black) through (m ^::: 2, 1=1) (eyas) to (m ^;::: l _s 1 ^:::: 2) (green), consistent with the model. Indeed, design pRO-2.5 (green curves), which has only one histidine network, is the least cooperative design tested and disassembles at approximatel pH 4 (Figure 3D bottom), despite having the lowest stability in chemical denaturation experiments (Figure 3B).

Context-dependence

While Equation 1 qualitatively accounts for the dependence of disassembly and cooperativity on m, n and /, the location of the histidine network layers also contributes. For example, pRO-2,3 and pRO-2.4 have Identical layer compositions (Figure 3 A) and nearly identical sequence compositions (Table 3), but pRO-2,4 disassembles at a higher transition pH and is less cooperative (Figure 3D). Overall, designs with a histidine network close to the termini have higher transition pH values and less cooperative transitions. Histidine residues close to the termini are likely more accessible and hence easier to ptetonaie, and this dynamic accessibility could better accommodate the destabilizing effect of profanation. Consistent with this hypothesis, designs pRO-2 and pRO-2,4, which have histidine networks closet to the termini, have higher flexibili ty as assessed by small-angle X-ray scattering (SAXS) measurements (29, 30) compared to designs pRO-2,1, pRO-2.3, pRO-2.5, and pRO 2-noHis, which do not have histidine networks close to the termini (Figure 12 and Table 5); a correlation betwee flexibility and reduced cooperativity is also observed when the ordered helix-connecting loops are replaced by a flexible GS-linker (Figure 13). Designs with histidine networks further away from the termini (and closer to the loop in the helical hairpin subunit) are presumably harder to initially protonate, but once protonated have a greater destabilizing effect that increases the accessibility of the other histidine positions, resulting in a more cooperative transition. pH-dependetif membrane disruption

The trimer interface contains a number of hydrophobic residues that become expose upon pll-induced disassembly; because atnpMpaihie helices can disrupt membranes (/ 7, 31), we investigated whether the designed proteins exhibit pH-dependent interactions with membranes. Purified protein with hexahisMne tag removed was added to synthetic liposomes containing the pH-insensitive fluorescent dye sulforhodamine B (SRB) at self- quenching concentrations over a range of pH values; leakage of liposome contents following disruption of the lipid membrane can be monitored through dequeochmg of the dye (32). Design pRQ-2 caused pH-dependent liposome disruption at pH valu s as high as 6, withmaximal acti vity around pH 5 (Figure 4A). Design pRO-2-noHis, which did not disassemble at low pH (Figure IC-D), showed no liposome activity at pH 5 (Figure 4S). Design pRO-2 also caused pH-dependent disruption of liposomes with more native-like lipid compositions, although increased cholesterol resulted in decreased activity (fig. 14). Design pRO-3 also caused pH-dependent liposome disruption (Figure 4C); however, design pRO-3 J , which is even more pH-sensitive than design pRG-3 (Figure 3D), did not exhibit any liposome disruption (Figure 4C). The. one major difference between pRO-3.1 compared to pRQ-3 and pRO-2 is the lack of a contiguous stretch of hydrophobic ami.no acids a t the C-termimrs (Figure 40), These putative membrane-interacting residues are sequestered in (he designed oligomeric state but likely exposed after pB- dueed disassembly. To test this hypothesis, a central isoleuc ine in this region of pRO-2 was mutated to asparagine (17GN), which resulte in atenuation of pH-induced liposome disruption (Figure: 4E). Our designs mirror the behavior of naturally occurring membrane fusion proteins, such as influenza HA, in undergoing conformational rearrangements that expose the hydrophobic faces of amphipathic a-helices, allowing them to interact with membranesiA-d).

To further increase the pH of disassembly without altering the putative membrane interacting residues, we tuned the pH-sensitivity by increasing or decreasing the overall interface affinity through .mutations in the hy drophobic layers (tuning AGhy&i,phi¾ii) of design pRO-2. Consistent with Eq, I, increasing DOnAor^ί through the A54M substitution decreases the transition pH, whereas weakening AGhWmpiwkewi h the 156 V substitution increases tire transi tion pH to approximately 5.8 (Figure 5A). Neither of the mutations substantially affect the cooper ti vity of the transition (Figure 5B). CD monitored denaturation experiments showed that A54M increases stability and 156V decreases stability, as expected (Figure 15). Similar tuning of tire heterodimer design pRO-4 with the destabilizing mutations L23A/V130A increased the pH transition point of disassembl from pH -4 to pH -4.6 (Figure 1G).

To characterize the physical interactions between protein and membranes, and the mechanism of membrane disruption, purified proteins were chemically conjugated to gold nanoparticies and visualized by cryo-eleetron microscopy ant! tomography. Desi n pR 0-2 156 V, which has a highest transition pH (Figure 5A), also has increased liposome

perrneabllizatlon. activity (Figure SB); it; directly interacts with liposomes at pH 5 bM not at pH 8 _V while the non-pH-responsive design pRO~2~noHis shows no interactions with liposomes at either pH (Figure 5C and fig. 16). We observed widespread membrane deformation and disruption of the lipid bilayer with design pRO- I56V and pRO-2 at pH 5along with association of protein conjugated gold nanoparticles to liposomes (Figure 5C and fig. 16), At either pH, pRO-2 -noHis and pRO-2 156V at pH 8, there were no signs of membrane deformation or disruption and protein conjugated gold nanopartic!es were well dispersed and did not associate to the membrane (Figure 5 C and fig, 16). At pH 5, design pRO-2 156 V causes significant deformation of the liposomal membrane and induces formation of tight extended interfaces between liposomes, we observe density at these interfaces that likely corresponds to pRO-2 156V (Figure SC and fig. 16).

