STATEMENT REGARDING FEDERAL FUNDING

The work described herein was at least partially funded by the United States through National Institutes of Health grant number RO1-HL097106. The United States may have certain rights in the subject matter disclosed.

BACKGROUND

A. FIELD OF THE DISCLOSURE

The present disclosure relates generally to modified proteins. Such proteins as well as assays for use therewith are provided .

B. BACKGROUND

Muscles serve numerous functions in animals. In addition to providing the tension that results in articulated movement of limbs and other physiological features, in mammals muscles are responsible for digestive peristalsis, respiration, dilation and contraction of the iris, and control of contraction and relaxation of blood vessels, to name a few examples.

The most critical function of mammalian muscles is the contraction and relaxation of the components of the heart. Not only must cardiac muscle function without cease throughout the life of the animal, but the portions of the heart must contract and relax in synchrony with one another to effectively pump blood. Even if a subject's cardiac muscles are actively contracting and relaxing, the heart cannot pump blood if they do so arrhythmically.

Muscle cells (myocytes) are composed of contractile fibrils (myofibrils) that are in turn composed of the basic unit of muscle, the sarcomere. The sarcomere is a subcellular structure composed of filamentous proteins that interact to contract or relax the sarcomere. The structure of the sarcomere involves interleaved "thick filaments" (composed of the protein myosin and other proteins) and "thin filaments" (composed of the proteins actin, troponin, and tropomyosin and other proteins). Contraction is triggered by the presence of a threshold amount of calcium in the cell, which causes the thin filament to change in conformation and allow myosin within the thick filament to associate with it. Because calcium binding occurs on the thin filament, understanding how the thin filament changes in response to calcium binding is of particular importance in understanding the functioning of the sarcomere (and by extension, the muscle).

The functioning of the thin filament (or lack thereof) affects numerous muscle-related disorders, perhaps the most notable of which is heart failure. Heart failure affects over 5 million Americans and is a leading cause of mortality in developed countries, and imposes a serious morbidity burden as well. Consequently, great importance is attached to the evaluation of drugs and other factors that can affect heart failure. Evaluating such drugs and other factors can be a time-consuming and expensive task. Animal models of human heart failure may be used, but maintaining captive animal populations is costly. In vitro cellular models of human heart failure allow much greater numbers of organisms (cell cultures) to be exposed to candidate drugs or other factors, but cell culture techniques require specialized equipment and materials, and are labor-intensive to maintain. Consequently there is a need in the art for acellular tools to observe changes in the thin filament in response to calcium binding.

SUMMARY

The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key or critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

Polypeptide derivatives of troponin C (TnC) and troponin I (Tnl) are provided. Such TnC and Tnl polypeptides may be used for constructing an artificial troponin complex. The derivative TnC and Tnl polypeptides, in one embodiment, comprise a mutation or series of mutations that results in a single cysteine residue being present in the polypeptides. A combination of the TnC and Tnl polypeptide derivatives disclosed is also described. Furthermore, a kit comprising a combination of the TnC and Tnl polypeptide derivatives disclosed is also described. Such a kit may be used in the preparation of an artificial troponin complex.

An artificial troponin complex is provided that is useful in the detection of calcium-dependent changes in troponin. A general embodiment of the artificial troponin complex comprises: a donor/acceptor pair of a first and a second chromophore for Forster resonance energy transfer (FRET); a troponin C protein (TnC) conjugated to the first chromophore; and a troponin I protein (Tnl) conjugated to the second chromophore in the C-terminal region. The artificial troponin complex may further comprise additional polypeptide components required for function of the troponin complex. Such additional polypeptides may be natural, wild-type (un-modified) or may be modified with respect to the natural sequence. In one embodiment, the additional polypeptide is troponin T (TnT). The artificial troponin complex can be used in the production of macromolecular components, such as, but not limited to, regulated actin complexes, artificial thin myofilaments and artificial sarcomeres. In one embodiment, the artificial troponin complex comprises TnC, Tnl and TnT. In one embodiment, the artificial troponin complex comprises TnC, Tnl and TnT, wherein at least one of the TnC or Tnl is a modified polypeptide as described herein. In one embodiment, the artificial troponin complex comprises TnC, Tnl and TnT, wherein both of the TnC and Tnl are a modified polypeptide as described herein. A method of screening a candidate factor for modulation of calcium-induced changes in troponin is also provided. A general embodiment of the method comprises: providing a troponin complex comprising a TnC and a Tnl; measuring the distance between the C-terminal region of Tnl to another portion of the troponin complex, such as a site of TnC, in the presence of the candidate factor to obtain a measured distance; and comparing the measured distance to a baseline distance.

The present disclosure also provides for nucleic acid constructs coding for any of the TnC and Tnl poylpetides described herein. The nucleic acid may be RNA, DNA, LNA, PNA, GNA, TNA or any other form of nucleic acid analogue known in the art. The present disclosure also provides for a nucleic acid that is complementary to any of the sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows scanned images of 12% (29:1) SDS-PAGE gels of purified thin filament proteins. The left gel shows molecular weight standards (lane 1) and reconstituted troponin consisting of wild type (w.t.) TnT, Tnl-211 C*ATTO550, and TnC-127C*ATT0655 (lane 2). The right gel shows molecular weight standards (lane 1), native tropomyosin, which is a mixture of a and β isoforms (lane 2), and native filamentous actin (lane 3).

FIG. 2 shows the normalized autocorrelation function of the fluorescence emission from TnC89C*ATTO550 in three contexts: non-associated (dotted line), within the Tn assembly (solid line), and within the regulated actin filament (dashed line). The traces were fit to a model of a single diffusing species (smooth solid lines) to recover the translational correlation times of 80 ns for non-associated TnC, 160 ns for reconstituted troponin, and 23,700 ns for regulated actin filaments. TnC and reconstituted troponin were measured at 500 pM in high salt working buffer (hsWB: 150 mM KCI, 50 mM MOPS pH 7.0, 2 mM EGTA, 5 mM BME, 5 mM MgC ). Regulated actin filaments were measured at 1 nM in working buffer (WB: 75 mM KCI, 50 mM MOPS pH 7.0, 2 mM EGTA, 5 mM BME, 5 mM MgCI ₂ ).

FIG. 3 shows epifluorescence images of surface-deposited regulated actin filaments containing Tnl151 C*Alexa546 (FRET donor) and TnC35C*ATT0655 (FRET acceptor) stained with phalloidin*AlexaFluor488. From left to right, the panels show the non-sensitized emission from donor, acceptor, phalloidin, and an overlay of each.

FIG. 4 shows the domain organization of cardiac TnC and Tnl. a helices and coils/turns are represented, respectively, as boxes and lines. TnC has four sites that may bind Ca ²⁺ or Mg ²⁺ . In cardiac TnC, site I is inactive, site II preferentially binds Ca ²⁺ , and sites III and IV preferentially bind Mg ²⁺ . Sites I and II are within the N-lobe, and sites III and IV are within the C-lobe. Cardiac Tnl contains an isoform specific N-terminal extension (residues 1-33), three helices— helix I (residues 44-80), helix II (residues 91-136), and helix III (residues 152-188)— and a C-terminal region (residues 138 - 211) that contains the inhibitory domain (residues 138-149), the switch I domain (residues 152-160), the mobile domain (residues 162-188) and the switch II domain (residues 189-211). Arrows identify the sites of single Cys mutants that were constructed. Residue numbering is for mouse cardiac Tnl.

FIG. 5 shows a molecular model of a portion of the troponin assembly under Ca ²⁺ -saturating conditions. The molecular model, adapted from Schwartz (Manning, E.P., et. al, Biochemistry 50, 7405-7413 2011), contains the switch II domain and the inhibitory domain of Tnl that are not resolved in the crystal structure of the 52 kDa core of troponin (Takeda, S., et. al, Nature 424, 35-41 , 2003). Residues 202- 276 of TnT, residues 40-211 of Tnl, and residues 1-161 of TnC are shown as ribbon traces in grey, dark grey and light grey, respectively. The Cys atoms of the single Cys mutants used in this study are shown as beads. Cys residues 151-189 (grey) in Tnl were labeled with the FRET donor dye AlexaFluor546. Cys residues 196-211 (light grey) in Tnl were labeled with the FRET donor dye ATTO550. Cys residues in TnC (dark grey) were labeled with the FRET acceptor dye ATT0655.

FIG. 6 shows the steady state emission spectra of regulated actin (50 nM in Tn) containing Tnl211 C*ATTO550 and w.t. TnC (donor-only sample, dashed lines), or regulated actin (50 nM in Tn) containing Tnl211 C*ATTO550 and TnC127C*ATT0655 (donor-acceptor sample, solid lines). Samples were prepared in WB (Mg ²⁺ ), WB supplemented with 1 mM free Ca ²⁺ (Ca ²⁺ ), or in WB supplemented with 1 mM free Ca ²⁺ and 3 M GnHCI (+Ca ²⁺ +GnHCI). The inset provides a magnified view of the emission from ATT0655 (acceptor dye).

FIG. 7 shows the photon counting histograms of ATTO550 (donor dye) in the donor-only sample in WB (D), the donor-only sample in WB supplemented with 1 mM free Ca2 ⁺ (D+Ca ²⁺ ), the donor-acceptor sample in WB (DA), and the donor-acceptor sample in WB supplemented with 1 mM free Ca ²⁺ (DA+Ca ²⁺ ). The data were collected by time correlated single photon counting (TCSPC) with a timing resolution is 16 psec/channel. The data were convolved with the instrument response function (trace descending near channel 150), and fit to a two exponential decay model (smooth black lines). The weighted residuals and goodness of fit (χ ² ) for each fit are shown on the right.

FIG. 8 shows amplitude weighted mean lifetime of the donor dye in the FRET constructs from reconstituted Tn assemblies (500 nM) in hsWB (open) or in hsWB supplemented with 1 mM free Ca ²⁺ (filled). Donor-only samples (squares) and donor-acceptor samples (triangles) are shown. In the donor- acceptor samples the acceptor dye was on TnC at position 35 (left panel), 89 (middle panel), or 127 (right panel).

FIG. 9 shows amplitude weighted mean lifetime of the donor dye in the FRET constructs from regulated actin filaments (50 nM in troponin) in WB (open) or in WB supplemented with 1 mM free Ca2+ (filled). Donor-only samples (squares) and donor-acceptor samples (triangles) are shown. In the donor- acceptor samples the acceptor dye was on TnC at position 35 (left panel), 89 (middle panel), or 127 (right panel).

FIG. 10 shows corrected transfer efficiencies for the FRET constructs. Transfer efficiencies and inter- dye distances calculated from the average lifetime of the donor (FIG. 8 and FIG. 9) are shown for samples without added Ca ²⁺ (open) or with 1 mM free Ca ²⁺ (closed). Samples consisted of Tn assemblies (triangles) and regulated actin filaments (circles) with acceptor dye on TnC at position 35 (left panel), 89 (middle panel), or 127 (right panel).

FIG. 11 shows the calculated inter-dye distances for the FRET constructs. Inter-dye distances calculated from the average lifetime of the donor (FIG. 8 and FIG. 9) are shown for samples without added Ca ²⁺ (open) or with 1 mM free Ca ²⁺ (closed). Samples consisted of Tn assemblies (upright triangles) or regulated actin filaments (circles) with acceptor dye on TnC at position 35 (left panel), 89 (middle panel), or 127 (right panel). The Co Ca distances for the molecular model in FIG. 5 (inverted triangles) are shown. Dashed lines mark the range of distances that are reliably measured (transfer efficiency range of 0.1 - 0.9) by the FRET construct.

FIG. 12 shows a summary of changes in the FRET transfer efficiency. Changes are from Ca ²⁺ -depleted (< 10nM free Ca2+) to Ca ²⁺ -saturating (1 mM free Ca ²⁺ ) conditions. Bars are colored according to the position of the acceptor dye on TnC: 35 (open), 89 (grey), or 127 (black). Data are shown for the Tn assembly (left) and for regulated actin filaments (right).

FIG. 13 shows the relative displacement of the inter-dye distance. Changes are from Ca ²⁺ -depleted (« 10nM free Ca ²⁺ ) to Ca ²⁺ -saturating (1 mM free Ca ²⁺ ) conditions. Bars are colored according to the position of the acceptor dye on TnC: 35 (open), 89 (grey), or 127 (black). Data are shown for the Tn assembly (left) and for regulated actin filaments (right).

FIG. 14 demonstrates the use of a FRET construct to identify agents that modulate the sensitivity of regulated actin filaments to Ca ²⁺ . Regulated actin (1 imL, 20 nM in Tn) containing ΤηΙ21 ΑΤΤΟ550 (FRET donor) and TnC127*ATT0655 (FRET acceptor) in WB were serially diluted with 2 μΙ_ of WB supplemented with 50 mM CaC . The volume-corrected, normalized emission intensity of the donor dye in regulated actin, and regulated actin supplemented with 200 μΜ bepridil, 200 μΜ Levosimendan, or 0.5% (v/v) DMSO drug vehicle solvent are shown. Each trace (dots) was fit (line) to the Hill equation to recover the sensitivity to Ca ²⁺ (pCaso) and apparent cooperativity ΠΗ.

FIG. 15 is a schematic showing the domain organization of cardiac TnC and Tnl. a helices and coils/turns are represented, respectively, as boxes and lines. Sites III and IV of TnC bind Mg ²⁺ constitutively. Site II of TnC binds regulatory Ca ²⁺ . The mobile element of Tnl (ME-Tnl) consists of (from N- to C-) the inhibitory (inh), switch, mobile, and C-terminal (c-tem) domains. To generate the FRET assays, single Cys were positioned at the numbered residues shown on TnC and Tnl. FIG. 16 is a cartoon illustrating the movement of ME-Tnl between actin and the N-lobe of TnC (N-TnC). FIG. 17 is a molecular model of the globular domain of Ca ²⁺ -activated troponin showing ME-Tnl bound to the N-TnC. In an embodiment of the FRET reporter assay, mutant Tnl189C is labeled with Alexa546 (FRET donor), and mutant TnC127C is labeled with ATT0655 (FRET acceptor).

FIG. 18 shows the spectral characterization of FRET-labeled regulated actin filaments. Steady state fluorescence emission spectra of regulated actin filaments (RF) are shown. RF (500 nM in troponin) were prepared from Tn labeled with FRET donor (RF-D, dashed lines) consisting of, Tnl189C*Alexa546, TnC127C, WT TnT or from Tn labeled with FRET donor and FRET acceptor (RF- DA, solid lines) consisting of Tnl189C*Alexa546, TnC127C*ATT0655, WT TnT) in 75 mM KCI, 50 mM MOPS pH 7.0, 5 mM MgCI ₂ , 2 mM EGTA, 5 mM BME. Emission spectra (excitation, 530 nm) are shown for samples prepared in WB (Mg ²⁺ ), WB supplemented with 1 mM free Ca ²⁺ (Ca ²⁺ ), or in WB supplemented with 1 mM free Ca ²⁺ and 3 M GnHCI (Ca ²⁺ +GnHCI). The inset provides a magnified view of the emission from ATT0655 (acceptor dye). Spectra were corrected as described in materials and methods for dilution and for concentration differences between RF-D and RF-DA samples.

FIG. 19 shows epifluorescence images of RF-DA showing aggregated filaments (top row, arrows) reconstituted with a 7:1 :1 mixture of actin:Tm:Tn and normal filaments (bottom row) reconstituted with a 7:5:1 mixture of actin:Tm:Tn. Emission from the FRET donor {excitation, 545/25; emission 605/70), directly excited FRET acceptor {excitation, 620/60; emission 700/75), and the merged images are shown. Scale bar, 5 μιπ

FIG. 20 shows images of RF-D and RF-DA for conditions identical to Fig. 19. Scale bar, 5 μιπ

FIG. 21 shows FCS analysis of incorporation of TnC into Tn and RF. The normalized autocorrelation function of free Alexa546 dye (dye, 500 pM), labeled TnC (troponin C, TnC127C*Alexa546, 500 pM), troponin reconstituted with TnC127C*Alexa546 (troponin, 10 nM), and RF reconstituted with TnC127C*Alexa546 (regulated actin, 10 nM in Tn) are shown. Smooth solid lines represent fits of Alexa546, TnC, and troponin samples to a model of a single diffusing species with translational correlation times 0.03, 0.10, and 0.16 ms, respectively, or fit of regulated actin to a model of two diffusing species with correlation times (fractional amplitudes) of 2.48 (0.44) and 18.23 (0.56) ms.