We next investigated the behavior of the designed proteins in the low pH environment of the mammalian cell endocyiie pathway, internalized proteins are either recycled back or destined for degradation through fusing with lysosomes that contain hydrolytic enzymes that are activated at around pH 5(55), To test their behavior in the endocyiie pathway, we expressed the pRO~2 trimers as fusions to +36GFP(5 35) to facilitate both fluorescent imaging and endocytosis; these fusions also showed signs of pH-induced liposome disruption by cryo-electron microscopy and tomography (Figure 5D). Following addition to U2-OS cells, +36GFP fusions of pRO-2 and 156V colocalize with lysosomal membranes and are not degraded, whereas pRO-2-noHis is not observed in lysosomes (Figure 5E-F). I56V, which is the most pH-sensitlve and membrane active design in this study (figure 5A-C), is the most strongly colocalized with the lysosomal membrane (Figure SF) We hypothesize that pRO~2 and 156V disassemble in the lower pH environment of the lysosome and endosonie, and interact, with membranes to cause proton leakage and neutralization, preventing degradation; pRO-2-noHis is not pH-responsi ve nor membrane acti ve and is presumably degraded by the lysosomes. To test this hypothesis, U2-OS loaded with dye to track pH (LysoSensor

Yeliow/Blne DND-160) were incubated ior one hour with pRQ-2 (5 pM), pRO-2 156V (5 pM), or pRO-2-toHis (5 pM); design pRO 2 156V raises the lysosomal pH compare to pRO~2 -noHis and normal cell controls (Figure 5G and fig. 17). Design pRO~2 156V

produces larger changes in lysosomal pH than two drugs, Bafilomycin A and Chloroquine, known to neutralize lysosomal pH (Figure SCI).

As shown in Figure 18, the increase in fluorescence between pH 8.0 and 5.3 is shifted towards lower pH For the 163.2(2+ i)-cpmoxCerulean;V v2 construct (cyan) compared with the (l56\ 163.2{2+l)<pn Cerulean3_y2 construct (blue), which supports the theoretical model that reduced .interface energy of hydrophobic layers (Dq¾> » _G>ύ no¾) in the helical bundle due to the isoleueinerto-valhte mutations increases the pH at which the helical bundle unfolding transition occurs.

As shown in Figure 1 , at high pH, the helical bundle trimer (grey) is associated, and the cpmoxCerulean3 v2 (cyan) acts as a FRET donor to the C-termkial cf$<3FP2 (green) which acts as a FRET acceptor, producing a quantifiable FRET signal. At low pH, the helical bundle trimer dissociates due to histidine residues at the trimer interface becoming protonated, the conformational change of which is coupled to the cpmoxCerolean3_v2 FRET donor increasing i fluorescence brightness. The cpmoxCer«Iean3__v2 has a low pK* of unfolding, while the efSGFP2 has a high pi¾ of unfolding, so at low pH the

cpmoxCerulean3 _ v2 remains folded and the cfSGFP2; unfolds reducing its ability to act as a FRET acceptor. Thus, at low pH, because the FRET donor increases in fluorescence brightness while the FRET acceptor decreases in fluorescence brightness, the overall FRET signal is reduced at low pH, The described mechanism allows the designed conformational change of the helical bundle upon pH change to be coupled to measureable fluorescence readouts,

pH-dependent membrane disruption ability can. be conferred to other proteins vi fusion at the « terminus of «symmetrized single-chain pH trimers. In this example,

Asym206TEVA«ti (magenta) was fused to a aanoparticle and is expressed and purfled fro E, Coli, Single-chain «symmetrized pM-responsive trimers fused to nanoparticles exhibited pH-dependent lipolysis equal to and greater than pR02,3 (data not shown). Proteins wer mixed with liposomes encapsulating self-quenching sulibrhodamine B (SRB) fluorescent: dye. Liposome disruption was measured by measuring fluorescence of released and dequenehed of dye leaked from d isrupte membranes on a spectre luoromeier.

Conclusions

It was not previously clear how to achieve the high cooperativity that allows proteins to dramatically alter function in response to small changes is the environment,. OUT results now clearly answer the latter question in the affirmative— The complete loss of trimer pRO- 2 over a very' narro pH range in the present disclosure demonstrates that such high cooperativity has been achieved. Furthermore, the disclosure further demonstrates die ability to; systematically tune the set point: and cooperativity of the conformational change. The modular and tunable pH set point and cooperaiivity of our designed homooligomers. together with their liposome permeabilizhig activity, makes them attractive for delivery of biologies into the cytoplasm throug endosomal escape. Delivery methods relying on cell penetrating peptides, supercharged proteins, and lipid-fusing chemical reagents can be toxic because of nonspecific interactions with many types of membranes in a pH-independent manner.

References:

1. C. B. Anfinsen, Principles that govern the folding of protein chains. Science. 181, 223- 230 {1973).

2. P.-S. Huang, S. E. Beyfcen, D. Baker, The coming of age of de novo protein design.

Nature 537 ^’ 320-327 (2016).

3. G. J. Rocklin ei a! , Global analysis of protein folding using massively parallel design, synthesis, and testing. Science 357, 168-175 (2017).

4. C. M Carr , P, S, Kim, A spring- loaded mechanism for the conformational change Of influenza hemagglutinin. Ceil. 73, 823-832 (1993).

5. J. M. White, S E. Delos, M. Brecher, K. Schomberg, Structures and mechanisms of viral membrane fusio proteins: multiple variations on a common theme. Crit. Rev Biochem. Mol. Bkrf. 4$, 189 219 (2008)

6. C. M. Mair, K. Ludwig, A. Herrmann, C. Siefaen, Receptor binding and pH stability— how influenza A virus hemagglutinin affects host-specific vires infection. Biochimica ei Biopfiyma Acta (BBA)-Btommkrmes. 1838, 1153-1168 (2014).

7. M L. Murtangh, S. W. Fanning, T. M. Sha mm, A. M. Terry, l R. Horn, A

combinatorial histidine scanning library approach to engineer highly pH-dependeut protein switches, Protein Set 20, 1619-163 ] (201 ! ).

8. E.-M. Siraueh, S J. Fleishman, D. Baker, Computational design of a pH-sensitive IgG binding protein Proc, Nail. Acad. Set. U. R A. Ill, 675-680 (2014).

9. N. Gera, A B. Hill, D P. White, R. G. Carbonell, B. M. Rao, Design of pH sensitive binding proteins fi¾m the hyperihennophilic Sso7d scaffold. PLoS One. 7, 048928

(2012)7

10. M. Daimau, S. Lim, S.-W. Wang, pH-niggered disassembly in a caged protein complex.

Biomacromolecules. 10, 31 9-3206 (2009).

1 i . T. Igawa <?/ a/., Antibody recycling by engineered pH-dependent antigen binding

improves the duration of antigen neutralisatio . Nat. Biotechnol 28, 1203-1:207 (2010).

12. K. Wada, T. Mizimo, J.-i. Oku, T Tanaka, pH-indueed conformational change in an alpha-helical eoiled-coiS is controlled by His residues in the hydrophobic core. Protein Pept Lett 10, 27-33 (2003).

13. R. Lkatovic el at., A De Novo Designed Coiled-Coil Peptide with a Reversible pH- induce Oligomerization Switch Structure . 24, 946-955 (2016)

14. K . Page! ei al , Random Coils, p-Sheet Ribbons, and «-Helical Fibers: One Peptide Adopting Three Different Secondary Structures at Will. J. Am Chem. Soe 128, 2196- 2197 (2006).

15. C. Minel!L J X. Drew, M. Muthu, H. Andresen, Coiled cod peptide -functionalized surfaces for reversible molecular binding. Soft Matter , 5119-5124 (2013).

16. J. Aupic, F Lapenta, R. leraia, SwitCCh; Metal-site design for controlling the assembly of a eoiled-coil homodimer. Chembioehem (2018), doi : 10.1002/cbic.201800578. Y Zhang et al., Oornpuiationa! design and experimental characterization of peptides intended for pH -dependent membrane insertion and pore formation, ACS Chem. Biol.

10, 1082-1093 (2015).

S. E. Boyken e( al., De novo design of protein homo-oiigomcrs with modular hydrogen- bond network-mediated specificity. Science 352, 680-687 (2016).

F, H. C. Crick, The Fourier transform of a coiled-coil. Acta Crystallogr, 6, 685-689 (1953).

G. Grigoryan, W, F. Degrade, Probing designabiiity vi a generalized model of helical bundle geometry. J. Mol. Biol 405, 1079-1 100 (201 1).

A, Leaver-Fay et al , in Methods in Emymohgy, M. L. Johnson, L. Brand, Eds.

(Academic Press, 201 1), vol 487, pp. 545-574.

B. Kuhlmaa, D. Baker, Native protein sequences are close to optimal for their structures P c. Natl Acad Set U. S. A. 97, 10383-10388 (2000).

P.-S. Huang ef al , High thermodynamic stabi lity of parametrically designed helical bundles. Science. 346, 481—485 (2014).

S. Mehmood, T. M. Allison, C. V, Robinson, Mass spectrometry of protein complexes: from origins to applications. Amu. Rev. Phys. Chem. 66, 453-474 (2015).

E. Boeri Erba, K. Barylyuk, Y. Yang, R, Zenobi, Quantifying protein-protein interactions within noncovaient complexes using electrospray ionisation mas spectrometry. Anal Chem. S3, 9251-9259 (2011).

N. Leloup et al , Low H-induc&d conformational change and dimerization of sortiliu triggers endocytosed ligand release. Nat, Commw. 8, 1708 (2017).

Y.-B. Hu, E, B, Hammer, R.-j, Ren, G, Wang, The endosomal- lysosomal system: from acidification and cargo sorting to neurodegeneration Trans L Neurodegeher. 4, .1 (2015).

M Grabe, G. Osier, Regulation of organelle acidity, J. Gen. Physiol 117, 329-344 (2001).

K. N Dyer et al. High-throughput SAXS for the characterization of biotnolecnles in solution: a practical approach. Methods Mol Biol 1091, 245-258 (20.14).

S, Classen et al , Implementation and performance of SIBYLS: a dual endstation small- angle X-ray scattering and macromolecular crystallography heamline at the Advanced Light Source. J. AppL Oysta!iogr. 46, 1-13 (2013).

E. Eirfksdottir, K, Ronate, U, Eangel, G. Diyita, S. Deshayes, Secondary structure of cell-penetrating peptides controls membrane interaction an insertion Biockim, Bi&phys. Acta. 1798, 11 19-1128 (2010),

L. Gni K. K. Lee, in .Influenza Virus: Methods and Protocols, Y, Yamauchi, Ed.

(Springer New York, New York, NY, 2018), pp, 261-279.

C. S, Piilay, E. Elliott, C. Dennison, Endoiysosomal proteolysis and its regulation.

Bioehem. J. 363, 4 I 7--429 (2002).

M. Li et a!.. Discover and characterization of a peptide that enhances endosomal escape of delivered proteins in vitro and in vivo. J Am. Chem. Soc : 137, 14084-14093 (2015). M. S. Lawrence, K. J. Phillips, D. R. Liu, Supercharging proteins ca impart unusual resilience. ,/ Am. Chem. Soc. 129, 10110-10112 (2007f

j . Monod, J. Wyman, i -P. Changeax, in Selected Papers in Molecular Biology by Jacques Mon d, A. Lwofi) A. Ulima n, Eds. (Academic Press, 1978), pp. 593-623. E, L. Snyder, S, F. Dowdy, Cell penetrating peptides in drug delivery. Pharm. Res. 21, 389-393 (2004).

Y. S. Choi, M, Y, Lee, A, B. David, Y, S. Park, Nanoparticles for gene delivery:

therapeutic and toxic effects. Molecular «£ Cellular Toxicology. 10, 1-8 {2014). Materials and Methods Computational Desi an Methods

Backbone sampling: Oligomeric protein backbones with an inner and outer ring of a- helices were produced by systematically varying helical parameters using the Crick generating equations (19. 20). ideal values were used for the supereoil twist (oo) and helical twist (&{)(! 9 ₁ 20). Starting points for the superhelical radii were chosen based on successful previous designs (18) and the helical phase (D h) was sampled from 0° to 90° with a step s e of 10° The offset along the « axis (Z-offset) for the first heli was fixed to 0 as reference point with the rest of the helices independently sampled from -1.51 A to 1.5 Ϊ A, with a step size of 1.51 A. For heterodimer designs, snpereod phases (D o> were fixed at 0°, 90°, 180° and 270°, respectively, for the four helices. The inner and outer helices were connected by short, structured loops as described previously (IS). To find backbones that could accommodate more than two histidine networks, a second round of parametric design was performed with finer sampling around the helical parameters of the initial designs (Note: because the inne and outer helice have different superhelical radii, the repeating geometric cross sections o f the heli cal bundle are not always perfect geometric repeats along the z~axis ; hence, because of the geometric sensitivity of hydrogen bonding, finer sampling was required to find backbones that could accommodate the same histidine hydrogen bond networks at multiple layers / cross sections).