FIG. 22 shows the sparse incorporation of the FRET donor dye into regulated actin filaments. The RF- DA was prepared from a 1 :5 mixture of troponin containing both FRET donor an FRET acceptor (Tnl189C*Alexa546, TnC127C*ATT0655, WT TnT) to troponin containing FRET acceptor (Tnl189C, TnC127C*ATT0655, WT TnT). RFs have been stained with phalloidin*Alexa488 to visualize F-actin {excitation, 475/35; emission, 550/88). Conditions, all samples in 75 mM KCI, 50 mM MOPS pH 7.0, 5 mM MgCI ₂ , 2 mM EGTA, 5 mM BME. FIG. 23 shows isometric force plotted against pCa (-log[Ca ²⁺ ]f _ree ). Force-pCa measurements of skinned fibers (n = 9) were performed at short (1.9 μιη, filled) and long (2.3 μιη, open) sarcomere lengths (SL) for fibers exchanged with WT Tn (grey) and mutant Tn (black). Mutant Tn was comprised of TnC(C35S, C84S, T127C), Tnl(C81 S, C98I, V189C), and c-myc-lnl. WT Tn was comprised of WT TnC, WT Tnl, and c-myc-TnT. Ca ²⁺ -dependent force from each fiber was fit to the Hill equation to recover Ca ²⁺ - sensitivity (pCaso) and maximum force F _max . Solid lines are drawn using the mean (pCa ₅₀ ) and maximum force (F _max ) from the ensemble of measurements.

FIG. 24 shows that there was no statistically significant difference in Ca ²⁺ -sensitivity between fibers exchanged with mutant Tn compared to fibers exchanged with WT Tn at SL = 1.9 μιη (open bars) or 2.3 μιη (solid bars) (P > 0.05, unpaired i-test). Data are represented as mean ± SEM.

FIG. 25 shows that there was no statistically significant difference in maximum force between fibers exchanged with mutant Tn compared to fibers exchanged with WT Tn at SL = 1.9 μιη (open bars) or 2.3 μιη (solid bars) (P > 0.05, unpaired i-test). Data are represented as mean ± SEM.

FIG. 26 shows pulsed interleaved excitation diffusion single pair FRET measurements of regulated actin with sparse (5%) incorporation of FRET-labeled Tn consisting of Tnl189C*Alexa546, TnC127C*ATT0655, WT TnT (sRF-DA) under Ca ²⁺ -saturated conditions (WB supplemented with 3mM CaC ). Histogram of photon delay times following dye excitation with interleaved pulses (~ 100 ps FWHM) of green (532 nm) and red (638 nm) light. Grey boxes represent the time gating used to identify photons within the DD, DA and AA channels. Data from Ca ²⁺ -saturated regulated actin are shown. FIG. 27 shows burst traces derived from the DD, DA, and AA channels in FIG. 26. The top panel shows burst traces of the DD (black) and DA (grey, shown inverted) channels. Vertical grey lines identify bursts within both donor- and acceptor-filters. The bottom panel shows a burst trace of the AA channel. Vertical grey lines identify bursts within the acceptor-filter.

FIG. 28 shows two-dimensional histograms of FRET efficiency E and donor lifetime (τ) of troponin in freely diffusing sRF-DA under relaxing (WB, apo), Ca ²⁺ -saturated (WB supplemented 3 mM CaC , +Ca), myosin-saturated (WB supplemented with 3.5 μΜ myosin, +S1), or Ca ²⁺ -saturated and myosin- saturated (WB supplemented 3 mM CaCl2 and 3.5 μΜ myosin, +Ca+S1) conditions. Overlays (smooth curved lines) are Eq. 1 with Ei = 0.24, Ei - 0.46, and TD = 4.6 nsec.

FIG. 29 shows histograms of E and global fit to two Gaussians (mean ± SD) with Ei, 0.22 ± 0.17 (smooth bell-shaped curve labeled actin) and Ei, 0.43 ± 0.20 (smooth bell-shaped curve labeled TnC) from samples in FIG. 28. Fractional area of Ei (fi , actin), fractional area of Ei [ , TnC), and mean E (fiEi + fiEi) are reported.

FIG. 30 shows use of pulsed interleaved excitation to select assemblies that contains both FRET donor and acceptor dyes. Two-dimensional E-S histograms were constructed form fluorescence bursts without (left) and with (right) applying the acceptor-selection filter derived from interleaved excitation with the red laser. Data are shown for apo, +Ca, +S1 , and +Ca+S1 samples in FIG. 28-29.

FIG. 31 shows Monte Carlo simulations of the structural dynamics of the mobile element of troponin in the Ca ²⁺ -saturated state. Two-dimensional E-S histograms are shown for simulations of the mobile element of troponin I toggling between actin (si) and N-TnC (S2). Simulations were performed for isomerization rates k _1→2 = k _2→1 = 10, 25, 50, 100, 250, 500, 1000, 2500, and 5000 sec- ¹ . See text for other simulation parameters.

FIG. 32 shows an assessment of sample purity. SDS-PAGE 12% (29:1) of thin filament proteins. Lanes, molecular weight markers (M), troponin composed of TnT, Tnl, and TnC (Tn), tropomyosin with a and β isoforms (Tm), F-actin (Act), and myosin subfragment 1 (S1) composed of the N-terminal proteolytic fragment of myosin heavy chain (MHC) in complex with either essential light chain 1 (ELC1) or ELC2.; pre-spin mixture (W) and pellet (P) of sedimented regulated actin composed of 1 :1 :7 or 1 :5:7 mixture of Tn:Tm:Act (5 μΜ in actin) without added S1 and with added S1 (10 μΜ). Conditions, all samples were in WB.

FIG. 33 shows Perrin analysis of translational correlation times from FCS measurements. Related to FIG. 22. Plot of translational correlation times vs. species molecular weight for free Alexa546 dye (circle), labeled TnC (TnC127C*Alexa546) (X) and troponin reconstituted with TnC127C*Alexa546 (triangle). The solid line represents the fit of the data to a power law, τ = a ^> MW< ^1/3) with a = 0.037 ms/kDa< ^1/3) .

FIG. 34 shows confirmation of single-pair resolution by FCS. The autocorrelation function of the FRET donor in fully dye-labeled RF-DA (triangles) and sparsely labeled RF-DA (circles) is shown. Fully labeled RF-DA with was prepared with FRET dye-labeled troponin (Tn-DA: Tnl189*AF546, TnC127*ATT0655, WT TnT). Sparsely labeled RF-DA was prepared from a 1 :20 a mixture of Tn-DA and unlabeled troponin (Tnl189, TnC127, WT TnT). Smooth black lines represent fits of the autocorrelation function to a model of two diffusing species. For fully labeled RF-DA, the correlation times (fractional amplitude) of 81.61 (0.54) and 4.16 (0.46) ms and a G(0) of 0.50 were recovered. For sparsely labeled RF-DA (sRF-DA), the correlation times (fractional amplitude) of 54.61 (0.47) and 2.48 (0.53) ms and G(0) of 4.35 were recovered. The average number of donor dye molecules in the confocal volume was calculated using (N) = 1/G(0). (N) for fully labeled RF-DA and sparsely labeled RF-DA are 2.00 and 0.23, respectively. Conditions, all samples were in WB at a concentration of 500 nM in Tn.

FIG. 35 shows efficiency of Tn exchange into cardiomyocytes. Related to Figures 23-25. Cardiomyocytes isolated from rat left ventricle were exchanged with Tn. A Western blot of n = 3 fiber exchange experiments using monoclonal anti-TnT JLT-12 is shown, c-myc-tagged TnT within exchanged Tn has increased molecular weight compared to endogenous TnT. Lanes: control (C) non- exchanged myocytes, myocytes exchanged with WT Tn (WT), and myocytes exchanged with mutant Tn (M). WT Tn is comprised of WT TnC, WT Tnl, c-myc-TnT. Mutant Tn is comprised of TnC(C35S, C84S, T127C), Tnl(C81 S, C98I, V189C), and c-myc-TnT. Fractional exchange in WT samples is 0.82, 0.81 , and 0.78, respectively. Fractional exchange in M samples is 0.70, 0.63, and 0.75, respectively. FIG. 36 shows photon counting histograms from RF-DA, Related to FIG. 26. Histograms of burst intensity in DD + DA channels (left) and the AA channel (right). Solid and dashed lines represent, respectively, the lower and upper thresholds used to create the donor- and acceptor-filters.

DETAILED DESCRIPTION

A. DEFINITIONS

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art of this disclosure. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well known functions or constructions may not be described in detail for brevity or clarity

The terms "about" and "approximately" shall generally mean an acceptable degree of error or variation for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error or variation are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. For biological systems, the term "about" refers to an acceptable standard deviation of error, preferably not more than 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

With reference to the use of the word(s) "comprise" or "comprises" or "comprising" in the foregoing description and/or in the following claims, unless the context requires otherwise, those words are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, and that each of those words is to be so interpreted in construing the foregoing description and/or the following claims. In all instances in which these words appear, embodiments of the disclosed subject matter are also contemplated that may "consist of or "consist essentially of the same listed elements, and claims that include such transitional phrases should be considered supported.

Articles such as "the" and "a" are not intended to limit a given element or step to only a single one of its type, and it is to be understood that when reference is made to "an element" or "a step" that more than one such element or step may be present unless specified to the contrary. Likewise, it is to be understood that when reference is made to "the element" or "the step" that more than one such element or step may be present unless specified to the contrary. Such articles should generally be read to refer to "at least one" element or step.

The terms "first" and "second" are used herein to describe various features or elements, but these features or elements should not be limited by these terms. These terms are only used to distinguish one feature or element from another feature or element. Thus, a first feature or element discussed below could be termed a second feature or element, and similarly, a second feature or element discussed below could be termed a first feature or element without departing from the teachings of the present disclosure.

The transitional phrase "consisting essentially of means that, in addition to the recited elements, what is claimed may also contain other elements (steps, structures, ingredients, components, etc.) that do not adversely affect the operability of what is claimed for its intended purpose as stated in this disclosure. Importantly, this term excludes such other elements that adversely affect the operability of what is claimed for its intended purpose as stated in this disclosure, even if such other elements might enhance the operability of what is claimed for some other purpose

B. TROPONIN COMPLEX AND COMPONENTS THEREOF

An artificial troponin complex is provided. The artificial troponin complex is useful for screening candidate agents for the modulation of calcium-induced regulation of the actin myofilament, as well as other purposes. A general embodiment of the troponin complex comprises: a donor/acceptor pair of chromophores; a TnC conjugated to one of the chromophores; and a Tnl conjugated to the other chromophore in the C-terminal region. In one embodiment, the donor and acceptor chromophores are adapted for FRET analysis. In another embodiment, the artificial troponin complex comprises polypeptides other than TnC and Tnl, such as but not limited to TnT. The additional polypeptides may contain additional donor and/or acceptor chromophores or the additional polypeptides may lack such donor and/or acceptor chromophores.

1. Donor/Acceptor Pair

FRET involves the non-radiative transfer of energy from an excited state donor fluorophore to a nearby acceptor. The energy transfer efficiency (E) is directly related to the distance separating a given donor and acceptor pair. The following equation describes the relationship between transfer efficiency and distance:

E = [1 +(r/Ro) ⁶ ]- ¹

The resolution of FRET is thus a function of the "Forster distance" of the chromophores pair (Ro), which is the distance at which E is 50%; and the final distance between the chromophores (r). The Forster distance depends of the extent of overlap between the donor emission spectrum and the acceptor excitation spectrum and several other factors. Given a known r (or a generally predictable range of r) one of ordinary skill in the art can select a donor/acceptor pair with a sufficiently high E to be useful in detecting a change in distance between the donor and acceptor of about r. The necessary E will depend on the FRET method employed, and can be determined by one of ordinary skill. For example, in some embodiments of the artificial troponin complex the donor/acceptor pair will be selected to have an E of at least about 15%. In further embodiments the donor/acceptor pair will be selected to have an E selected from 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99%, about any of the foregoing, or a range between any two of the foregoing.

The donor/acceptor pair of chromophores in the artificial troponin complex may be any known in the art. Some embodiments of the chromophore pair will have an Ro of about 4-9 nm. Further embodiments of the chromophore pair may have Ro selected from: 5-8 nm, 5-7 nm, 6-7 nm, 6 nm, and about any of the foregoing.

Commonly used donors include naphthalene, tryptophan, Alexa dyes (e.g., AlexaFluor 350, 488, 546, 555, 568, 594, 647), ATTO dyes (e.g., ATTO 390, 425, 465, 488, 495, 514, 520, 532, 550, 565, 590, 594, 610, 620, 633, 647, 647N, 655, 680, 700, 725, 740), green fluorescent protein (GFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), rhodamine, fluorescein, Lucifer yellow, dansyl, fluorscein-5-isothiocyanate, and B-phycoerythrin. Commonly used acceptors include dansyl, trinitrophenyl-ATP, octadecylrhodamine, eosin maleimide, carboxymethylindocyanine, tetramethylrhodamine, Alexa dyes, ATTO dyes, and non-fluorescent quenchers (e.g., QSY 35, 7, 9, 21). As is known in the art, fluorescent molecules may serve as both donor and acceptors in FRET and similar assays as all that is required is that the absorption spectra of one of the pair overlaps with the emission spectra of the other member of the pair.

Examples of potentially suitable donor/acceptor pairs include (donor listed first, acceptor listed second): AlexaFluor546 and oxazine dye ATT0655; oxazine dye ATTO550 and oxazine dye ATT0655; AlexaFluor 350 and AlexaFluor 488; AlexaFluor 488 and any one of AlexaFluors 546, 555, 568, 594, and 657; AlexaFluor 546 and any one of AlexaFluors 568, 594, and 647; AlexaFluor 555 and any one of AlexaFluors 594 and 647; AlexaFluor 568 and AlexaFluor 647; fluorescein and tetramethylrhodamin; Cy3 and carboxymethylindocyanine; Cy3 and Cy5, CFP and either one of YFP and GFP; BFP and GFP; GFP and YFP; naphthalene and dansyl; Lucifer yellow and either of trinitrophenyl-ATP and eosin maleimide; dansyl and octadecylrhodamine; fluorscein-5-isothiocyanate and eosin maleimide, and B- phycoerythrin and carboxymethylindocyanine.

2. Troponin C

The described TnC polypeptides may be provided as isolated components, as a component of a kit, as part of an artificial troponin complex or as a part of a larger artificial complex, such as a regulated actin complex. The TnC may be any functional version of the polypeptide. There are two general types of TnC: slow skeletal and cardiac TnC ("cardiac TnC"), and fast skeletal TnC. The TnC will in some embodiments be cardiac TnC; this has the advantage of providing a superior model for modulators of cardiac troponin activity. Cardiac TnC is a 161 residue polypeptide that is highly conserved among mammals and birds; like all TnC, it functions to bind calcium in the thin myofilament. Consequently TnC functions as the allosteric calcium sensor. The binding of calcium to TnC abolishes the inhibitory action of Tnl, thus allowing the interaction of actin with myosin, the hydrolysis of adenosine triphosphate, and the generation of tension.

The cardiac TnC may comprise a canonical structure for cardiac TnC from any of various species. For example, the cardiac TnC may comprise a canonical structure for cardiac TnC from a species in which the entire amino acid sequence has been determined and confirmed, such as Homo sapiens (human), Mus musculus (house mouse), Lagus cuniculus (rabbit), Sus scrofa (pig), Bos taurus (cattle), Gallus gallus (chicken), and Coturnix japonica (Japanese quail). The GenBank accession numbers of each such canonical sequence and SEQ ID NOS in the attached sequence listing are provided in Table 1 , below (all such GenBank sequences are incorporated by reference in their entireties into this application).

Table 1 : Canonical Cardiac TnC Sequences for Selected Species

Species GenBank Accession No. SEQ ID NO

Homo sapiens P63316 1

Mus musculus P19123 2

Lagus cuniculus P02591 3

Sus scrofa P63317 4

Bos taurus P09860 5

Gallus gallus P09860 6

Coturnix japonica P05936 7

Accordingly, some embodiments of the cardiac TnC comprise the sequence of any one of SEQ ID NOS: 1-7 or functional derivatives thereof. A cardiac TnC functional derivative as defined herein refers to a cardiac TnC polypeptide that includes one or more fragments, insertions, deletions or substitutions. The cardiac TnC derivative may have an activity that is comparable to or increased (in one embodiment, 50% or more) as compared to a wild-type cardiac TnC activity and as such may be used to increase a cardiac TnC activity.

A fragment of cardiac TnC is any polypeptide consisting of any number of adjacent amino acid residues having the same identity and order as any segment of cardiac TnC. Conservative modifications to the amino acid sequence of any fragment are also included (conservative substitutions are more fully discussed below). Such fragments can be produced for example by digestion of cardiac TnC with an endoprotease (which will produce two or more fragments) or an exoprotease. Fragments may also be generated artificially. A fragment may be of any length up to the length of cardiac TnC. A fragment may be, for example, at least 145 residues in length.