Design of histidine networks: the HBNet (18) method in Rosetta™ (21) was ex tende to include program code that all owed for the selection of hydrogen bond networks that contain at least one histidine at oligomeric interfaces, and also the option to select for cases where the histidine residue accepts a hydrogen bon across the oligomeric interface KBNei ^iM was used to select backbones that coul accommodate 1-4 such networks in die hcmottimerie and heterodtmenc backbones

Rosetid ^m design eakidalions: To design the sequence an sidechain roiamer conformations for the rest of the protein surrounding the hydrogen bond networks, the network residues were constrained using AtemPair ^iM constraints on the donors and acceptors of the hydrogen bonds and RosettaDesign™ calculations carried out, and best designs selected.

Design strategy to tune pH se paint and cooper at mty via modular placement of the histidine networks: Once successful designs were identified, HBNet ^lM was used to generate all possible combinations of hydroge bond network placement for the existing network within the backbone of that design; for each _» the ammo acid sequence an d side c hain rotamer conformations were optimized around those placed networks as described above. Fro these combinations for pRO-2, designs pRO-2 , 1 -2.5 (Figure 3) were selected based on placement of networks m and / relative to the hydrophobic layers, n, to test our tuning strategy. Design pRO-2 mutants |56V and A54M were designed rationally without any computational design.

Protei expression and purification

Plasmids containing synthetic genes that encode the designed proteins were ordered through Genseripl Inc, (Piscaiaway. N.J., USA), cloned into the Ndel and Xhol sites of either pET2T-NESG or pET~2 h vectors (see tabled). Plasmids were transformed into chemically competent E. coif express ion strains BL2i(DE3)Star (inviteogen) or

Lemo ^TM 21(DE3) (New England Biolabs). Following transformation, single colonies were picked from agar plates and grown overnight in 5 ml starter cultures ofLu -Bertani (LB) medium containing 50 pg/ L carbeiiicillin (for p£T21-N£SG vectors) or kanamycin (for pBT-28b vectors) with shaking at 225 s m for 12-18 honts at 37 ^a C. 5 ml starter cultures were added to 500 ml TBM-5052 with antibiotic for expression by autoinduction; cells were grown at 37 ^C C for 4-7 hours and temperature was dropped to 18°C overnight. After 18-24 hours, cells were harvested by centrifugation for 15 minutes at 5000 ref at 4°C and

resuspended la 20 nil lysis buffer (25 mM Tris pH 8.0 at room temperature, 300 tsM NaCI, 20 mM Imidazole).

Cells were lysed by microfluidization in the presence of 1 M PMSF Lysates were clarified by centrifugation at 24,000 ref at 4X for at least 30 minutes. Proteins were purifie by Immobilized metal affinity chromatography (IMAC): supernatant was applied to Ni-NTA (Qiagen) colnmns pre-equiiibmted in lysis buffer. The column was washed twice with 15 column volumes (CV) of wash buffer (25 mM Tris pH 8.0 at room temperature, 300 mM HaCl, 40 M Imidazole), followed by 3-5€V of high-salt wash buffer (25 mM Tris pH 8.0 ai room temperature, I M NaCI, 40" M Imidazole) then an additional 15 CV of wash buffer. Protein was eluted with 250 M Imidazole, and buffer-exchanged into 25 mM Tris pH 8 0 and 150 m NaCI without imidazole for cleavage of the N-ter mal bexahlstidine tag by purified hexahisiidme-tagged TEV protease (with the exception of design pRO-1 , which was cleaved using restriction grade thrombin (EMD Millipore 69671-3) at room temperature for 4 hours or overnight, using a 1:5000 dilution of enzyme into sample solution) A secon Ni- NTA step was used to remove hexahistidlne tag, uncleaved sample and the hexaMstidlne- tagged TEV protease, and the cleaved proteins were then concentrated an further purified by gel filtration using FPLC and a Superdex ^¼ 75 Increase 10/300 GL (CiE) size exclusion column In 25 mM Tris pH 8.0 at room temperature, 150 mM Had, and 2% glycerol

Buffers for varvine pH

For low-pH experiments in solving circular dkhroisni (CD), small-angle X-ray scattering (SAXS), and size exclusion chromatography (SBC), NajPCk-Citrate buffer was used to ensure that a single buffer system could be used that was stable over the entire pH range to be tested. Buffers were made using established ratios, of stock solutions of 0.2 _: M NajPGi and 0.1. M Citrate; final pH was adjusted using hydrochloric add {HO) or sodium hydroxide (NaOH) if needed. For S AXS and SBC, 150 mM NaCl and 2% glycerol were added. Nati ve mass spectrometry experiments required the use of ammonium acetate buffer, and pH was adjusted using acetic acid, with the final pH value measured (see Native Mms Spectrometry section below). For liposome disruption assays, 10 mM Iris, 150 mM NaCl 0.02% NaNj, pH 8.0 was used and pH was change by rapid acidification using 10 mM HEPES, 150 M NaCI, 50 M Citrate and 0.02% NaNj buffer at pH 3.0 as described previously! iP). and final pH values were measured (see Fluorescence Dequmching

Liposome Leakage X.way section below)

Hexahistidiue fag was removed for all experiments that tested the effect of pH

Circular Dichtoism (CD)

CD wavelength scans (260 to 195 nm) and temperature melts (25 to 95 ^C C) were measure using a JAS€O J-l 500 or an AVIV™ model 420 CD spectrometer. Temperature melts monitore absorption signal at 222 nm and were carried out at a beating rate of 4 ^¾ C/min; protein samples were at 0 25 mg/niL in either phosphate buffered saline (PBS) pH 7.4 or NasPOi-Citrate at indicated pH values (see Buffers systems fi>r varying pH).

Guanidioiom chloride (GdmCl) titrations were all performed on an AVIV 420 spectrometer with an automated titration apparatus using either PBS pH 7.4 or NasPOs-Citrate buffers at indicated pH at room temperature, monitoring helical signal at 222 nm, using a protein eoaceniration of ^' 0.025 rag/raL in a \ cm cuvette ith stir bar. Each titration consisted of at least 30 evenly distributed concentration points with one minute mixing time for each step, Titrani solution consisted of the same concentration of protein in the same buffer system pins GdmCl; GdmCl concentration of starting solutions was determined by refractive index.

Native Mass Spectrometry Samples were buffer exchanged twice into 200 siM ammonium acetate (NFHAe; Mil.UporeSigma) using Micro Bio-Spin P-6 columns (Bio-Rad). Protein concentrations were determined by UV absorbance using a Nanodro 2000c spectrophotometer (Thermo Fisher Scientific) and diluted to make up a 1 -fold stock solution (50 uM and 16.7 mM monomer and trimer concentration, respectively). 1 pL of this solution was mixed with 9 mB 2O0 thM NHaAc / 50 s¾M Uiethylasnmonium acetate (TEAA; MifliporeSignia), adjusted with acetic acid (Fisher Scientific) to obtain the desired final pH an incubated on ice for 30 min. For experiments to test for the reversibility of disassembly , the pH was subsequently increased either by addition of ammonia or b buffer-exchange to 200 mM NFHAe / 50 niM TEAA (pH 7.0) via uitrafiitraiioii (Anricon Ultra, MWCO 3 kDa). 5 pL samples were filled into an in- house pulled glass papillary and ionfoed by nESI at a monomer or a trimer concentration of 5 mM or 1.67 mM, respecti vely. All pH titration data were acquired on an in-house modified SYMAPT™ G2 HDMS (Waters Corporation) with a surface-induced dissociation (SID) device incorporated between a truncated trap traveling wave ion guide and the ion mobility cell(59). The following instrument parameters were used; spray voltage 0.S .3 kV ;

sampling cone, 20 V; extraction cone, 2 V; source temperature, room temperature; trap gas flow, 4 mL/min; trap bias, 45V. The data were processed with MassLynx™ v4,i and DriftScope v2.1. Smoothed mass spectra (mean; window 20; number of smooths 20) are shown in figs. 9 and 20. For relati ve quantification, charge slate series were extracted from DriftScope ^1M , and smoothed spectra (mean; window 20; number of smooths 20) were integrated.

Small-angle x-ray scattering tSAXS i

Samples were purified by gel filtration in either 25 mM I ris pH 8,0 at room temperature, 150 mM NaCl, and 2% glycerol, or KasPCb-Citrate buffer at indicated pH with iSCtrnM NaCl and 2% glycerol. For each sample, data as collected for at least two different concentrations to test lor concentration-dependent effects; "high” concentration samples ranged from 4-10 mg/ml and ^'‘ low” concentration samples ranged from 1-5 mg/mi (table 5), Fractious preceding the void volume of the column, or from the flow-through during concentration using spin concentrators (Millipore), were used as blanks for buffer

subtraction. SAXS measurements were made at the SlBYLS ^iM 12.3.1 heam!ine at the

Advanced Light Source. The X-ray wavelengt (l) was 1 ,27 A and the sampie-io-detector distance of the Marl 65 detector was 1.5 m, corresponding to a scattering vector q ( ^:::

dxAsinfW ) where 20 is the scattering angle) range of 0.01 to 0 59 A ¹ . Data sets were collected using 34 0 2 second exposures over a period of 7 seconds at 11 keV with protein at a concentration of 6 mg rnL. The light path is generated by a super-bend magnet to provide a 1012 photons/sec flux (I A wavelength) and detected on a PilatosS 2M pixel array detector. Each sample is collected multiple times with the same exposure length, generally every 0.3 seconds for a total of 10 seconds resulting in 30-34 femes per sample. These individual spectra were averaged together o ver each of the Gunier, Parod, and Wide-q regions depending on signal quality over each region and frame using the Frames lice™ web server. The average spectra for each sample were analyzed using the ScAtier™ software package as previously described (29, 40). FoXS™ (41, 42) was used to compare design models to experimental scattering profiles an calculate quality of fit (X) values.

X-ray crystallography

Purified protein samples were concentrated to 13 mg/m! for pRO-2 3 and 17 mg/.ml for pRO-2.5 in 20 mM Tris pH 8.0 at room temperature with 100 inM NaCl. Samples were screened with a 5-position deck Mosquito crystallization robot (ttplabtech) with an active humidity chamber, utilizing JCSG Core ^tM HV screens (Qiagen) Crystals were obtained after 2 to 14 days by the sitting drop vapor diffusion method with the drops consisting of a 1:1, 2:1 and 1:2 mixture of protein solution and reservoir solution. The conditions that resulted in the crystals used for structure determination are as follows: pRO-2.3 crystallized in JCSG-I B7, which consists of 0.2M di-sodium tartrate and 2034 w/v PEG 3350; pRO-2.5 crystalrzed in JCSG-I A9, which consists of 0.2 M Potassium acetate and 20% w/ PEG 3350.

X-rav data collection and structure determination

Protein crystals were loope an place in reservoir solution containing 20% (v/v) glycerol as a eryoproteetani, and flash-frozen in liquid nitrogen. Datasets were collected at the Advanced Light Source at Lawrence Berkeley National Laboratory with beamlines 8 2.1 and 8 2.2 Data sets were indexed and scaled using XDS (43). Phase information was obtained by molecular replacement using the program PHASER ^{1 M} (44) from the Phenix software suite (45); computational design models were used for the initial search. Following molecular replacement, the models were improved usin Phenix™ autobuiid (46); efforts were made to reduce model bias by setting rebuild-in-piace to false, and using simulated annealing and piime-and- switch phasing. Iterative rounds of manual building in COOT ^{1 M} (47) and refinement in Phenix™ were used to produce the final models. Due to the high degree of self-similarity inherit in cotled-cotHike proteins, datasets for the reported structuressuffered from a high degree of pseudo translational non-crystallographic symmetry, as report by Phen * %Xtriagfi, which complicated structure refinement and may explain the higher than expected R- values reported, RMSDs of bond lengths, angles and dihedral s from ideal geometries were calculated using Phenk ^SM (4$). The overall Quality of the final models was assessed using MOLPROBITY (48). Table 4 summarizes diffraction dat and refinement, statistics.