Derivatives to cardiac TnC may account for known variants of a cardiac TnC polypeptide. The existence of a natural variant with a substitution or deletion at a certain position in a polypeptide evidences that the native residue at that location is not required for proper functioning of the polypeptide. For example, the following are among known human variants of cardiac TnC: A8V, L29Q, C84Y, E134D, D145E, and G159R. One embodiment of the derivative of human cardiac TnC may have any residue in at least one of positions 8, 29, 84, 134, 145, and 159 (SEQ ID NO: 8). A further embodiment of the derivative of human cardiac TnC may have one or more substitutions selected from A8V, L29Q, C84Y, E134D, D145E, and G159R (SEQ ID NO: 8 in which at least one of X ₈ is A, X ₈ is V, X29 is L, X29 is Q, Xs4 is C, Xs4 is Y, X134 is E, X134 is D, X145 is D, X145 is E, X159 is G, and X159 is R). In a yet further embodiment of the derivative of human cardiac TnC, the sequence is SEQ ID NO: 8, wherein: Xs is A or V, X29 is L or Q, Xs4 is C or Y, X134 is E or D, E145 is D or E, and X159 is G or R.

For identifying suitable areas of the molecule that may be changed without destroying activity, one skilled in the art may target areas not believed to be important for activity. For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of a cardiac TnC to such similar polypeptides. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of a cardiac TnC that are not conserved relative to such similar polypeptides would be less likely to adversely affect the biological activity and/or structure of the cardiac TnC. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity (conservative amino acid residue substitutions). Therefore, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

For example, the consensus sequence of canonical sequences of cardiac TnC between H. sapiens, M. musculus, L cuniculus, S. scrofa, B. taurus, G. gallus, and C. japonica is

MDDIYKAAVE QLTEEQKNEF KAAFDIFVLG AEDGCI STKE LGKVMRMLGQ NPTPEELQEM IDEVDEDGSG TVDFDXFLVM MVRCMKDDSK GKXEEELSDL

FRMFDKNADG YIDLXELKXM LQATGETITE DDIEELMKDG XKNNDGRIDY DEFLXFMKGV E (SEQ ID NO: 9).

In one embodiment of the consensus sequence X ₇ 6, X93, X115, X119, Xi4i , and X155 of SEQ ID NO: 9 may each be independently selected from any amino acid. A further embodiment of the consensus sequence is SEQ ID NO: 9, comprising at least one of: E at position 76, Q at position 76, S at position 93, T at position 93, D at position 115, E at position 115, 1 at position 119, M at position 119, D at position 141 , N at position 141 , E at position 155, and Q at position 155. In a yet further embodiment of the consensus sequence, the consensus sequence is SEQ ID NO: 9, wherein: X ₇ 6 is E or Q, X93 is S or T, X115 is D or E, X119 is I or M, X141 is D or N, and X155 is E or Q.

The consensus sequence may further take into account the presence of natural human variants. For example, the consensus sequence of SEQ ID NO: 9 may be broadened to account for the known human variants of cardiac TnC discussed above, as shown below:

MDDIYKAXVE QLTEEQKNEF KAAFDIFVXG AEDGCI STKE LGKVMRMLGQ NPTPEELQEM IDEVDEDGSG TVDFDXFLVM MVRXMKDDSK GKXEEELSDL

FRMFDKNADG YIDLXELKXM LQATGETITE DDIXELMKDG XKNNXGRIDY DEFLXFMKXV E (SEQ ID NO: 10)

In one embodiment of the consensus sequence Xs, X29, X76, Xs4, X93, X115, X119, X134, Xi4i , X145, Xi5i , and X159 of SEQ ID NO: 10 may each be independently selected from any amino acid. A further embodiment of the consensus sequence is SEQ ID NO: 10, in which at least one of: Xs is A, Xs is V, X29 is L, X29 is Q, X ₇ 6 is E, X ₇₆ is Q, X ₈ 4 is C, X ₈ 4 is Y, X93 is S, X ₉₃ is T, Xn ₅ is D, Xn ₅ is E, Xn ₉ is I, Xn ₉ is M, X134 is E, X134 is D, Xi4i is D, X _M i is N, X145 is D, X145 is E, X151 is 151 , X151 is 151 , X159 is G, and X159 is R. A yet further embodiment of the consensus sequence is SEQ ID NO: 10, in which all of: Xs is A or V, X29 is L or Q, X ₇ 6 is E or Q, Xs4 is C or Y, X93 is S or T, X115 is D or E, X119 is I or M, X134 is E or D, Xi4i is D or N, X145 is D or E, X155 is E or Q, and X159 is G or R.

It has been shown that the initiator methionine is often removed in post-translational modification of the polypeptide; therefore some embodiments of the cardiac TnC may comprise a fragment of at least 145 residues from positions 2-161 of any of SEQ ID NOS: 1-10. A specific embodiment of the TnC comprises postions 2-161 of any of SEQ ID NOS: 1-10.

Derivatives of cardiac TnC will have some degree of homology with a given cardiac TnC sequence, such as any of SEQ ID NOS: 1-10. For example, those skilled in the art would expect that most derivatives having from 95-100% homology with native cardiac TnC would retain the function of cardiac TnC. It is also within the abilities of those skilled in the art to predict the likelihood that functionality would be retained by a homolog to cardiac TnC within any one of the following ranges of homology: 75-100%, 80-100%, 85-100%, 90-100%, and 95-100%. Persons having ordinary skill in the art will understand that the minimum desirable homology can be determined in some cases by identifying a known non-functional homolog to cardiac TnC, and establishing that the minimum desirable homology must be above the homology between cardiac TnC and the known non-functional homology. Persons having ordinary skill in the art will also understand that the minimum desirable homology can be determined in some cases by identifying a known functional homolog to cardiac TnC, and establishing that the range of desirable homology must encompass the percent homology between cardiac TnC and the known functional homology.

The deletions, additions and substitutions can be selected, as would be known to one of ordinary skill in the art, to generate a desired cardiac TnC derivative. For example, it is not expected that deletions, additions and substitutions outside of the calcium-binding region of a cardiac TnC would alter a cardiac TnC activity. Likewise conservative substitutions or substitutions of amino acids with similar properties are expected to be tolerated in the calcium-binding region, and a cardiac TnC activity may be conserved. Of course non-conservative substitutions in these regions would be expected to decrease or eliminate a cardiac TnC activity. In addition, specific deletions, insertions and substitutions may impact, positively or negatively, a certain cardiac TnC activity but not impact another cardiac TnC activity.

Conservative modifications to the amino acid sequence of any of SEQ ID NOS: 1-10, including combinations thereof (and the corresponding modifications to the encoding nucleotides) will produce cardiac TnC derivatives having functional and chemical characteristics similar to those of naturally occurring cardiac TnC. In contrast, substantial modifications in the functional and/or chemical characteristics of cardiac TnC may be accomplished by selecting substitutions in the amino acid sequence of any of SEQ ID NOS: 1-10, including combinations thereof, that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the binding site for a binding target, or (c) the bulk of a side chain. The nature of these conservative amino acid substitutions are further described below.

3. Troponin I

The described Tnl polypeptides may be provided as isolated components, as a component of a kit, as part of an artificial troponin complex or as a part of a larger artificial complex, such as a regulated actin complex. Tnl functions to bind to actin in the thin myofilament to hold the actin-tropomyosin complex in place, which prevents the strong binding of myosin to actin in relaxed muscle. Portions of Tnl are dislocated in response to allosteric changes caused by calcium binding to TnC; ultimately this results in the removal of tropomyosin from the binding site for myosin on actin leading to contraction of the sarcomere. There are three isoforms of Tnl: slow-twitch skeletal muscle, fast-twitch skeletal muscle, and cardiac. Some embodiments of the Tnl in the artificial troponin complex are the cardiac isotype of Tnl, which provides a superior model for screening cardiotonic agents.

Human cardiac Tnl comprises a TnC binding region at about positions 43-79 and an actin binding region at about positions 137-148 and about positions 172-180. It has been unexpectedly discovered that a portion of the C-terminal region of Tnl undergoes very significant conformational changes relative to TnC upon calcium activation of the troponin complex when troponin is associated with tropomyosin and filamentous actin as occurs in the native thin filament. The C-terminal region corresponds to about positions 138-211 in mouse, rat, and dog cardiac Tnl (corresponding to about positions 137-210 in human and cat cardiac Tnl, and to about positions 139-212 in bovine cardiac Tnl). The portion of the C-terminal region that appears to undergo the most significant conformational changes is referred to herein as the switch II region and corresponds to about positions 189-211 in mouse, rat, and dog cardiac Tnl (corresponding to about positions 188-210 in human and cat cardiac Tnl, and to about positions 190-212 in bovine cardiac Tnl).

The cardiac Tnl may comprise a canonical structure for cardiac Tnl from any of various species. For example, the cardiac Tnl may comprise a canonical structure for cardiac Tnl from a species in which the entire amino acid sequence has been determined and confirmed, such as Homo sapiens (human), Mus musculus (house mouse), Rattus norvegicus (Norway rat), Canis familiaris (dog), Felis catus (cat), Equus ferus (horse), Xenopus laevis (African clawed frog), Bos taurus (cattle), Gallus gallus (chicken), and Coturnix japonica (Japanese quail). The GenBank accession numbers of each such canonical sequence and SEQ ID NOS in the attached sequence listing are provided in Table 2, below (all such GenBank sequences are incorporated by reference in their entireties into this application).

Table 2: Canonical Cardiac Tnl Sequences for Selected Species

Species GenBank Accession No. SEQ ID NO

Homo sapiens P19429 11

Mus musculus P48787 12

Rattus norvegicus P23693 13

Canis familiaris Q8MKD5 14

Felis catus Q863B6 15

Equus ferus Q5PYI0 16

Xenopus laevis P50754 17

Bos Taurus P08057 18

Gallus gallus P27673 19

Coturnix japonica P27672 20

Accordingly, some embodiments of the cardiac Tnl comprise the sequence of any one of SEQ ID NOS: 11-20 or functional derivatives thereof. A cardiac Tnl functional derivative as defined herein refers to a cardiac Tnl polypeptide that includes one or more fragments, insertions, deletions or substitutions. The cardiac Tnl derivative may have an activity that is comparable to or increased (in one embodiment, 50% or more) as compared to a wild-type cardiac Tnl activity and as such may be used to increase a cardiac Tnl activity. A fragment of cardiac Tnl is any polypeptide consisting of any number of adjacent amino acid residues having the same identity and order as any segment of cardiac Tnl. Conservative modifications to the amino acid sequence of any fragment are also included (conservative substitutions are discussed below). Such fragments can be produced for example by digestion of cardiac Tnl with an endoprotease (which will produce two or more fragments) or an exoprotease. A fragment may be generated by artificial means. A fragment may be of any length up to the length of cardiac Tnl. A fragment may be, for example, at least 186 residues in length.

Derivatives to cardiac Tnl may account for known variants of a cardiac Tnl polypeptide. The existence of a natural variant with a substitution or deletion at a certain position in a polypeptide evidences that the native residue at that location is not required for proper functioning of the polypeptide. For example, the following are among known variants of human cardiac Tnl: A2V, K36Q, P82S, A116G, R141Q, L144Q, R145G, R145W, A157V, R162Q, R162P, A171T, deletion at 177, K178E, N185K, R186Q, D190H, R192H, D196N, R204H, and K206Q (SEQ ID NO. 23). One embodiment of the derivative of human cardiac Tnl may have any residue in at least one of positions 2, 36, 82, 116, 141 , 144, 145, 157, 162, 171 , 177, 178, 185, 186, 190, 192, 196, 204 and 206 of SEQ ID NO: 23. A further embodiment of the derivative of human cardiac Tnl may have one or more substitutions selected from A2V, K36Q, P82S, A116G, R141 Q, L144Q, R145G, R145W, A157V, R162Q, R162P, A171T, deletion at 177, K178E, N185K, R186Q, D190H, R192H, D196N, R204H, and K206Q (SEQ ID NO: 23) in which at least one of X ₂ is A, X ₂ is V, X ₃₆ is K, X ₃₆ is Q, X ₈₂ is P, X ₈₂ is S, Xii6 is A, Xi i6 is G, Xi ₄ i is R, Xi ₄ i is Q, Xi ₄₄ is L, Xi ₄₄ is Q, Xi ₄ 5 is R, Xi ₄ 5 is G, Xi ₄ 5 is W, X157 is A, X157 is V, Xi62 is R, Xi62 is Q, X162 is P, X171 is A, X171 is T, X177 is absent, Xi ₇ s is K, Xi ₇ s is E, Xiss is N, Xiss is K, Xiss is R, X186 is Q, X190 is D, X190 is H, Xig ₂ is R, X192 is H, X196 is D, X196 is N, X ₂₀₄ is R, X ₂ o4 is H, X ₂ o6 is K, and X ₂ o6 Q). In a yet further embodiment of the derivative of human cardiac Tnl, the sequence is SEQ ID NO: 23, wherein: X ₂ is A or V, X ₃₆ is K or Q, X ₈₂ is P or S, Xne is A or G, X _M i is R or Q, X _w is L or Q, X145 is R, G, or W, X157 is A or V, X162 is R, Q, or P, X171 is A or T, X177 is V or is deleted, X178 is K or E, Xiss is N or K, X186 is R or Q, X190 is D or H, X192 is R or H, X196 is D or N, X ₂₀₄ is R or H, and

For identifying suitable areas of the molecule that may be changed without destroying activity, one skilled in the art may target areas not believed to be important for activity. For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequences of a cardiac Tnl to such similar polypeptides. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of a cardiac Tnl that are not conserved relative to such similar polypeptides would be less likely to adversely affect the biological activity and/or structure of the cardiac Tnl. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity (conservative amino acid residue substitutions). Therefore, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

For example, the consensus sequence of canonical sequences of cardiac Tnl between H. sapiens, M. musculus, R. norvegicus, C. familiaris, F. catus, E. ferus, and B. Taurus is shown below:

1 NYRAYATEPH AKKKSKI SAS RKLQLKTLXL QIAKQEXERE AXERRGEKGR 51 XLXTRCQPLX LXGLGFXELQ DLCRQLHARV DKVDEERYDX EAKVTKNITE

101 IADLXQKIXD LRGKFKRPTL RRVRI SADAM MQALLGXRAK EXLDLRAHLK

151 QVKKEDXEKE NREVGDWRKN IDALSGMEGR KKKFEX (SEQ ID NO: 21)

Residue one of the consensus sequence (SEQ ID NO. 21) corresponds to residue 25 of the human sequence (SEQ ID NO. 11) and residue 26 of the mouse sequence (SEQ ID NO. 12).

In one embodiment of the consensus cardiac Tnl sequence, each X may be independently selected from any amino acid. Further embodiments of the consensus cardiac Tnl sequence, each X may be selected from those amino acids found at the corresponding position in one of the canonical sequences shown in Table 3; referring to SEQ ID NO: 21 , such embodiments comprise one or more of M or L at position 29; M or L at position 37; E or V at position 42; V or A at position 51 ; S or R at position 53; V or E at position 60; D or A at position 62; A or E at position 67; V or I at position 90; T or N at position 105; F or Y at position 109; T or A at position 137; S or T at position 142; I or T at position 157; and G or S at position 186.

The consensus sequence may further take into account the presence of natural human variants. For example, the consensus sequence of SEQ ID NO: 21 may be broadened to account of the known human variants of cardiac Tnl discussed above, as shown below:

1 NYRAYATEPH AXKKSKISAS RKLQLKTLXL QIAKQEXERE AXERRGEKGR

51 XLXTRCQXLX LXGLGFXELQ DLCRQLHARV DKVDEERYDX EXKVTKNITE

101 IADLXQKIXD LRGKFKXPTX XRVRI SADAM MQXLLGXXAK EXLDLRXHLK

151 QVKXEDXEKE NREVGXWXKN IXALSGMEGX KXKFEX (SEQ ID NO: 22).

Residue one of the consensus sequence (SEQ ID NO. 22) corresponds to residue 25 of the human variant 1 sequence (SEQ ID NO. 23) and residue 26 of the mouse sequence (SEQ ID NO. 12).

In one embodiment of the consensus sequence that takes into account natural human variants,

Xi2, X58, X92, X117, X120, X121 , X133, Xi38, X147, X154, X161 , Xi62, X166, X168, Xi72, X180, and Xi82 are each independently selected from any amino acid. A further embodiment of the consensus sequence that takes into account natural human variants comprises SEQ ID NO: 22 in which at least one of X12 is K,

X12 is Q, X29 is M, X29 is L, X ₃₇ is M, X ₃₇ is L, X42 is V, X42 is E, X51 is V, X51 is A, X53 is R, X53 is S, X ₅ s is P, X ₅ 8 is S, Xeo is V, X ₆ o is E, X ₆ 2 is D, X ₆ 2 is A, X ₆₇ is E, X ₆₇ is A, X ₉₀ is V, X ₉₀ is I, X ₉₂ is A, X ₉₂ G, X105 is N, X105 is T, X109 is Y, X109 is F, Xn ₇ is R, Xn ₇ is Q, X120 is L, X120 is Q, X121 is R, X121 is G, X121 is W, X133 is A, X133 is V, X137 is T, X137 is A, X138 is R, X138 is Q, X138 is P, X142 is T, Xi ₄ 2 is S, Xi ₄₇ is A, Xi ₄₇ is T, X153 is absent, X154 is K, X154 is E, X157 is I, X157 it T, X161 is N, X161 K, X162 is R, X162 Q, X166 is D, X166 is H, X168 is R, X168 is H, Xi ₇ 2 is D, X172 is N, Xiso is R, Xiso is H, X182 is K, X182 is Q, X186 is G and Xise is S.