Liposome Preparation and Characterization

liposomes composed of DOPC (1 ,2-dioleoyl-sn-glycero-3-phosphocholine.), DOPC with 25% cholesterol (molar ratio to DOPC), 3; 1 DOPCTPOPS (l-paimiioyS-2-oieoyl-sn- glycero-3-phoxpho-L-serine), and 3 :1 DOPC: POPS with 25% cholesterol were prepared identically to a final concentration of 5 M total lipid as previously described (32); lipids from Avanti Polar Lipids. Lipids solubilized in chloroform were dried under nitrogen gas and stored under vacuum for a minimum of 2 hours to remove residual solvent. The dried lipid film was the resuspended in Tris buffer (10 mM Tris, 150 mM NaCi, and 0.02% NaN pH 8.0) containing 25 mM Sul forhodamme B (SRB) fluorophore (Sigma) and subjected to 10 sequential freeze thaw cycles in liquid nitrogen. Liposomes were extruded 29 times through 100 ntn pore size polycarbonate filters (Avanti Polar Lipids) and purified from free

fluorophore using a PD- 10 gel filtration column (GE Healthcare) into storage buffer (10 mM Tris, 150 mM NaCI, and 0.02% Nabb pH 8 0) Liposome size and homogeneity was analyzed by dynamic light scattering (DLS) using a Dynapro Nanostar™ DLS (Wyatt Technologies). On average liposome diameter ranged From 120- .130 nm with low

polydispersity Liposomes were store at 4°C and used within 5 days of preparation.

Fluorescence Dequenching Liposome Leakage Assay

Liposome disruption and content leakage was analyzed by fluorescence spectroscopy as previously described (32). Liposomes containing SRB fluorophore at self-quenching concentrations were incubated with 2.5 pM peptide, with respect to monomer, at 24°C and pH 8.0 in Tris buffer (10 mM Tris, 150 M NaCi, 0 02% NaNs, pH 8.0) for 10 minutes. Die solution was rapidly acidified to the target pH by addition of a fixed volume of acidification buffer an incubated for 20 minutes. Acidification buffers are mixtures of the Tris pH 8.0 buffer and citrate buffer pH 3.0 (10 mM HEPES, 150 mM NaCi, 50 mM Citrate and 0.02% NaNs pH 3.0) in empirically determined ratios to achieve the target pH. SRB fluorescence i independent of pH within the ranges used here. Finally _, , Triton X- 100 (Sigma) was added to a final concentration of 1% to fully disrupt liposomes. Liposome disruption as indicate by content leakage and SRB dequenching was normalized using the formula [F«>-F<»)}/[F(M*<r F(0)j where F< > is the average fluorescence intensity before acidification and F(Ma _*} is the average fluorescence intensity after addition of Triton X-l 00. All measurements were collected on a Varian Cary Eclipse spectrophotometer using an excilation/emission pairing of 1 565/586 and 2.5 nm sli t widths at 24°C Any data plotted together was collected using the sam batch of liposomes.

Designs pRO-2, pRO-2 156 V, and pRO-2 -noHis were chemically conjugated to 10 nm Gold tanoparticies according to manufacturers instaictions, ensuring all gold nanoparticles were conjugated to protein _* The conjugation reactions were performed immediately prior to use for electron microscopy imagings For each design pRQ-2, pRO-2 I56V, and pRO-2-noHis a solution of 2.5 mM purified protein, 0.125 mM gold-conjugate protein, and 1 mM DOPC liposomes was applied to glow-discharged C-Flat 2/2-2C-T holey carbon grids (Protochips, Inc,) and acidified on the grid by addition of HEPES-citrate buffer. The grids were prepared using a Vitrobot Mark IV (FEl) at 40 and 100% humidity before being plunge-frozen in ethane cooled with liquid nitrogen.

Electron micrographs were collected using a Tecnai G2 Spirit™ Transmission Electron Microscope· (FEl) operated at 120kV and equipped with a 4k x 4k Gatan Ultrascan CCD camera a a nominal magnification of 26,000x or a Tecnai TF-20 Transmission Electron Microscope (FEl) operated at 200kV equipped with a K2 Summit Direct Electron Detector (Gatan).

Projection micrographs collecte on the TF-20 were captured with the detector operating in counting mode. Specimens were imaged at 14,500 magnification, giving a pixel size of0.254nm, with a dose ofH 8e-/ A" across 75 200ms movie frames. Data were collected in a semi-automated fashion using Legmen™ (4.9) and micrograp movie frames were aligned using: MotionCor2 ^iM (SO): Leginon™ was used to collect tomography tilt series from -48 to +48 degrees bidirectionally in 3 degree increments with a total accumulated dose of ~ 100 e-/A ² , Reconstructions were processed using etomo in the iM0D software suite (51) with CTF parameters estimated from CTFFIND4 ^IM (52), Reconstructed tomogfams were visualized and measurements were made using Image) ^lM (55). Ceil Culture, Plating, and Transfection

U-2 OS (ATCCj cells were cultured in DMEM supplemented with 10% (y/vj inactivated PBS (Corning), 2 mM glutamine, penicillin (100 lU/mL), and streptomycin (iOO pg/mL) at 37 ^t C and 5%€02. The glass-botom eoverslip chambers were pre-coate with 500 ug/rnL (>f Matrigel (Corning). Transfection of LAMPl-HaloTag™ was performed using Lanza Nncieofector system according to the Manufacturer's specifications. After overnight of recovery and expression, the eelis expressing LAMH-HaioTag ^~iM were labeled with 100 siM 3F646-HTL for 30 minutes and washed three times with pre-warmed DMEM medium.

Live Cell Experiments

The final concentration of 5 mM 1-36GFP fusion proteins was incubated with the LAMPi-HaloTag™ expressing U-2 OS ceils on a pre-coated coversKp for 1 hr. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature (RT) and quenched/rinsed with PBS supplemented with 30 mM glycine. The», the coverslips were mounted on FiuoroSave™ (Millipore). For pH measurement of the Iysosorae, LysoSensor™

Yeilow/Blue DND-16Q was incubated at I mg/m overnight and washed twice prior to iroaging(5 ). Tire final concentration of 5 pM protein was incubated with the LAMP1- HaloTag expressing U2-OS cells that were loaded with 1 mg/mL LysoSensor™ Yellow/Blue DND-160 for .1 hr. In separate chambers, LysoSensor ^m Yellow/Blue DND-160 loaded tells were incubated with bafilomycin A1 (1 mM) and chloroqui e (50 pM) for 1 hr as a control.