Derivatives of cardiac Tnl will have some degree of homology with a given cardiac Tnl sequence, such as any of SEQ ID NOS: 11-23. For example, those skilled in the art would expect that most derivatives having from 95-100% homology with native cardiac Tnl would retain the function of cardiac Tnl. It is also within the abilities of those skilled in the art to predict the likelihood that functionality would be retained by a homolog to cardiac Tnl within any one of the following ranges of homology: 75-100%, 80-100%, 85-100%, 90-100% and 95-100%. Persons having ordinary skill in the art will understand that the minimum desirable homology can be determined in some cases by identifying a known non-functional homolog to cardiac Tnl, and establishing that the minimum desirable homology must be above the homology between cardiac Tnl and the known non-functional homology. Persons having ordinary skill in the art will also understand that the minimum desirable homology can be determined in some cases by identifying a known functional homolog to cardiac Tnl, and establishing that the range of desirable homology must encompass the percent homology between cardiac Tnl and the known functional homology.

The deletions, additions and substitutions can be selected, as would be known to one of ordinary skill in the art, to generate a desired cardiac Tnl derivative. For example, it is not expected that deletions, additions and substitutions outside of the calcium-binding region of a cardiac Tnl would alter a cardiac Tnl activity. Likewise conservative substitutions or substitutions of amino acids with similar properties are expected to be tolerated in the calcium-binding region, and a cardiac Tnl activity may be conserved. Of course non-conservative substitutions in these regions would be expected to decrease or eliminate a cardiac Tnl activity. In addition, specific deletions, insertions and substitutions may impact, positively or negatively, a certain cardiac Tnl activity but not impact another cardiac Tnl activity.

Conservative modifications to the amino acid sequence of any of SEQ ID NOS: 11-23, including combinations thereof (and the corresponding modifications to the encoding nucleotides) will produce cardiac Tnl derivatives having functional and chemical characteristics similar to those of naturally occurring cardiac Tnl. In contrast, substantial modifications in the functional and/or chemical characteristics of cardiac Tnl may be accomplished by selecting substitutions in the amino acid sequence of any of SEQ ID NOS: 11-23, including combinations thereof, that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the binding site for a binding target, or (c) the bulk of a side chain. The nature of these conservative amino acid substitutions are further described below.

4. Modification for Conjugation to a Chromophore

As discussed herein, the TnC and Tnl polypeptides of the present disclosure each contain one of a donor and acceptor chromophore. Any of the Tnl polypeptides disclosed herein may be modified to facilitate conjugation with one of the donor/acceptor chromophore. Any modification known in the art to conjugate or attach a chromophore to a polypeptide may be used. In one embodiment of such an approach, the modification will introduce a reactive group at a location where the chromophore will be attached. In one particular embodiment, the reactive group will generally not be found elsewhere in the molecule to facilitate specificity in the location of the chromophore. Numerous such reactive groups are known in the art. In some embodiments the modification will take the form of substituting a cysteine (C) for another amino acid at the position to which the chromophore is attached. In certain embodiments, the TnC and Tnl polypeptides comprise exactly one cysteine residue. In such embodiments, the cysteine residue may be naturally occurring, the cysteine may be a specifically introduced substitution for another amino acid that occurs in any of the TnC or Tnl sequences described herein, or the cysteine residue may be a specific addition to any of the TnC or Tnl sequences described herein. Furthermore, in such embodiments when the cysteine residue is a specifically introduced substitution for another naturally occurring amino acid or an added residue, any native cysteine residues that are present in the TnC and/or Tnl polypeptide may be deleted or replaced by a substitution with another amino acid. Suitable substitutions are described herein, specifically in Section 5. For example, the cysteine may be substituted with isoleucine, alanine or serine. If multiple cysteine residues are present, the substituting amino acid may be the same or may be different.

Certain embodiments of the TnC polypeptide comprise exactly one cysteine. The cysteine residue may be positioned across the TnC polypeptide. In certain embodiments of the TnC polypeptide, the TnC polypeptide comprises exactly one cysteine, the cysteine being at a position corresponding to one of the following positions in SEQ ID NOS. 1-10: 35, 84, 89, and 127. In certain embodiments of the TnC polypeptide, the TnC polypeptide comprises exactly one cysteine, the cysteine being at a position corresponding to one of the following positions in SEQ ID NOS. 1-10: 35, 89, and 127. In certain embodiments of the TnC polypeptide, the TnC polypeptide comprises exactly one cysteine, the cysteine being at a position corresponding to one of the following positions in SEQ ID NOS. 1-10: 127. In such embodiments, when a cysteine residue is introduced into a TnC polypeptide, such as at positions 89 or 127, the native cysteine residues will be substituted with another amino acid, such as, but not limited to, isoleucine, serine or alanine. In addition, when a native cysteine residue is retained and used as the site of attachment, the additional cysteine residue(s) will be substituted with another amino acid, such as, but not limited to, isoleucine, serine or alanine. Such native cysteine residues are found at positions 35 and 84 of SEQ ID NOS. 1-10.

Therefore, in one embodiment, a suitable TnC sequences for use in the present disclosure includes SEQ ID NOS. 1-10 wherein one of residues 35, 84, 89, and 127 is a cysteine residue and wherein residues 35 is an amino acid other than cysteine (such as but not limited to isoleucine, alanine and serine) when one of residues 84, 89, and 127 is cysteine and residue 84 is an amino acid other than cysteine (such as but not limited to isoleucine, alanine and serine) when one of residues 35, 89, and 127 is cysteine. In certain embodiment, the cysteine residue is at position 35, 89, and 127 of SEQ ID NOS. 1-10 or position 127 of SEQ ID NOS. 1-10.

Certain embodiments of the Tnl polypeptide comprise exactly one cysteine, said exactly one cysteine being in the C-terminal region (CTD). Further embodiments of the Tnl polypeptide comprise exactly one cysteine, said exactly one cysteine being in the CTD. In certain embodiments, the Tnl polypeptide comprises exactly one cysteine, the cysteine being at a position corresponding to one of the following positions in mouse, rat, and dog cardiac Tnl (SEQ ID NOS:12-14, respectively): 81 , 98, 151 , 160, 167, 174, 177, 182, 189, 196, 200, 204, 208, and 211 ; at a position corresponding to one of the following positions in human (SEQ ID NOS: 11 and 23) and cat cardiac Tnl (SEQ ID NO.: 15): 80, 97, 150, 159, 166, 173, 176, 181 , 188, 195, 199, 203, 207, and 210; or at a position corresponding to one of the following positions in the consensus sequences (SEQ ID NOS: 21 and 22): 56, 73, 126, 135, 142, 149, 152, 157, 164, 171 , 175, 179, 183, and 186. In certain embodiments, the Tnl polypeptide comprises exactly one cysteine, the cysteine being at a position corresponding to one of the following positions in mouse, rat, and dog cardiac Tnl (SEQ ID NOS:12-14, respectively): 189, 196, 200, 204, 208, and 211 ; at a position corresponding to one of the following positions in human (SEQ ID NOS: 11 and 23) and cat cardiac Tnl (SEQ ID NO.: 15): 188, 195, 199, 203, 207, and 210; or at a position corresponding to one of the following positions in the consensus sequences (SEQ ID NOS: 21 and 22): 164, 171 , 175, 179, 183, and 186. In certain embodiments of the Tnl polypeptide, the Tnl polypeptide comprises exactly one cysteine, the cysteine being at a position corresponding to one of the following positions in mouse, rat, and dog cardiac Tnl (SEQ ID NOS:12-14, respectively): 210; at a position corresponding to one of the following positions in human (SEQ ID NOS: 11 and 23) and cat cardiac Tnl (SEQ ID NO.: 15): 209; or at a position corresponding to one of the following positions in the consensus sequences (SEQ ID NOS: 21 and 22): 186. In such embodiments, when a cysteine residue is introduced into a Tnl polypeptide, the native cysteine residues will be substituted with another amino acid, such as, but not limited to, isoleucine, serine or alanine. In addition, when a native cysteine residue is retained and used as the site of attachment, the additional cysteine residue(s) will be substituted with another amino acid, such as, but not limited to, isoleucine, serine or alanine. Such native cysteine residues are found at positions 81 and 98 of mouse, rat and dog cardiac Tnl (SEQ ID NOS: 12-14, respectively); at positions 80 and 97 of human (SEQ ID NOS: 11 and 23) and cat cardiac Tnl (SEQ ID NO.: 15); at positions 82 and 99 of bovine cardiac Tnl (SEQ ID NO: 18); at positions 75 and 92 of horse cardiac Tnl (SEQ ID NO:16); at positions 105 and 122 of frog cardiac Tnl (SEQ ID NO: 17); at positions 35 and 52 of chicken cardiac Tnl (SEQ ID NO: 19); and positions 74 and 91 of quail cardiac Tnl (SEQ ID NO: 20) additionally such cysteine residues are found at positions 56 and 73 of the consensus sequences (SEQ ID NOS: 21 and 22).

Therefore, in one embodiment, a suitable Tnl sequences for use in the present disclosure includes SEQ ID NOS. 12-14 wherein one of residues 81 , 98, 151 , 160, 167, 174, 177, 182, 189, 196, 200, 204, 208, and 211 is a cysteine residue and wherein residues 81 is an amino acid other than cysteine (such as but not limited to isoleucine, alanine and serine) when one of residues 98, 151 , 160, 167, 174, 177, 182, 189, 196, 200, 204, 208, and 211 is cysteine and residue 98 is an amino acid other than cysteine (such as but not limited to isoleucine, alanine and serine) when one of residues 81 , 151 , 160, 167, 174, 177, 182, 189, 196, 200, 204, 208, and 211 is cysteine. In certain embodiment, the cysteine residue is at position 189, 196, 200, 204, 208 or 211.

In another embodiment, a suitable Tnl sequences for use in the present disclosure includes SEQ ID NOS. 11 , 23 and 15 wherein at least one of residues 80, 97, 150, 159, 166, 173, 176, 181 , 188, 195, 199, 203, 207, and 210 is a cysteine and wherein residues 80 is an amino acid other than cysteine (such as but not limited to isoleucine, alanine and serine) when one of residues 97, 150, 159, 166, 173, 176, 181 , 188, 195, 199, 203, 207, and 210 is cysteine and residue 97 is an amino acid other than cysteine (such as but not limited to isoleucine, alanine and serine) when one of residues 80, 150, 159, 166, 173, 176, 181 , 188, 195, 199, 203, 207, and 210 is cysteine. In certain embodiment, the cysteine residue is at position 188, 195, 199, 203, 207 or 210.

In another embodiment, a suitable Tnl sequences for use in the present disclosure includes SEQ ID NOS: 21 and 22 wherein at least one of residues 56, 73, 126, 135, 142, 149, 152, 157, 164, 171 , 175, 179, 183, and 186 is a cysteine and wherein residue 56 is an amino acid other than cysteine (such as but not limited to isoleucine, alanine and serine) when one of residues 73, 126, 135, 142, 149, 152, 157, 164, 171 , 175, 179, 183, and 186 is cysteine, and residue 73 is an amino acid other than cysteine (such as but not limited to isoleucine, alanine and serine) when one of residues 56, 126, 135, 142, 149, 152, 157, 164, 171 , 175, 179, 183, and 186 is cysteine. In certain embodiment, the cysteine residue is at position 164, 171 , 175, 179, 183, or 186.

5. Conservative Substitutions

For example, a "conservative amino acid substitution" of TnC or Tnl may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine.

Conservative amino acid substitutions also encompass non-naturally occurring amino acid residues which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics, and other reversed or inverted forms of amino acid moieties. It will be appreciated by those of skill in the art that nucleic acid and polypeptide molecules described herein may be chemically synthesized as well as produced by recombinant means.

Naturally occurring residues may be divided into classes based on common side chain properties: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, He; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.

In making such changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (Kyte et al., J. Mol. Biol., 157:105-131 , 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity.

In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within +/- 2 may be used; in an alternate embodiment, the hydropathic indices are with +/- 1 ; in yet another alternate embodiment, the hydropathic indices are within +/- 0.5.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. The greatest local average hydrophilicity of a polypeptide as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within +/- 2 may be used; in an alternate embodiment, the hydrophilicity values are with +/- 1 ; in yet another alternate embodiment, the hydrophilicity values are within +/- 0.5.

Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the cardiac TnC, or to increase or decrease the affinity of the cardiac TnC with a particular binding target in order to increase or decrease a cardiac TnC activity.

Exemplary amino acid substitutions are set forth in Table 3.

Table 3: Exemplary and Preferred Conservative Substitutions

Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a cardiac TnC that corresponds to amino acid residues that are important for activity or structure in similar polypeptides. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues of cardiac TnC.

One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of that information, one skilled in the art may predict the alignment of amino acid residues of a cardiac TnC with respect to its three dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test cardiac TnC derivatives containing a single amino acid substitution at each desired amino acid residue. The derivatives can then be screened using activity assays known to those skilled in the art and as disclosed herein. Such derivatives could be used to gather information about suitable substitution. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, derivatives with such a change would be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

Numerous scientific publications have been devoted to the prediction of secondary structure from analyses of amino acid sequences (see Chou et al., Biochemistry, 13(2):222-245, 1974; Chou et al., Biochemistry, 113(2):211-222, 1974; Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148, 1978; Chou et al., Ann. Rev. Biochem., 47:251-276, 1979; and Chou et al., Biophys. J., 26:367-384, 1979). Moreover, computer programs are currently available to assist with predicting secondary structure of polypeptides. Examples include those programs based upon the Jameson-Wolf analysis (Jameson et al., Comput. Appl. Biosci., 4(1 ):181 - 86, 1998; and Wolf et al., Comput. Appl. Biosci., 4(1):187-191 ; 1988), the program PepPlot.RTM. (Brutlag et al., CABS, 6:237-245, 1990; and Weinberger et al., Science, 228:740-742, 1985), and other new programs for protein tertiary structure prediction (Fetrow. et al., Biotechnology, 11 :479-483, 1993).

Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural data base (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure (see Holm et al., Nucl. Acid. Res., 27(1):244-247, 1999). Additional methods of predicting secondary structure include "threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87, 1997; Suppl et al., Structure, 4(1):15-9, 1996), "profile analysis" (Bowie et al., Science, 253:164-170, 1991 ; Gribskov et al., Meth. Enzym., 183:146-159, 1990; and Gribskov et al., Proc. Nat. Acad. Sci., 84(13): 4355-4358, 1987), and. "evolutionary linkage" (See Home, supra, and Brenner, supra).

6. Exemplary Troponin Complexes

As discussed herein, artificial troponin complexes are provided. In a general embodiment, the troponin complex described comprise a donor/acceptor pair of chromophores, a TnC conjugated to one of the chromophores and a Tnl conjugated to the other chromophore in the C-terminal region. The troponin complex may further comprise at least one additional polypeptide, such as, but not limited to, TnT, as well as other components required for the functioning of the troponin complex. Any TnC or Tnl polypeptide described herein may be used in such troponin complexes. In one embodiment, the TnC polypeptide has the sequence of any one of SEQ ID NOS. 1-10 and the Tnl polypeptide has the sequence of any one of SEQ ID NOS. 11-23. In another embodiment, the TnC and Tnl polypeptides have any of the sequences described in Section 5 herein. Furthermore, the TnC and Tnl polypeptides may be functional variants of any of the sequences described herein.

In one embodiment, the TnC and Tnl polypeptides are each from the same species. In another embodiment, the TnC and Tnl polypeptides are from a species identified in Tables 1 and 2 herein. In another embodiment, the TnC and Tnl polypeptides are from a mammal, such as a human or mouse. In still another embodiment, the TnC and Tnl polypeptides are from human. In a further embodiment, the TnC and Tnl polypeptides are from mouse.

As discussed herein, each of the TnC and Tnl polypeptides contain one of a donor/acceptor pair. Any of the donors and acceptors described herein may be used. In one embodiment, TnC contains the acceptor and Tnl contains the donor. Furthermore, the locations of the donor/acceptor molecules on TnC and Tnl can encompass any of the locations described herein. In one embodiment the donor/acceptor on the TnC polypeptide is present at any one of positions 35, 84, 89 and 127 with reference to SEQ ID NOS. 1-10 and the donor/acceptor on the Tnl polypeptide is present at any one of: (i) positions 81 , 98, 151 , 160, 167, 174, 177, 182, 189, 196, 200, 204, 208, and 211 in mouse, rat, and dog cardiac Tnl (SEQ ID NOS:12-14, respectively); (ii) positions 80, 97, 150, 159, 166, 173, 176, 181 , 188, 195, 199, 203, 207, and 210 in human (SEQ ID NOS: 11 and 23) and cat cardiac Tnl (SEQ ID NO.: 15); or (iii) positions 56, 73, 126, 135, 142, 149, 152, 157, 164, 171 , 175, 179, 183, and 186 of the consensus sequences of SEQ ID NOS: 21 and 22.

In another embodiment the donor/acceptor on the TnC polypeptide is present at any one of positions 35, 89 and 127 with reference to SEQ ID NOS. 1-10 and the donor/acceptor on the Tnl polypeptide is present at any one of: (i) positions 189, 196, 200, 204, 208, and 211 in mouse, rat, and dog cardiac Tnl (SEQ ID NOS:12-14, respectively); (ii) positions 188, 195, 199, 203, 207, and 210 in human (SEQ ID NOS: 11 and 23) and cat cardiac Tnl (SEQ ID NO.: 15); or (iii) position 164, 171 , 175, 179, 183, and 186 of the consensus sequences of SEQ ID NOS: 21 and 22.

In still another embodiment the donor/acceptor on the TnC polypeptide is present at position

127 with reference to SEQ ID NOS. 1-10 and the donor/acceptor on the Tnl polypeptide is present at any one of: (i) positions 189, 196, 200, 204, 208, and 211 in mouse, rat, and dog cardiac Tnl (SEQ ID NOS:12-14, respectively); (ii) position 188, 195, 199, 203, 207, and 210 in human (SEQ ID NOS: 11 and 23) and cat cardiac Tnl (15); or (iii) positions 164, 171 , 175, 179, 183, and 186 of the consensus sequences of SEQ ID NOS: 21 and 22.

In one embodiment, particular combinations of donor/acceptor positions are used. In one embodiment, the donor/acceptor on TnC is present at position 35 with reference to SEQ ID NOS. 1 , 2 or 9-10 and the donor/acceptor on Tnl is present at positions 200, 204 or 210 with reference to SEQ ID NO. 12. In another embodiment, the donor/acceptor on TnC is present at position 89 with reference to SEQ ID NOS. 1 , 2 or 8-10 and the donor/acceptor on Tnl is present at positions 200, 204, 208 or 211 with reference to SEQ ID NO. 12. In still another embodiment, the donor/acceptor on TnC is present at position 127 with reference to SEQ ID NOS. 1 , 2 or 8-10 and the donor/acceptor on Tnl is present at positions 189, 196, 204 or 211 with reference to SEQ ID NO. 12. In one embodiment, reference is made to the positions with respect to mouse TnC (SEQ ID NO. 2). In another embodiment, reference is made to the positions with respect to human TnC (SEQ ID NO. 1). In another embodiment, reference is made to the positions with respect to human TnC variant 1 (SEQ ID NO. 8). In still another embodiment, reference is made to the positions with respect to the consensus TnC (SEQ ID NOS. 9 and 10).

In a further embodiment, the donor/acceptor on TnC is present at position 89 with reference to SEQ ID NOS. 1 , 2 or 8-10 and the donor/acceptor on Tnl is present at positions 199, 203 or 210 with reference to SEQ ID NO. 11. In another embodiment, the donor/acceptor on TnC is present at position 89 with reference to SEQ ID NOS. 1 , 2 or 8-10 and the donor/acceptor on Tnl is present at positions 199, 203, 207 or 210 with reference to SEQ ID NO. 11. In still another embodiment, the donor/acceptor on TnC is present at position 127 with reference to SEQ ID NOS. 1 , 2 or 8-10 and the donor/acceptor on Tnl is present at positions 188, 195, 203 or 210 with reference to SEQ ID NO. 11. In one embodiment, reference is made to the positions with respect to mouse TnC (SEQ ID NO. 2). In another embodiment, reference is made to the positions with respect to human TnC (SEQ ID NO. 1). In another embodiment, reference is made to the positions with respect to human TnC variant 1 (SEQ ID NO. 8). In still another embodiment, reference is made to the positions with respect to the consensus TnC (SEQ ID NOS. 9 and 10).

In still another embodiment, particular combinations of donor/acceptor positions are used. In one embodiment, the donor/acceptor on TnC is present at position 127 with reference to SEQ ID NOS. 1 , 2 or 8-10 and the donor/acceptor on Tnl is present at positions 175, 179, or 186 with reference to SEQ ID NOS. 21 and 22. In another embodiment, the donor/acceptor on TnC is present at position 89 with reference to SEQ ID NOS. 1 , 2 or 8-10 and the donor/acceptor on Tnl is present at positions 175, 179, 183, or 186with reference to SEQ ID NOS. 21 and 22. In still another embodiment, the donor/acceptor on TnC is present at position 127 with reference to SEQ ID NOS. 1 , 2 or 8-10 and the donor/acceptor on Tnl is present at positions 164, 171 , 179, or 186 with reference to SEQ ID NOS. 21 and 22. In one embodiment, reference is made to the positions with respect to mouse TnC (SEQ ID NO. 2). In another embodiment, reference is made to the positions with respect to human TnC (SEQ ID NO. 1). In another embodiment, reference is made to the positions with respect to human TnC variant 1 (SEQ ID NO. 8). In still another embodiment, reference is made to the positions with respect to the consensus TnC (SEQ ID NOS. 9 and 10).

7. Assemblies Comprising the Troponin Complex

Further macromolecular structures are provided comprising a troponin complex. Such structures can be used to assay for calcium dependent changes in muscle. These include an artificial regulated actin complex comprising any of the artificial troponin complexes described herein, including those described in Section 6. The regulated actin complex also comprises actin. The actin may be a native actin, a variant of a native actin, or a derivative of any of the foregoing. In some embodiments the actin is a native actin from a mammal or bird. In further embodiments the actin is a native actin from a mammalian species, the Tnl is a native Tnl from the same species that has been modified to facilitate conjugation to a chromophore, and the TnC is a native TnC from the same species that has been modified to facilitate conjugation to the other chromophore. In a specific embodiment the actin is mouse actin. In another specific embodiment the actin is human actin.

An artificial thin myofilament and an artificial sarcomere are provided, comprising any of the artificial troponin complexes above. The artificial thin myofilament further comprises a plurality of regulated actin complexes, one or more of which may be the artificial regulated actin complex described above. The artificial sarcomere further comprises a plurality of thin myofilaments and thick myofilaments. Some embodiments of the artificial sarcomere comprise one or more of the artificial thin myofilaments described above.

C. MEASURING CALCIUM-DEPENDENT CHANGES IN TROPONIN A method of measuring calcium-dependent changes in the troponin complex is provided, comprising: providing a troponin complex comprising a TnC and a Tnl, wherein each of the TnC and Tnl components comprise a chromophore; measuring the distance between the CTD of Tnl to another portion of the troponin complex in the presence of the candidate factor to obtain a measured distance; and comparing the measured distance to a baseline distance. The method is useful for example as an assay for calcium-induced regulation of the thin myofilament. Additional embodiments of the method may be used as assays for candidate cardiotonic factors, candidate anti-arrhythmic factors, candidate treatments or preventatives for muscular disorders, candidate treatments or preventatives for cardiac disorders, candidate treatments or preventatives for heart failure, candidate treatments or preventatives for congestive heart failure, and candidate treatments or preventatives for diastolic congestive heart failure.

The term "candidate factor" as used herein refers to a factor to be tested for a certain effect, for example, an effect on the prevention of disease, or an effect on the treatment of disease. The term may be narrowed to specify the effect to be tested, for example "candidate cardiotonic factor." The candidate factor may be any type of factor, including a physical factor, a chemical factor, a genetic factor, a biochemical factor, or an ecological factor. Examples of physical factors include temperature, oxidation potential, pressure, and radiation. Examples of chemical factors include osmotic potential, concentration of inorganic compounds, organic compounds, H, and salinity; either of the inorganic or organic compounds may be candidate drugs. Examples of biochemical factors include a nutrient concentration, a prion, and a toxin concentration. Examples of ecological factors include population density, intrinsic rate of growth, carrying capacity, predation, parasitism, and growth rate. Examples of genetic factors include a genotype, a mutation, an episome, a transposable genetic element, a virus, and a viroid. However, as the purpose of the assay is to identify putative agents for modulation of calcium-induced regulation of the thin myofilament that were previously unknown, it should be emphasized that the candidate factor may be any factor to which the troponin complex can be subjected.

In some embodiments of the method the artificial troponin complex may be any of the artificial troponin complexes described in the previous section.

The method may further comprise performing the measurement in the presence of an amount of calcium ions sufficient to cause structural changes in the troponin complex when the agent is absent.

The baseline value may be determined by any of various means understood in the art. The baseline may be the observed distance in the artificial troponin complex in the experimental system prior to the introduction of the candidate factor. It may also be the observed distance in the troponin complex established by prior measurements. In some embodiments of the method the baseline is calculated by modeling the structure of the troponin complex.

D. KITS

The present disclosure further provides kits. Kits include one or more compositions useful for the creation of an artificial troponin complex, in suitable packaging. In some embodiments kits provide at least one of a Tnl polypeptide and a TnC polypeptide, wherein each polypeptide provided is adapted to receive a chromophore. Any TnC and Tnl polypeptide described herein may be used as a component of the kit. In one embodiment, the Tnl and TnC polypeptides described in Section B6 are used. In one embodiment, the kit comprises both a Tnl and TnC polypeptide.

The Tnl and/or TnC polypeptides may be provided with or without an attached chromophore.

When the Tnl and/or TnC polypeptides are provided with a chromophore, the chromophore can be selected from any such molecules known in the art, including those described herein. When the Tnl and/or TnC polypeptides are provided without a chromophore the chromophore can be selected from any such molecules known in the art, including those described herein, and may be optionally provided as a component of the kit.

Such kits may further comprise additional components to create an artificial troponin complex. Such components include, but are not limited to, TnT, actin and tropomyosin (Tm). Any form of the foregoing may be provided, including those forms described herein.

The kits may additionally include reagents useful in the methods described, such as, but not limited to, buffers and other reagents, washes, buffers or other reagents for preconditioning the instrument on which assays will be run and control material. Kits may include one or more standards, e.g., standards for use in the assays of the invention. Kits may further include instructions.

E. NUCLEIC ACID CONSTRUCTS

The present disclosure also provides for nucleic acid constructs coding for the TnC and Tnl poylpetides described herein. In one embodiment, the nucleic acid constructs code for a polypeptide having the sequence of any one of SEQ ID NOS. 1-10 for TnC, provided that such constructs code for a polypeptide containing a single cysteine residue or any one of SEQ ID NOS. 11-23 for Tnl, provided that such constructs code for a polypeptide containing a single cysteine residue. The nucleic acid may be RNA, DNA, LNA, PNA, GNA, TNA or any other form of nucleic acid analogue known in the art. The present disclosure also provides for a nucleic acid that is complementary to any of the foregoing.

F. EXAMPLES

Working Example 1

Materials and Methods

Purification of native proteins Actin and tropomyosin (Tm) were purified as described (Frederiksen DW, Cunningham LW. Methods in Enzymology. Pardee, JA. Purification of muscle actin. Academic Press, New York London. 1982; 85: 161-184) from acetone powder derived from the left ventricles of bovine hearts. Tm consisting of a native mixture (9:1) of α:β isoforms was aliquated, lyophilized and stored at -80°C. Filamentous actin was stored at 4°C in 50 mM KCI, 2 mM Tris-HCI pH 8.0, 1 mM Na ₂ ATP, 0.2 mM CaCI ₂ , 2 mM MgC , 0.005% NaN3. Myosin was purified from chicken pectoralis muscle as described (Margossian and Lowey (1982), Methods Enzvmol 85 Pt B:55-71), suspended in 600 mM NaCI, 10 mM Na ₃ P0 ₄ pH 7.0, 1 mM EDTA, 1 mM DTT, diluted to 50% glycerol and stored at -20°C for up to one year.

Preparation of recombinant proteins

Recombinant wild type (WT) chicken skeletal troponin C (TnC), WT mouse cardiac troponin I

(Tnl), and WT adult rat cardiac TnT cDNAs were sub-cloned into pET3a (Novagen) plasmids for expression. The single-Cys mutant TnC(C84S), abbreviated TnC35C, and the Cys-less mutants TnC(C35S, C84S) and Tnl(C81 S, C98I) were constructed by QuikChange Lightning Site-Directed Mutagenesis (Agilent). Cys-less TnC was used as a template to construct the single-Cys mutants TnC(C35S, C84S, S89C) and TnC(C35S, C84S, T127C), abbreviated TnC89C and TnC127C, respectively. Cys-less Tnl was used as a template to construct single-Cys mutants Tnl(C81 S, C98I, XXX), where XXX represents the following: S151 C, L160C, S167C, L174C, V177C, I182C, V189C, I196C, S200C, G204C, K208C, and G211 C, abbreviated TnlXC, where X represents the mutated residue. Single Cys-containing and w.t. TnC and Tnl, and w.t. TnT were expressed in E. coli and purified as described (Robinson, J. M., et al. (2004), J Mol Biol 340(2): 295-305). Purity was analyzed with SDS-PAGE, and protein molecular weights were verified by MALDI-TOF MS. Proteins were lyophilized and stored at -80°C.

Preparation of dye labeled proteins

Cys residues of proteins were selectively labeled with maleimide fluorophores. Cys residues were reduced with 5 mM DTT, with subsequent removal of DTT with three steps of dialysis against deoxygenated LB (LB: 50 mM MOPS pH 7.2, 3 M urea, 100 mM KCI, 1 mM EDTA). Reduced proteins were reacted with a 5-fold excess of dye molecule for 12 hr at 4°C or 2 hr at RT under nitrogen with stirring. Labeling was terminated with the addition of 10 mM DTT. Unreacted dye molecules were removed by size-exclusion chromatography (Sephacryl S-100 HR, AKTAprime Plus, GE Life Sciences) in LB. The labeling procedure was repeated up to 3 times to increase the labeling efficiency. The labeling efficiency was determined by comparing the ratio of dye to protein concentration, with protein concentrations determined by Bradford assay. The dye concentrations were determined by absorption spectroscopy using the following molar extinction coefficients: AlexaFluor546 (abbreviated AF546) (Life Technologies) 104,000 at 555 nm; ATTO550 (ATTO-Tec GmbH) 120,000 at 554 nm; ATT0655 (ATTO- Tec GmbH) 125,000 at 663 nm. The following dye-labeled proteins were prepared: TnC35C*ATT0655, TnC89C*ATT0655, TnC127C*ATT0655, TnC89C*ATTO550, Tnl151 C*AF546, Tnl160C*AF546, Tnl167C*AF546, Tnl174C*AF546, Tnl177C*AF546, Tnl182C*AF546, Tnl189C*AF546, Tnl196C*AF546, Tnl200C*ATTO550, Tnl204C*ATTO550, Tnl208C*ATTO550, Tnl211 C*ATTO550. Aliquots of the labeled proteins were at t -80°C for up to 1 year.

Preparation of reconstituted troponin

Tn was prepared by incubating TnC (10 μΜ), Tnl (12 μΜ) and TnT (14 μΜ) on ice for 1 hr, then dialyzed stepwise into high salt working buffer (hsWB: 150 imM KCI, 50 mM MOPS pH 7.0, 5 mM MgCI ₂ , 2 mM EGTA, 5 mM BME) as described (Robinson, J. M., et al. (2004), J Mol Biol 340(2): 295- 305). Uncomplexed Tnl and TnT precipitate in hsWB, and were removed by centrifugation at 10,000x g for 1 min. Aliquots of Tn were stored at -80°C for up to 1 year.

Preparation of regulated actin filaments

Regulated actin filaments were prepared by incubating filamentous actin (7uM), Tn (1 μΜ) and Tm (1 μΜ) in 75 mM KCI, 50 mM MOPS pH 7.0, 5 mM MgCI ₂ , 2 mM EGTA, 5 mM BME on ice for 1 hr. Regulated actin filaments were stored at 4°C for up to 7 months.