C Microscopy

For fixed cell confocal microscopy, a customized Nikon TIE inverted scope outfitted with a Yokogawa spinning-disk scan head (#C$U-X1) along with an Aador iXon™ EM- CCD camera (DU _' 897) wilh MX ⁾ -tns exposure time was used to collect 3D images using an SR Apo TIRF 100X 1.49 oil-immersion objective. Mender’s coefficients were calculated in 3D with JF646 signal (LAMP! -HaioTag) and +36GFP signal {corresponding proteins) using Imaris software with thresholding Zeiss 880 equipped with AirySean™ was also used to obtain high resolution images using a Pkm-Apocthomatic 63x/i .4 oil DIG objective.

For live cell confocal microscopy, Zeiss 880 was used to collect LysoSensor* ^M Yellow Blue signal LysoSensor™ Yellow/Blne was excited with a 405 am laser, and its emission was collected into the two regions (Blue = 410-499 nm Yellow - 500-600 nm) using a Plan~Apocl«omat 63x/1.4 oil DIG objective. The ratio of the two channels was calculated using the home-built software in Matlab™. Visualization and figures

All structural images for figures were generated using PyMOL™(55).

Theoretical modeling ar d fitting to native mass spectrometry data

Python scripts were written to generate theoretical models according Equation 1 , and curve-fitting to native mass spectrometry data (Figures 1, 3, 5) according to Equation 2 by nonlinear least squares usin curve fit from scipy.optimke. The free energy estimates for individual n, m and l layers used in Equation 1 modeling were estimated by solving linear equations as follows; values for the free energy of folding for designs pRQ-2 and variants were estimated from GdmCl denaturation experiments (fig. 1 1); each of these designs have different numbers of «, nr, and l layers, thus series of linear equations relating the number of each layer type to the total free energies of folding were solved to estimate dG values of the individual lay ers of each type. These dG estimates for the individual », m, and I layers were then used in the theoretical modeling (Eq. 1) shown in Figure 3C.

Table 4: X~r»y crystaltography date collection »*nl retlmmmt statistics.

Statistics for the highest-resohition shell are shown in parentheses.

References

i . CL E. Anfnisen, Principles that govern the folding of protein chains. Science. 181, 223- 230 (1973). 2. R-S. Huang, S. E. Boyken, D. Baker, The coming of age of de novo protein design. Nature. 537, 320-327 (2016),

3. G J Rocklin et al, Global analysis of protein folding using massively parallel design, synthesis, and testing. Science. 357, 168-175 (2017),

4. C. Carr, P. S. Kim, A spring-loaded mechanist» lor the conformational change of influenza hemagglutinin. Cell 73, 823-832 (1993).

5. J. M. White, S. E, Delos, M. Brecher, K. Sehomherg, Structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme Crit. Rev. Biockem . Mot Biol. 43, 189-219 (2008).

6. C M. Mair, K Ludwig, A. Herrmat , C. Sieben, Receptor binding and pH stability . how influenza A vims hemagglutinin affects host-specific vims infection. Mo mnica et Biophymea Acta {BBA)~BwmemBmms 1838, 1153-1 168 (2014).

7. M. L. Muxtaugh, S. W. Fanning, T. M. Sharma, A. M Terry, J. SR Horn, A

combinatorial histidine scanning library approach to engineer highly pH-dependent protein switches. Protein Set 2Q, 1619-1631 (201 1 ).

8. E,~M. Strauch, S. J. Fleishman, D. Baker, Computational design of a pH-sensitive IgG binding protein. Proc. Nad. Acad Set U. S. L. I ll , 675-680 (2014).

9. N. Gera. A. B. Hill .D. P. White, R. G. CarhoneiL B. M. Rao, Design of pH sensitive binding proteins from the hyperthermophilic Sso7d scaffold PLoS One . 7, e48928 (2012).

10. M. Dalmau, S. Lim, S.-W. Wang, pH-triggered disassembly in a caged protei complex Biomacromotecuim . IP, 3199-3206 (2009).

11. T. igawa & al. , Antibody recycling by engineered pH-dependent antigen binding

improves the duration of antigen neutralization, Nat Bkmehml. 28, 1203-1207 (2010).

12. K Wada, T. Mizuno, 1-1. Oku, T. Tanaka, pH-mdueed conformational change in a» alpha-helical coiled-coil is controlled by His residues in the hydrophobic core. Protein Pept. led. i0, 27-33 (2003).

13. R. Lizatovic ei al, A De Novo Designed Coiled-Coil Peptide with a Reversible pH- induced Oligomerization Switch. Structur . 24, 46-955 (2016).

14. K. Page! ei al. Random Coils. fi-Sheet Ribbons, and «-Helical Fibers: One Peptide Adopting Three Different Secondary Structures at Will. J. Am. Chem. Soc. 128, 2196- 2197 (2006).

15. C Minelli, J. X blew, M Muthu, H. Andresen, Coiled coil peptide -functionalized surfaces for reversible molecular binding. Soft Matter. 9, 5119-5124 (2013).

16. J Aupib, F. Lapenia, R . Jerala, SwitCCh: Metal-she design for controlling th assembly of a coiled-coii ho odimer. Chmbiochm (2018), dot: 10.1 02/chic.201800578.

17. Y. Zhang ei al. , Computational design and experimental characterization of peptides intended for pH-dependent membrane insertion and pore formation. ACS Chem . Biol 10 1082-1093 (2015).

. S E Boyken et al, De novo design of protein homo-oligomers with modular hydrogen- bond network-mediated specificity. Science. 352 680-687 (2016)

. F. H C. Crick. The Fourier transform of a coiled-coi!. Acta CmtaUogr. 6, 685-689 (1953)

. G. Grigoryan, W, F Degrade, Probing designability vi a generalized model of helical bundle geometry. J. Mol Biol. 405 1079-1 100 (2011).

. A. Leaver-Fay et al, in Methods in Enzymology, M. L. Johnson, L. Brand, Eds.

(Academic Press, 2011 ), voi 487, pp. 545-574

. B. Knhhnan, 0. Baker, Native protein sequences ate close to optimal for their structures Proc. Nail Acad. Sci. (J. S. A. 97, 10383-10388 (2000),

. P.-S. Huang et al. , High thermodynamic stability of parametrically designed helical bundles. Science. 346, 481-485 (20.14)

. S. Mehmood, T. M Allison, C V. Robinson, Mass spectrometry of protein complexes: from origins to applications, Amm. Rev. Phys. Chem. 66, 453-474 (2015)

E Boers Erba, K Baryfyuk, Y Yang, R. Zesiobi, Quantifying protein-protein interactions within noneovaleni complexes using electrospray ionization mass spectrometry. Anal. Chem. 83 9251-9259 (201 1 ).

, N. LeSoup et al , Low pH-induced conformational change and dimerization of sortilin triggers endocylosed ligand release. Nat. Commun. 8, 1708 (2017).

Y.-B. Hu, E. B Dammer, R.-J, Ren, G Wang, The endosoma!- lysosomal system: from acidification and cargo sorting to neurodegeneration. 7’ransl . Neutodegemr. 4, 18 (2015).

. M Grabe, G Osier, Regulation of organelle acidity J. Gen. Physiol 117, 329-344

(2001 ).

. K. N Dyer et al , High-throughput S AXS for the characterisation o f biorao lecu!es in solution: a practical approach. Methods Mol Biol. 1091, 245-258 (2014)

, S Classen et al , Implementation and performance of SIBYLS: a dual endstation small- angle X-ray scattering and acromolecular ctystallography beamline at the Advanced tight Source. J. Appl OystaUogr. 46, 1-13 (2013).

E EiriksdottiiyK . Konale, U Langel, G, Divita, S. Deshayes, Secondary structure of ee!i-penetrating peptides controls membrane interaction and insertion. Mochitn. Bioplm. Acta. 1798, 1 1 19-1128 (2010).

. L. Gut, K. K. Lee, In Influenza Virus: Methods and Protocols, Y. Yamauebi, Ed.

(Springer New York, New York, NY, 2018), pp 261-279. €. S. Pillay, E. Elliot, C. Dennison, Endolysosomal proteolysis and its regulation. Biockcm. J. 363, 417-429 (2002).

M Li et ai, Discovery and characterization of a peptide that enhances endosomal escape of delivered proteins in vitro and in vivo. J. Am. Chem. Soc. 137, 14084-14093 (2015). M S. Lawrence, K. J. Phillips, D. R U«, Supercharging proteins can impart unusual resilience. J. Am. Chem Soc. 129, 10110-101 12 (2007).

1. Monod, X Wyman, l.~P. Change-ox, in Selected Papers in Molecular Biology by Jacques Monad, A. Lwoff, A. Ullmann, Eds. (Academic Press, 1978), pp. 593-623. E. L, Snyder, S. F. Dowdy, Cell penetrating peptides in drug delivery. Phartn. Res. 21 , 389-393 (2004).

Y S Choi, M. Y. Lee, A. E, David, Y. S Park, Nanoparticles for gene delivery;

therapeutic and toxic effects. Molecular & Cellular Toxicolog . 10, 1 -8 (2014).

M Zhou, C Huang, Y. H Wysocki, Surface-induced dissociation of ion mobility- separated noneova!ent complexes in a quadrupoie/fi e-of-flight mass spectrometer.

Arml. Chem. 84, 010-0023 (2012).

R. P. Ra bo, J. A. Tainer, Characterizing flexible and intrinsically unstructured biological macromolecules by SAS «sing the Pored-Dehye law Biopolymers. 95, 559-

571 (2011 ).

D, Schneid.mau~Duhov.ny . M. Ha el, A. Sail, FoXS: a web sewer for rapid computation and fitting Of SAXS profiles; Nucleic Acids Res. 38, W540-4 (2010). D. Schneidman-Duilovny, M. Hainrael, I. A. Tamer, A. Stdi, Accurate SAXS profile computation and its assessment by contrast variation experiments. Biopkys, J. 105, 962-

974 (2013).

W. absefe, XDS, A Crystallogh. IX Biol. Crymlhgr. 66, 125-132 (2010).

A. 3. McCoy m ai, Phaser crystallographic software. J. Appl. Crys ihgr. 40, 658-674 (2007).

P. D. Adams ef at., PHENIX: a comprehensive Python-based system for

macromoiecnlar structur solution, Acta Crystallbgr. I) Mol Crpsiallogr. 66, 213-221 (2010).

T. C. Terwilliger et ai, Iterative model building, structure refinement and density modification with the PHENIX AitioBuild wizard. Acta Crystal lagr. D Biol.

Oyslallogr. 64, 61-69 (2008).

P. Emsley, K. Cowtan, Coot: model-building tools for molecular graphics, Acta

Ctystallogr. b Biol, Crystal lagr. 60, 2126-2132 (2004).

I. W, Davis et ai, MoIProbity: all-atom contacts and structure validation for proteins and nucleic a ds. Nucleic Acids Res. 35, W375-83 (2007). C. Suloway et at , Automated molecular microscopy; the new Lesinon system. J. Struct l. 151, 41 -« (2005).

S Q Zheng &t at, MofionCor2: anisotropic correction of beam-induced motion for improved cryo-eieetron microscopy, Nat Methods. 14. 331-332 (2017).

D. N. astronarde, S. R. Held, Automated lilt series alignment and tomographic reconstruction in IMOD. /, Struct Biol. 197 ₁ 102-113 (2017).

A Rohou, N. Grigorieil CTFPIND4: Fast and accurate defocus estimation from electron micrographs J. Struct Biot 192, 216-221 (2015).

C. A. Schneider, W. S. R&sband, . W. Ehceirl Nil! Image to image) : 25 years of image analysis. Nat Methods. 9, 671-675 (2012)

Z. Dtwu, CL S. Chen, C. L Zhang, D. H. Klaubert, R. P. Haugland, in ABSTRACTS OF PAPERS OF WE AMERICAN CHEMICAL SOCIETY (AMFR CHEMICAL S DC 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 1999), vol. 218, pp. U27-U27, L L. C. Sciinxiinger, The PyMOL molecular graphics system, version 1 7. 6 6 (2015). F. Busch ei at . Localization of protein complex bound ligands by surface-induced dissociation high-resolution mass spectrometry. Anal Chem. (201B ,

doi; 10.1021/aes.analchem.8l>03263.