Imaging of regulated actin filaments

Regulated actin filaments were stained with phalloidin*AlexaFluor488 (Alexa488, Life Technologies) at a 20:1 molar ratio of g-actin:phalloidin, diluted to 10 nM in WB and deposited on aminosilanized glass coverslips. Fluorescence images were collected on an inverted microscope (1X71 , Olympus ) with TE cooled interline CCD camera (Clara, Andor) using a 100x (N.A. 1.4) oil immersion objective (UPlanSApo, Olympus) with excitation using a xenon lamp (X-Cite 120PC, Lumen Dynamics). For Alexa488, the filters (excitation: dichroic: emission) 475/35: 495: 550/88 (Semrock) were used. For Alexa546 and ATTO550, the filters 545/25: 565: 605/70 (Chroma) were used. For ATT0655, the filters 620/60: 660: 700/75 (Chroma) were used.

Time-resolved fluorescence spectroscopy

Time correlated single photon counting measurements were performed at room temperature (18 ± 2°C) using a MicroTime 200 confocal fluorescence lifetime microscope (PicoQuant, GmbH; Berlin, Germany) based on an inverted microscope (1X71 , Olympus). Excitation light from 532 nm pulsed diode laser (LDH-P-FA-530-B, PicoQuant) was passed, respectively, through a quarter wave plate, a single-mode fiber optic, laser clean-up filter (534/635-25, Semrock), principle dichroic mirror (DC1) (ZT532/638rpc, Chroma), and 100x (N.A. 1.3) oil immersion objective (UPlanFLN, Olympus). Emitted light passed through the objective and DC1 , then through a 550 nm long-pass filter (HQ550lp, Chroma), 50 μιη pinhole, a secondary dichroic (ZT532/638PC, Chroma), a bandpass filter (HQ580/70, Chroma) and recorded an avalanche photodiode (MPD PDM series Micro Photon Devices, Italy), respectively. Timing resolution was set to 16 psec. per channel. Laser power was maintained at approximately 50μ\Λ/. Data was collected until the maximum count per channel exceeded 10,000 (typically 10 minutes). Background intensity averaged 80 counts per second.

Fluorescence correlation spectroscopy

FCS measurements were performed in working buffer (WB: 75 mM KCI, 50 mM MOPS pH 7.0,

2 mM EGTA, 5 mM MgCI ₂ , 5 mM BME) or high salt working buffer (hsWB: 150 mM KCI, 50 mM MOPS pH 7.0, 2 mM EGTA, 5 mM MgCI ₂ 5 mM BME). Freely diffusing TnC89C*ATTO550 (500 pM in hsWB), troponin containing TnC89C*ATTO550 (500 pM in hsWB), and regulated actin filaments containing TnC89C*ATTO550 (10 nM of Tn in WB) were examined in the time resolved confocal microscope described above with a 0.4 fL excitation volume positioned 50 urn above the top surface of the glass coverslip. Excitation was from the pulsed 532 nm laser attenuated to 50 uW. Photon arrival times were collected for approximately 10 minutes then analyzed by calculating the autocorrelation function

(F(t)F(t+At-))

G (t) =

<F(t)> ² which compares fluorescence intensity F at time t, with the intensity at t+At,. The data were fit to a model of a single diffusing species to recover the translational correlation time and translational diffusion coefficient of the freely diffusing species.

Time-resolved FRET analysis

Using SymphoTime software, the photon counting histograms of TCSPC data were convolved with the instrument response function and fit to a two-exponential decay model /(t) = a ₁ e ^~t/Tl + a ₂ e ^~t/T2 , where l(t) is the intensity of the donor as a function of time t, and a, is the fraction of fluorophores with lifetime τ,. Amplitude-weighted average lifetimes (mean lifetime) (τ) =∑ _£ α _£ τ _£ /∑ _£ α _£ of the donor in the presence of the acceptor dye (T _DA ) and the donor in the absence of the acceptor dye (T _D ), were used to obtain the transfer efficiency

where f _A is the labeling efficiency with the acceptor dye. The distance R between the donor dye and acceptor dye is given by the Forster relation

where Ro is the Forster distance of the donor-acceptor dye pair.

Calcium titrations

Steady-state measurements were performed at 22.0°C on a Fluorolog-3 spectrofluorometer (Horiba) equipped with a MicroLab500 syringe dispenser (Hamilton). Regulated actin filaments containing Tnl21 1 C*ATTO550 (FRET donor) and TnC127C*ATT0655 (FRET acceptor) were prepared in WB (20 nM in 1 imL). Fluorescence emission from the FRET donor was monitored (570 nm emission, 550 nm excitation, 5 nm slit width) following 100 serial injections (2 uL) with WB supplemented with 50 mM CaCl2. Using Prism (GraphPad), the dilution-corrected fluorescence intensities were fit to the Hill equation

„ _ „ Fmax ^~ Fmin

fmin + (1 _{+ 10} η(ρεα ₅₀ -ρε _Ά )γ

where n is the Hill coefficient, pCa = - \og[Ca ^{2 +} ] , and pCaso is the negative log of the apparent dissociation constant. The free Ca ²⁺ concentrations were obtained as follows.

Description

A FRET assay with 12 individual donor positions on Tnl (positions 151 , 160, 167, 174, 177,

182, 189, 196, 200, 204, 208, and 21 1 with respect to SEQ ID NO. 12), and 3 individual acceptor positions on TnC (positions 35, 89 and 127 with respect to SEQ ID NO. 2), was constructed in order to measure conformational sub-states of the Tn complex within the thin filament under Mg ² - and Ca ²⁺ saturating conditions using time-correlated single photon counting (TCSPC) to determine donor lifetimes in the presence and absence of acceptor. Combinations of individual donor and acceptor positions were analyzed. The donor positions span the switch I rgion, the mobile region, and the switch II region of Tnl. The acceptors are positioned in Site I, the loop between the D and E helices, and the loop between helices F and G of TnC.

Experimental Design

A design was conceived and implemented to measure the Ca2+-induced internal rearrangements within the troponin assembly. Recombinant proteins were prepared as described in the Methods section. SDS page of the recombinant proteins used to construct the troponin complex are shown in FIG. 1 . The left gel shows molecular weight standards (lane 1 ) and reconstituted troponin consisting of wild type (w.t.) TnT, Tnl-21 1 C*ATTO550, and TnC-127C*ATT0655 (lane 2). The right gel shows molecular weight standards (lane 1), native tropomyosin, which is a mixture of a and β isoforms (lane 2), and native filamentous actin (lane 3).

Tnl was labeled with Alexa546 at positions 151 (Tn-l151 C*-Alexa546), 160 (Tnl-160*C- Alexa546), 167 (Tnl-167C*-Alexa546), 174 (Tnl-174C*-Alexa546), 177 (Tnl-177C*-Alexa546), 182 (Tnl- 182C*-Alexa546), or 189 (Tnl-189C*-Alexa546). Tnl was labeled with ATTO550 at positions 196 (Tnl- 196C*-ATTO550), 200 (Tnl-200C*-ATTO550), 204 (Tnl-204C*-ATTO550), 208 (Tnl-208C*-ATTO550), or 21 1 (Tnl-21 1 C*-ATTO550). TnC was labeled with ATT0655 at positions 35 (TnC-35C*-ATT0655), 89 (TnC-89C*-ATT0655) or 127 (TnC-127C*-ATT0655). Tn assemblies were prepared as described in the methods section. Both donor labeled only Tn (Tn-D) and donor and acceptor labeled Tn (Tn-DA) were prepared. Tn-D was prepared with donor labeled Tnl, unlabeled TnC and wild type TnT. Tn-DA was prepared with donor labeled Tnl, acceptor labeled TnC and wild type TnT. Regulated actin filaments were prepared as described in the methods section from Tn-D and Tn-DA constructs.

Steady state fluorescence and time-resolved fluorescence data were collected. Representative data are shown in FIGS. 6-7. FIG. 6 shows the steady state emission spectra of regulated actin filaments (50 nM in Tn) containing Tnl211 C*ATTO550 and w.t. TnC (donor-only sample, dashed lines), or regulated actin (50 nM in Tn) containing Tnl211 C*ATTO550 and TnC127C*ATT0655 (donor- acceptor sample, solid lines). Samples were prepared in WB (Mg2+), WB supplemented with 1 mM free Ca2+ (Ca2+), or in WB supplemented with 1 mM free Ca2+ and 3 M GnHCI (Ca2+ +GnHCI). The inset provides a magnified view of the emission from ATT0655 (acceptor dye). FIG. 7 shows the photon counting histograms of ATTO550 (donor dye) in the donor-only sample in WB (D), the donor-only sample in WB supplemented with 1 mM free Ca2 ⁺ (D +Ca ²⁺ ), the donor-acceptor sample in WB (DA), and the donor-acceptor sample in WB supplemented with 1 mM free Ca2 ⁺ (DA +Ca ²⁺ ). The data was collected by time correlated single photon counting (TCSPC) with a timing resolution is 16 psec/channel. The data were convolved with the instrument response function (trace decending near channel 150), and fit to a two exponential decay model (smooth black lines). The weighted residuals and goodness of fit (χ ² ) for each fit are shown on the right.

For each construct, the amplitude-weighed mean lifetime was calculated from the fitted lifetimes. FIGS. 8-9 show amplitude weighted mean lifetime profiles of the donor dye in the FRET constructs. FIG. 8 shows data from reconstituted Tn assemblies (500 nM) in hsWB (open) or in hsWB supplemented with 1 mM free Ca ²⁺ (filled). FIG. 9 shows data from regulated actin filaments (50 nM in troponin) in WB (open) or in WB supplemented with 1 mM free Ca ²⁺ (filled). Regulated actin was prepared as described in the Methods section. The Tnl and TnC constructs used were as described above.

For each FRET construct, the transfer efficiency (TE) was calculated from the mean lifetime of the donor-only and donor-acceptor sample (FIG. 4). For each FRET construct, inter-dye distance (IDD) was calculated from the TE, assuming a Forster critical distance Ro of 5.5 nm for the dye pair Alexa546/ATT0655 and an R ₀ of 6.4 nm for the dye pair ATTO550/ATTO655.

FIG. 10 shows the TE and IDD for samples without added Ca ²⁺ (open) or with 1 mM free Ca ²⁺

(closed). Samples consisted of Tn assemblies (triangles) and regulated actin filaments (circles) with acceptor dye on TnC at position 35, 89 , or 127. Tn assemblies and regulated actin filaments were prepared as described in the Methods section. FIG. 10 shows the TE for each of the constructs described under Ca ²⁺ -depleted (< 1 nM free Ca ²⁺ ) and Ca ²⁺ -saturating (1 mM free Ca ²⁺ ) conditions. FIG. 11 shows the IDD for each of the constructs described.

FIG. 6 shows a summary of Ca ²⁺ -induced changes the FRET transfer efficiency and the inter- dye distance. The constructs used were those described above for FIG. 5. Changes are from Ca ²⁺ - depleted (< 1 nM free Ca ²⁺ ) to Ca ²⁺ -saturating (1 mM free Ca ²⁺ ) conditions. Bars are shaded according to the position of the acceptor dye on TnC: 35. Figure 12 shows the Ca ²⁺ -induced change in the FRET transfer efficiency. FIG 13 shows the relative displacement of the inter-dye distance. Data are shown for the Tn assembly (left) and for regulated actin filaments (right). As can be seen from FIG. 13, Ca ²⁺ - induced structural changes in Tn differ when Tn is incorporated into regulated actin filaments. The most pronounced Ca2+-induced changes in IDD are observed between the switch II region of Tnl and residue 127 within the C-lobe of TnC. The IDD within regulated actin are taken as representative of the operation of Tn under native conditions.

FIG. 7 shows the use of a FRET construct to identify agents that modulate the sensitivity of regulated actin filaments to Ca ²⁺ . Regulated actin filaments (1 imL, 20 nM) containing ΤηΙ-21 ΑΤΤΟ550 (FRET donor) and TnC-127*ATT0655 (FRET acceptor) in WB were serially diluted with 2 \il of WB supplemented with 50 mM CaCl2. FIG. 14 shows the volume-corrected, normalized emission intensity of the donor dye in regulated actin (red), and regulated actin supplemented with 200 μΜ bepridil, 200 μΜ Levosimendan, or 0.5% (v/v) DMSO drug vehicle solvent. Each trace (dots) was fit (line) to the Hill equation to recover the sensitivity to Ca ²⁺ (pCaso) and apparent cooperativity n. Table 4 shows the recovered parameters (mean ± standard deviation). As can be seen from FIG. 7, use of the constructs described herein can be used to identify agents that modulate the sensitivity of regulated actin filaments to Ca ²⁺ .

Table 4

Sampie pCa _5Q n _H

regulated actin 5.19 ± 0.04 1.58 ± 0.19

+ DMSO 5.20 ± 0.04 1.85 ± 0.30

+ bepridil 5.43 ± 0.05 1 .63 ± 0.30

+ Levosimendin 5.14 ± 0.05 1.97 ± 0.37

Working Example 2

Materials and Methods

Purification of native proteins

Actin (27) and the tropomyosin dimer (28) (Tm) were purified as described from acetone powder derived from the left ventricles of bovine hearts. Bovine Tm consists of a 9:1 mixture of α:β isoforms that approximates the isoform mixture within the non-failing human left ventricle, Tm was aliquoted, lyophilized and stored at -80°C. Filamentous actin (F-actin) was stored at 4°C in 50 mM KCI, 2 mM Tris-HCI pH 8.0, 1 mM Na ₂ ATP, 0.2 mM CaCI ₂ , 2 mM MgCI ₂ , 0.005% NaN ₃ . Fast skeletal myosin was purified from chicken pectoralis muscle as described (29), suspended in 600 mM NaCI, 10 mM Na ₃ P0 ₄ pH 7.0, 1 mM EDTA, 1 mM DTT, 50% glycerol, and stored at -20°C for up to one year.

Preparation of myosin subfragment-1

Myosin S1 was prepared from chicken pectoralis major muscle as described (12) with some modifications. Myosin was dialyzed against 20 mM MOPS (pH 7.0), 50 mM KCI, 1 mM EDTA and digested with 1 :100 (w/w) of chymotrypsin to myosin for 10 minutes on ice. The digestion was terminated with 5 mM phenylmethylsulfonyl fluoride (PMSF). 5 mM Mg-ATP was added, and insoluble components were pelleted by centrifugation at 180,000 x g for 15 min. Soluble S1 in the supernatant was dialyzed into WB and stored at 4°C for up to 1 week. Catalytically inactive S1 (dead heads) were removed by ultracentrifugation at 64,000 rpm for 15 minutes (Beckman TLA-100 rotor) in the presence of 5 mM ATP and F-actin at a molar ratio of 1 S1 : 2 actin. ATP was removed using dialysis against working buffer (WB: 75 mM KCI, 50 mM MOPS pH 7.0, 5 mM MgCI ₂ , 2 mM EGTA, 5 mM BME). S1 was used within 24 hours of dead head removal.

Cloning, mutagenesis, recombinant protein expression, and purification

The cDNAs from wild type (WT) rat cardiac troponin C (TnC) (30) whose gene product is identical to mouse cardiac TnC, WT mouse cardiac troponin I (Tnl) (31), and WT adult rat cardiac TnT (12) were sub-cloned into pET3a (Novagen) plasmids for expression, c-myc-tagged mouse cardiac TnT in pSBETa (32), was used without modification. The single-Cys mutant TnC(C84S), abbreviated TnC35C, and the Cys-lite mutants TnC(C35S/C84S) and Tnl(C81 S/C98l) were constructed by QuikChange Lightning Site-Directed Mutagenesis (Agilent). Cys-lite TnC was used as a template to construct the single-Cys mutants TnC(C35S/C84S/S89C) and TnC(C35S/C84S/T127C), abbreviated TnC89C and TnC127C, respectively. Cys-less Tnl was used as a template to construct single-Cys mutants Tnl(C81 S/C98l/X), where X represents the following: S151 C, L160C, S167C, L174C, V177C, I182C, V189C, I196C, S200C, G204C, K208C, and G211 C, abbreviated Tnl(X), where X represents the mutated residue. Single Cys-containing and WT TnC and Tnl, and WT TnT were expressed in E. coli and purified as described (12). Proteins were analyzed for purity by SDS-PAGE, and their molecular weights were verified by ESI-MS. All proteins were lyophilized and stored at -80°C.

Dye labeling of proteins

Cys residues of proteins were selectively labeled with maleimide-containing fluorescent dye molecules. Cys residues were reduced by dialysis against labeling buffer (LB: 50 mM MOPS pH 7.2, 3M urea, 100 mM KCI, 1 mM EDTA) containing 5 mM DTT. The DTT was removed 3 steps of dialysis against LB. Reduced proteins (100 μΜ) were reacted with a 5-fold excess of dye molecule for 12 hr at 4 °C under nitrogen with stirring. Labeling was terminated by the addition of 10 mM DTT. Unreacted dye molecules were removed by size exclusion FPLC (Sephacryl S-100 HR, AKTAprime plus, GE Life Sciences) in LB. The dye-labeling procedure was repeated (up to 3 times) until the labeling efficiency was greater than 90%, the molar ratio of dye to protein. Protein and dye concentrations were determined by absorption spectroscopy using the following extinction coefficients (M- ¹ cm- ¹ ): TnC, 4,480 at 280 nm; Tnl, 11 ,460 at 280 nm; TnT, 15,470 at 280 nm; Tm, 21 ,760 at 280 nm; F-actin, 43,960 at 280 nm; myosin-S1 , 90,850 at 280 nm; Alexa546 (Life Technologies) 104,000 at 555 nm; ATT0655 (ATTO-Tec GmbH) 125,000 at 663 nm. TnC127C was labeled with ATT0655. Tnl189C was labeled with Alexa546. Aliquots of labeled proteins were stored at -80°C for up to 1 year.

The concentration of dye-labeled proteins was determined by absorption spectroscopy using [protein] = (/ 280 - λ A _max )lz, where A _max is absorption maximum of the dye and λ is the correction factor for dye absorption at 280 nm, which is 0.12 for Alexa546 and 0.08 for ATT0655. The labeling efficiency is the concentration of dye divided by the concentration of the protein.

Preparation of troponin

Troponin (Tn) was reconstituted from TnC, Tnl, and TnT, in a molar ratio of 1 :1.2:1.4, using stepwise dialysis into storage buffer (SB: 150mM KCI, 50mM MOPS, pH 7.2, 5mM MgCI ₂ , 2mM EGTA, 5mM BME) as described (12). Uncomplexed Tnl and TnT, which precipitate in SB, were removed by centrifugation at 10,000x g for 1 min. Aliquots of Tn were stored at -80 °C.

Preparation of regulated actin filaments

Regulated actin filaments (RF) were prepared by incubating F-actin (7 μΜ) with Tm and Tn at a molar ratio of 7:5:1 in working buffer (WB: 75 mM KCI, 50 mM MOPS pH 7.0, 5 mM MgCI ₂ , 2 mM EGTA, 5 mM BME) on ice for 1 hr then at 4°C for at least 1 week. Imaging revealed that during the 4°C incubation the filaments elongated and straightened. RF were stable for up to 3 months when stored at 4 °C. For spFRET measurements we prepared RF using a 20:1 mixture of WT Tn and doubly-labeled Tn (Tnl189C*Alexa546/TnC127C*ATT0655/TnT). This produced sparsely labeled RF that minimized the likelihood of more than one FRET dye-pair would be in the confocal volume of the microscope at any given time.

Pulsed interleaved single pair FRET (PIE-spFRET) measurements

Time tagged time resolved (TTTR) data were collected at room temperature (20 ± 2°C) on a MicroTime 200 confocal fluorescence lifetime microscope (PicoQuant, GmbH; Berlin, Germany) based on an inverted microscope (1X71 , Olympus). Excitation light from interleaved 532 nm and 638 nm pulsed diode lasers (LDH-P-FA-530-B and LDH-D-C-640, PicoQuant) were passed, respectively, through a quarter wave plate, a single-mode fiber optic, a triple notch laser clean-up filter (FF01- 485/537/627-25, Semrock), a custom principle dichroic mirror (DC1) (ZT532/638rpc, Chroma), and a 100x (N.A. 1.3) oil immersion objective (UPlanFLN, Olympus). Emitted light was passed through the objective and DC1 then through a 550 nm long-pass filter (550lp, Chroma), a 50 μιη pinhole and a secondary dichroic mirror (DC2) (T660LPXR, Chroma). The non-reflected light from DC2 was passed through a bandpass filter (ET 700/75, Chroma) and recorded on an avalanche photodiode (APD1) (MPD PDM series Micro Photon Devices, Italy). The reflected light from DC2 was passed through a bandpass filter (HQ580/70, Chroma) and recorded on a second avalanche photodiode

(APD2) (MPD PDM series Timing resolution was set to 16 ps per channel. The laser power from the 532 and 635 lasers was 5 μ\Λ/ and 2 μ\Λ/, respectively.

Analysis of PIE-spFRET data

The PIE-spFRET data were analyzed by custom software written in MATLAB (33) based on the previously published methods (34-36) with modifications. The color (APD2 records green photons; APD1 records red photons), arrival time, and delay time were extracted from the TTTR data. Histogram of local photon arrival times, also called the time-correlated photon counting (TCSPC) histogram, were calculated for the green (APD2) and red (APD1) emission channels. Time gates were defined as channel ranges for emitted photons from excitation with the 532 nm and 638 nm lasers. Donor excited- donor emitted (DD), donor excited-acceptor emitted (DA), and acceptor excited-acceptor emitted (AA) photons are identified in the channel ranges. Burst traces (1.2 msec binning window time) were calculated from the global arrival time of the DD, DA, and AA photons within the channel range limits defined in the TCSPC histogram. The burst traces show the time-dependent intensities (number of collected photons) HDD, HDA, and ΠΑΑ, from DD, DA, and AA, respectively as a function of time. We determined that a single donor dye was in the confocal volume when HDD + HDA was between 14 and 70 counts (donor-selection filter). We determined that an FRET acceptor dye was present in the confocal volume when ΠΑΑ was between 14 and 70 counts (acceptor-selection filter). For each burst within the donor-filter, we calculated the FRET efficiency

E = n _DA / (yn _DD + n _DA ),

dye intensity ratio

S = Yn _DD /n _DA ,

and the mean of the local photon arrival times from DD

where γ is the ratio of apparent brightness of the acceptor vs. donor dyes (24), which depends on the quantum yield of the FRET donor and FRET acceptor and the detection efficiencies of the two channels, to is the timing delay of the instrument, and n _DA = n _DA - (a + β)η _ΟΑ is the intensity of DA corrected, respectively, for spectral crosstalk a and for direct excitation of the FRET acceptor β by the 532 laser. Crosstalk is the appearance of photon emitted by the donor in the acceptor detection channel. For our instrument with Alexa546 (FRET donor) and ATT0655 (FRET acceptor) dyes, γ = 0.40, a = 0.02, and to = 2.4 nsec. In the analysis, we set a = 0 and β=0.

Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) measurements were performed on the MicroTime 200 microscope described above with DC2. Dyes were excited with the 532 nm laser at a power of 50 μ\Λ/. Emitted photons were collected using APD2 after passage through the HQ580/70 bandpass filter. Data were collected for approximately 10 minutes then analyzed by calculating the autocorrelation function

where 5F(t) = F(t) - (F) is the fluctuation in fluorescence from the temporal average

(F) = - J ₀ F t)dt. To recover the translational correlation times, the data were fit to a pure diffusion model with one or two speci

where∑ ₌₁ p. = 1/(N), where (N) is the average number of molecules in the confocal volume; κ = z ₀ /w ₀ , where zo is the effective focal radius along the optical axis at 1/e ² intensity, and wo is the effective lateral focal radius at 1/e ² intensity, r, is the diffusion time of the ith diffusing species. The effective confocal volume (0.4 fL) was determined fitting the autocorrelation function of free Alexa546 dye (1 nM) with fixed correlation time (0.029msec).

Steady state fluorescence measurements

Steady-state measurements were performed at 22.0°C on a Fluorolog-3 spectrofluorometer (Horiba) equipped with a MicroLab500 syringe dispenser (Hamilton). The excitation and emission monochromator slit width was 5 nm. To provide the relative concentration of the FRET donor dye in RF- DA and RF-D samples, each sample was denatured with 3 M guanidine hydrochloride (GnHCI) and the emission spectrum was re-measured. All intensity data were corrected for dilution. The intensity of the RF-DA sample /DA was corrected for concentration mismatch with the corresponding RF-D sample using _DA = I _DA {I _D * /ID*A) _> ^{WNERE 1} D ^{is tne} intensity of the denatured RF-D sample and l^ _A is the intensity of the denatured RF-DA sample.

Ensemble time-resolved fluorescence measurements

Time correlated single photon counting (TCSPC) measurements were performed at room temperature (20 ± 2°C) with 500 nM RF (in Tn) using the MicroTime 200 confocal fluorescence lifetime microscope configured as described for the FCS measurements. All data sets had peak intensity of 10,000 counts or more. The TCSPC histograms were fit using SymPhoTime Ver. 5.1 (PicoQuant,

GmbH; Berlin, Germany).

Imaging

RF were stained with phalloidin*Alexa488 (Life Technologies) at a 20:1 molar ratio of g- actin:phalloidin, diluted to 10 nM in WB and deposited on aminosilanized glass coverslips. Fluorescence images were collected on an inverted microscope (1X71 , Olympus) with a sCMOS camera (Zyla, Andor) using a 100x/1.4 oil immersion objective (UPlanSApo, Olympus) with excitation using a Mercury Vapor Arc lamp (X-Cite 120PCQ, Lumen Dynamics). For Alexa488 the filters (excitation: dichroic: emission) FF01-475/35: FF495: FF01-550/88 (Semrock) were used. For Alexa546 the filters ET545/25: T565lpxr: ET605/70 (Chroma) were used. For ATT0655 the filters ET620/60: T660lpxr: ET700/75 (Chroma) were used. Length was calibrated by imaging a dual axis linear scale (Edmund Industrial Optics). Images were pseudo-colored and merged using image J 1.47v (National Institutes of Health, Bethesda, MD). Length was calibrated by imaging a dual axis linear scale (Edmund Industrial Optics).

Simulations of structural dynamics

We used the Markov chain Monte Carlo method (37) to simulate the dynamic movement of the mobile element of Tnl (ME-Tnl) between actin and TnC. The intensity of donor-excited emission, including donor excited-donor emitted photons (DD) and donor excited-acceptor emitted photons (DA), the detector noise from the DD channel, and detector noise from the DA channel were Poisson distributed random variables with mean intensity of 20, 2, and 2 counts/msec, respectively. Simulations were performed using custom written code in MATLAB (33).

Muscle mechanics experiments

Cardiac myocytes were prepared and analyzed for the relationship of force-pCa as previously described (Barefield 2013, Salhi 2014). Briefly, myocytes were isolated from rat left ventricular tissue snap frozen and stored in -80°C. Frozen tissue was homogenized and filtered through a 70 μιη cell strainer and pelleted by centrifugation at 120 x g for 1 min at 4°C. Cells were skinned by resuspending the pellet in relaxing solution (97.92 mM KOH, 6.24 mM ATP, 10 mM EGTA, 10 mM Na ₂ CrP, 47.57 mM Kprop, 100 mM BES, and 6.54 mM MgCb) supplemented with 1 % Triton X-100, and incubated at room temperature for 15 min. on a rocking table. Triton was washed out in two centrifugation steps using relaxing solution. Exchange of Tn into skinned rat myocytes and an analysis of Tn exchange percentage by Western blot was performed as previously described.

Tn-exchanged rat ventricular myocytes were transferred to a culture dish coated with 0.1 % BSA, and attached to two metal micro-needles using UV-sensitive glue (Norland, Cranbury NJ) with the use of an inverted microscope (Leica DM IRB) under brightfield at 40X magnification. Myocytes were selected based on uniformity of the cell and clear striation patterns, and were perfused via a closely placed perfusion pipette with relaxing solution. Subsequent perfusion used a mixture of relaxing and activating solutions with varying calcium concentrations (pCa 10.0-pCa 4.5) to measure force development at sarcomere lengths (SL) of 1.9 μιη and 2.3 μιπ Maximal activating calcium solution was administered at the start of the experiment to ensure proper cell attachment. SL was measured using FFT analysis of video images using custom-made LabView software (National Instruments, Austin, TX). Force-pCa curves were fit using a modified Hill equation (P/P ₀ )=[Ca ²⁺ ]" ^H /( Caso ^NH +[Ca ²⁺ ] ^nH )), wher ΠΗ is the Hill coefficient. Cell cross-sectional elliptical area was calculated by buckling the cell, followed by measurement using calibrated screen monitor. Developed force was measured at both SLs at each activating cycle, with the baseline value of developed force subtracted from subsequent measures. Data were not considered if total rundown was greater than 205 after final maximal activation at the end of each activating cycle at both SLs. Three hearts were used, with 3 cells per heart. All data were acquired by custom-made LabView software and analyzed using Origin Pro 8.0.

All data are represented as means+/-SEM. Statistical analysis was performed using GraphPad Prism (version 6.0), and data were analyzed using two-way ANOVA with a Bonferroni post hoc test. Statistical significance was defined as P <0.05.

Description

A FRET troponin assay was constructed with green-excitable FRET donor Alexa546 on the mobile element of Tnl and red-excitable FRET acceptor ATT0655 on the C-lobe of TnC to examine the structural dynamics of cardiac thin filament activation by Ca2+ and myosin motors at single dye-pair resolution.

Two color FRET design

Mouse cardiac TnC is a 161 -residue protein with globular N- and C-lobes connected by a flexible linker. Each lobe possesses paired EF-hand structural motifs that bind divalent cations (Figure 15). Under physiological conditions, sites III and IV in the C-lobe constitutively bind Mg ²⁺ (38). In cardiac TnC, Site I is unable to bind Ca ²⁺ in the physiological range due to mutations in residues that coordinate Ca ²⁺ . Site II in the N-lobe selectively binds Ca ²⁺ , and it operates as the Ca ²⁺ sensor of the myofilament (17,39).

Mouse cardiac Tnl is a 211-residue protein that possesses three helices (I, II, and III) and a cardiac isoform-specific 33-residue N-terminal extension (Figure 15) (40). Helix II of Tnl forms a coiled- coil with TnT (the IT-helix) that along with Helix I grasp the C-lobe of TnC like a pair of chopsticks. The C-terminus of Tnl (residues 138-211), called the mobile element of Tnl (ME-Tnl), consists of an extended helix (helix III, residues 152-188) that is flanked by two intrinsically disordered regions: reward is the inhibitory region (residues 138-149); C-ward is the C-terminal domain (c-term, residues 189- 211). Helix III is subdivided into the switch (residues 152-160) and mobile (residues 162-188) domains. Under relaxing conditions ME-Tnl binds to actin to suppress XB cycling. Cryo-EM reconstructions suggest that in the relaxed state, ME-Tnl extends laterally across the actin filament to interact with the opposing strand of actin (41). Studies from NMR (42), FRET (43), and X-ray crystallography (6) suggest when Ca ²⁺ binds to site II of TnC, the N-lobe of TnC structurally "opens" through a helical rearrangement of the B/C helices and binds the proximal portion (switch domain) of Tnl-helix III. Figure 16 illustrates the activation of troponin, where ME-Tnl releases from the opposing strand of actin and binds to the N-lobe of TnC.

To monitor the switching of ME-Tnl between actin and TnC, 3 single Cys-containing mutants of TnC were engineered, with Cys positioned in the N-lobe (residue 35), positioned between the N-lobe and C-lobes (residue 89), and positioned within the C-lobe (residue 127) (Figure 15). Residue 127 is in a loop that is not part of the paired EF-hand motifs within the C-lobe of TnC . In each TnC mutant, the Cys residue was labeled with ATT0655 (FRET acceptor). 12 single Cys-containing mutants of Tnl were engineered with Cys positioned at sites spanning ME-Tnl (residues 151 , 160, 167, 174, 177, 182, 189, 196, 200, 204, 208, and 211) (Figure 15). In each Tnl mutant, the Cys residue was labeled with either Alexa546 (FRET donor) or ATTO550 (FRET donor).

Spectral characterization of FRET-labeled regulated actin filaments

Tn, Tm, and F-actin self assemble to form regulated actin filaments (RF), which are a simplified biochemical model for native thin filaments within the cardiac sarcomere (10). RF was reconstituted by co-incubating F-actin, Tm and dye-labeled Tn in a stoichiometry of 7:5:1 actin:Tm:Tn (see below for rationale). In all, 12 singly-labeled filaments containing the FRET donor were prepared, and 36 unique doubly-labeled RF containing both FRET donor and FRET acceptor were prepared. Using the steady state fluorescence emission of the donor, the 36 constructs were screened for large Ca ²⁺ -induced increases in the FRET efficiency. The largest Ca ²⁺ -induced change in FRET efficiency was observed in the FRET construct TnC(C35S, C84S, T127C) labeled with ATT0655, abbreviated TnC127C*ATT0655, and Tnl(C81S, C98I, V189C) labeled with Alexa546, abbreviated Tnl189C*Alexa546 (Figure 17). RF containing the FRET donor only (RF-D) were reconstituted from Tn consisting of TnC127C, Tnl189C*Alexa546, and WT TnT. Filaments containing the FRET donor and acceptor (RF-DA) were reconstituted from Tn consisting of TnC127C*ATT0655, Tnl189C*Alexa546, and WT TnT.

Figure 18 shows the steady state emission spectra of RF-D and RF-DA under Ca ²⁺ -depleted (no added Ca ²⁺ ), Ca ²⁺ -saturated (+ 3 mM CaCI ₂ ), and denatured (+ 3 mM CaCI ₂ , + 3M GnHCI) conditions. In RF-D and RF-DA, no change in fluorescence was observed when the concentration of GnHCI was increased beyond 3M, suggesting that 3M GnHCI was sufficient to maximally denature RF RF-D and RF-DA samples (data not shown). The emission intensity of RF-D was relatively insensitive to the addition of 3 mM CaCl2, while the emission intensity of RF-DA showed a 35% decrease in the emission of the FRET donor (575 nm peak) and a corresponding 15% increase in the emission from the FRET acceptor (675 nm peak). Denaturation with 3M GnHCI served two purposes. In the RF-DA sample under Ca ²⁺ -depleted and Ca ²⁺ -saturated conditions, denaturation eliminated the emission peak from the FRET acceptor (Figure 18, inset). This strongly suggests that the emission peak at 675 nm is fluorescent emission from the acceptor due to photons transferred from the donor dye to the acceptor dye through FRET (sensitized emission). As described in the Materials and Methods, denaturation allowed correction for concentration differences between RF-D and RF-DA samples since the emission spectrum in the presence of 3M GnHCI provides the relative concentration of the FRET donor dye in the sample.

Ensemble time resolved fluorescence measurements of the FRET donor in RF-D and RF-DA were performed under Ca ²⁺ -depleted (no added Ca ²⁺ ) and Ca ²⁺ -saturated (+ 3 mM CaC ) conditions. The time correlated single photon counting (TCSPC) histograms were fit to a multi-exponential decay model to recover the amplitude weighted fluorescence lifetime of each sample (Table 5).

Table 5

Lifetimes (ns) ^a

Sample Condition Xr ²

RF-D 0.78 (0.07) 3.80 (0.93) 3.58 1.04

+ Ca ²⁺ 0.80 (0.09) 3.76 (0.91) 3.48 1.02

RF-DA 0.20 (0.07) 1.44 (0.10) 3.60 (0.83) 3.15 1.04

+ Ca ²⁺ 0.26 (0.13) 1.50 (0.23) 3.50 (0.64) 2.60 1.01 aLifetime (fractional amplitude).

bAmplitude-weighted mean lifetime.

Physical characterization of reconstituted regulated actin filaments

Reconstituted RF were routinely imaged to screen for filament aggregation (bundling). Reconstituting RF with an excess of Tm (7:5:1 , actin protomer:Tim:Tn) vs. the native stoichiometry of the thin filament (7:1 :1), eliminated bundling in more than 95% of the samples. Figure 19 shows bundled filaments prepared with 7:1 :1 actin:Tm:Tn stoichiometry and non-bundled (normal) RF-DA prepared with 7:5:1 actin:Tm:Tn stoichiometry. Normal RF-DA appear as thin chains. In images of RF (stored at 4 °C) taken at 1 week intervals, it was observed that the filaments lengthened and straightened, suggesting that regulated actin remodels during storage. Figure 20 shows epifluorescence images from normal RF-D and RF-DA stored at 4 °C at for 3 weeks. The mean filament length is about 10 μιπ In RF-D, the absence of fluorescence from the TnC (red) channel suggests that spectral bleed through from the FRET donor is minimal. In the merged image of RF-DA, fluorescence from TnC and Tnl co-localize, suggesting that FRET-labeled Tn incorporates into RF as an intact assembly.

Single molecule studies require that the sample be studied at low concentrations, which through mass action favors filament disassembly. To optimize the experimental conditions that promote stable filaments, RF containing Tn labeled with Alexa546 (Tn*Alexa546: TnC127C*Alexa546, WT Tnl, and WT TnT) were prepared in solutions of varying pH and ionic strength. Imaging and fluorescence correlation spectroscopy (FCS) were used to monitor the stability of the RF. Among the conditions that were tested, RF were most stable when suspended in 75 mM KCI, 50 mM MOPS pH 7.0, 5 mM MgCl2, 2 mM EGTA, 5 mM BME (working buffer, WB). Figure 21 shows FCS measurements of samples in WB. The autocorrelation function of Alexa546 emission of free Alexa546 dye (500 pM), dye-labeled TnC (TnC127C*Alexa546, 500 pM), dye-labeled Tn (Tn*Alexa546, 10 nM) and dye-labeled regulated actin (RF Tn*Alexa546, 10 nM in Tn) are plotted. The autocorrelation function of the free dye, TnC and Tn were fit to a model of a single diffusing species with translational correlation times T _c = 0.03 msec, 0.10 msec, 0.16 msec, respectively. The autocorrelation function for regulated actin filaments was fit to a model of two diffusing species with correlation times (fractional amplitude) T _c = 2.48 msec (0.44) and 18.23 msec (0.56). The mean correlation time (T _C ) was 11.3 msec. The shorter of the two correlation times for regulated actin (2.48 msec) is much greater than the correlation time of Tn (0.10 msec). Thus, the FCS analysis did not detect unbound Tn. Therefore, at least 95% of Tn is bound to regulated actin. A plot of the correlation time vs. molecular weight (Fig. 33) showed that correlation times of free dye, TnC, and Tn follow a power law dependence on their molecular weight, T _C = with drag coefficient a = 0.037 msec/kDa ^1/3 and shape (Perrin) factor β = 1. Approximating regulated actin as a rod with shape factor β = 4 (44), the calculated average molecular weight of RF is MW = (τ ₀ /αβΥ = 445 MDa.

To achieve single dye pair resolution in RF requires minimizing the likelihood that more than one FRET dye-pair would be in the -0.4 fl effective confocal volume (x-, y-, z-dimensions, 0.2, 0.2 and ~1.2 μιη, respectively). In RF, Tn are positioned at -38 nm intervals on both strands of F-actin. Single pair FRET resolution would not be achievable by simply diluting the concentration of RF since one RF- DA would deliver multiple dye-labeled Tn assemblies into the confocal volume of the microscope. To achieve single-pair resolution Tn labeled were sparsely incorporated with FRET donor and FRET acceptor (Tn-DA: TnC127C*ATT0655, Tnl189C*Alexa546, and WT TnT) into RF by preparing RF with a 20:1 excess of unlabeled Tn (TnC127C, Tnl189C, and WT TnT) to Tn-DA using a 7:5:1 stoichiometry of actin :Tm:total Tn. FCS measurements of the sparsely labeled (5% dye-labeled Tn) RF-DA (Fig. 34) showed that (N) = 0.23 for RF at a concentration of 500 nM in total Tn. This is below the value of 0.5 that is required for single molecule resolution. Therefore, the slow translational diffusion of the massive (445 MDa) RF assembly, the excluded volume effect of worm-like RF, and sparse incorporation of dye- labeled Tn into RF would allow PIE-dspFRET measurements of RF-DA to be performed at the relatively high concentration of 500 nM total Tn, which discourages filament disassembly.

Images confirmed that labeled Tn was incorporated sparsely and randomly into the RF. The strategy of constructing sparsely labeled RF is illustrated in Figure 22, which shows images of RF-DA prepared from a 1 :5 mixture Tn-DA to Tn labeled with FRET acceptor (Tn-A: TnC127C*ATT0655, Tnl189C, and WT TnT). The RFs were also stained with phalloidin*Alexa488 to visualize F-actin. Emission from Alexa546 dyes attached to Tnl superimpose on confluent emission from ATT0655 attached to TnC and Alexa488 attached to phalloidin/actin, suggesting random incorporation of FRET donor dye into RF.

Functional Characterization of the engineered troponin

The engineered FRET assay involved triple mutants of TnC and Tnl. To assess the effect of mutagenesis on function, the function of detergent-treated cardiomyocytes isolated from left ventricles rats exchanged with either mutant Tn or WT Tn (control) were compared. Mutant Tn consisted of TnC(C35S, C84S, T127C), Tnl(C81 S, C98I, V189C), and c-myc TnT. WT Tn consisted of WT TnC, WT Tnl, and c-myc-tagged TnT {c-myc TnT). The efficiency of exchange of mutant Tn (80.6±1.0%) and WT Tn (69.2±3.5%) were estimated from Western blots of the exchanged cells (Figure 35). Figure 23 shows measurements of force at different Ca ²⁺ concentrations. During measurements, the sarcomere length was maintained at either 1.9 μιη or 2.3 μιπ At 1.9 μιη and 2.3 μιη sarcomere lengths, peak force (Figure 24) and Ca ²⁺ sensitivity (pCaso) (Figure 25) were not statistically different between fibers exchanged with WT and mutant Tn (P > 0.05). Therefore, the mutagenesis required as part of the FRET assay design did not significantly alter the function of Tn.

Single-pair FRET measurements of regulated actin filaments

Pulsed-interleaved excitation diffusion single pair FRET (PIE-dspFRET) measurements were performed on freely diffusing sparsely labeled (5% dye-labeled Tn) RF-DA. Measurements were performed on a confocal epi-illuminated microscope by exciting the FRET donor and FRET acceptor dye molecules with alternating pulses (20 MHz) of green (532 nm) and red (638 nm) light. A 100x 1.3 NA oil immersion objective was used to concentrate incident photons into an -0.4 fl confocal volume positioned ~5 μιη above the top surface of a glass coverslip. Emitted photons from the FRET donor and FRET acceptor were collected by the same objective before being passed through a 50 μιη pinhole then resolved by a second dichroic mirror and channeled into different avalanche photodiode detectors. FR-DA diffusing freely through the solution occasionally passed through the detection volume, producing brief (~ 10 ms) bursts of fluorescence. The detector channel (green or red color), arrival time, and delay time (time between the laser pulse and detected photon) of each detected photon were stored for subsequent analysis.

The RF-DA were examined under Ca ²⁺ -depleted (apo), Ca ²⁺ -saturating (+Ca), myosin- saturating (+S1), and both Ca ²⁺ - and myosin-saturating (+Ca+S1) conditions. Results of the PIE- dspFRET measurements are shown in Figure 14. Panels A and B show data from the +Ca sample, which is representative of the other samples. Figure 26 shows histograms of the delay times (fluorescence decays) of green (donor)-excited, donor-emitted (DD) photons, donor-excited, acceptor- emitted (DA) photons, and red (acceptor)-excited, acceptor-emitted (AA) photons. Acceptor-excited, donor-emitted (AD) photons are not appreciated in the donor trace. Photons within selected time gates (boxes) comprise a channel. Figure 27 plots burst traces of the DD, DA, and AA channels obtained by binning (1.2 msec window) the photon arrival times. The top panel plots fluorescence derived from excitation with the green laser. Intensity from the DD channel (black), and intensity from the DA channel (grey, inverted) are shown. The bottom panel plots the intensity from the AA channel that from emission from the FRET acceptor that has been directly excited with the red laser. Clearly defined bursts are observed in the DD, DA and AA channels as dye molecules enter the confocal volume of the microscope, suggesting that single molecule resolution has been achieved.

Bursting events were selected for further analysis by constructing selection filters for the donor and acceptor dyes. The donor-filter selects bins where the sum of the DD and DA channels is between upper and lower thresholds as described (45). The acceptor-filter was constructed by thresholding the AA channel. In Figure 27, vertical grey bars in the donor excitation panel indicate bins within the donor- filter. Vertical grey bars in the acceptor excitation panel indicate bins within the acceptor-filter. Figure 36 plots the histogram of bin intensities from DD + DA and AA channels. Also shown are the upper and lower thresholds that were used to construct the donor- and acceptor-filters.

In bins where the donor dye is present (i.e., bins within the donor-filter) the FRET efficiency E, relative dye intensity S, and the mean lifetime of the donor dye (τ) were calculated as described in

Materials and Methods. E is related to inter-dye distance r by E = (1 + (r/# ₀ ) ⁶ ) ^-1 (46), where Ro =

5.5 nm is the Forster critical distance for the Alexa546-ATT0655 dye pair (assumed, κ ² = 2/3).

When a system interconverts between two states (si, S2, with FRET efficiencies Ei and E2, respectively) slower than the inter-photon time (1/20 MHz for our instrument), (τ) is related to E by (47)

{E ₂ - E) {E - E ₁ )

(τ)/τ ₀ = 1 - E +

1 — E

[Eq. 1]

Figure 28 shows two-dimensional (2D) E - (τ) histograms of the apo, +Ca, +S1 , and +Ca+S1 samples. It was observed that the mean values «r)/nsec, E) for the apo (3.46, 0.25), +Ca (3.22, 0.32), +S1 (2.83, 0.39), and +Ca+S1 (2.85, 0.40) samples are related by Eq. 1 (red line) with Ei = 0.24, E ₂ = 0.46, and TD = 4.1 nsec.

To resolve states si and S2 the 1 D E histograms of the apo, +Ca, +S1 , and +Ca+S1 samples were globally fit to a two state model, with each state parameterized by a Gaussian distribution of FRET efficiencies (Figure 29). The first two bins of the 1 D FRET histogram were excluded from the fits, since these contain data from troponin that lack FRET acceptor (see the 2D E-S histograms below). The two states were interpreted according to the structural model in Figure 16, with ME-Tnl bound to actin (red, state si) or to TnC (blue, state S2). The actin- and TnC-bound states have mean FRET efficiencies Ei and E2, respectively. The fraction of inactive Tn— Tn with ME-Tnl bound to actin (state si)— under apo, +Ca, +S1 , and +Ca+S1 conditions is 94%, 56%, 19%, and 13%, respectively. Therefore, about 50% of Ca ²⁺ -bound troponin are inactive. Maximal activation of troponin requires myosin. Myosin binding alone is almost sufficient to maximally activate troponin.

Rejection of samples lacking a FRET acceptor

The relative dye intensity S is the corrected ratio of emission intensity from the donor and acceptor dyes following excitation of the donor. S is a scalar measure of the opposing changes in the donor and acceptor dyes that occur in FRET as seen in Figure 18. Fig. 30 shows the 2D E-S histograms of the apo, +Ca, +S1 , and +Ca+S1 samples. E-S histograms are shown for bins without application of the acceptor-filter (left) and with application of the acceptor-filter (right). Assemblies that lack a FRET acceptor produce the so-called "zero peak" with E = 0 and log(S) = 1.5. The ability of the acceptor-filter to reject assembles that do not contain the FRET acceptor dye is appreciated from the projection of the distribution onto the log(S) axis (left box). Note that the acceptor filter also rejects the peak near E = 0.15, which appears to be a shoulder of the zero peak caused by noise in the DD channel. With application of the acceptor-selection filter, for all samples, the number of counts in the zero peak is less than 5% of the total counts in the 2D histogram. Therefore, the acceptor-selection filter that was applied to the FRET histograms in Figure 14 efficiently overcomes the issue of incompletely labeled or incompletely assembled samples. Nevertheless, remnants of the zero peak occur in the first two bins of the E histogram. These bins were excluded from the fitting in Figure 14.

Structural Dynamics of Troponin

Fitting of the PIE-spFRET data for the +Ca sample suggested that troponin exists as a mixture of two conformational states si and S2 with FRET efficiencies Ei = 0.22, E2 = 0.47 (Figure 29). When a system isomerizes between two states, and the isomerization rates are slow compared to the binning window, the intensity of DA in the burst trace is anti-correlated with intensity of DD (48), producing a characteristic splitting of peaks in the 2D E-S histogram (25). However, in the +Ca sample, a unimodal distribution was observed in the 2-D E-S histogram (Fig. 30). Furthermore, inspection of bursts within the burst trace of the +Ca sample showed that the DA intensity tracked the DD intensity.

To better understand the nature of the unimodal distribution in the 2-D E-S histogram of +Ca sample, the switching of ME-Tnl was modeled as a two-state transition between actin (si) and TnC (S2) with isomerization rate constants k _l→z and k _2→1 . Markov chain Monte Carlo (MCMC) simulations were performed with switching modeled as exponentially distributed isomerization events governed by rate constants k\ = = k. Simulations were performed for k = 10, 25, 50, 100, 250, 500, 1000, 2500, and 5000 S ^"1 with parameters Ei = 0.22, Ei - 0.47. Additional parameters were color intensity correction factor Y =1 , binning time window At = 1.2 msec, spectral crosstalk a = 0, and simulation time step At = 1 \is. The simulation corresponds to RF-DA with a single dye-pair that has been immobilized in the center of the confocal volume of the microscope.

Fig. 31 shows the 2D E-S histogram of the Monte Carlo simulations. When the isomerization rates between states si and S2 were slow compared to the binning time window (slow kinetic regime) the 2D E-S histogram resolves peaks from states si and S2, as observed previously (25). In plots showing the time evolution of the system, it was observed that when the isomerization rates between states Si and S2 were fast compared to the binning time window (fast kinetic regime) the system made several transitions between states si and S2 during the binning time. This produced a single "kinetically blurred" peak in the 2D E-S histogram. Comparing the experimental 2D E-S histogram of the +Ca sample (Figure 14) with the simulated 2D E-S histogram (Fig. 31), in the +Ca state, the mobile element is in dynamic equilibrium between actin and TnC with isomerization rates greater than 1000 sec- ¹ .

G. CONCLUSIONS

It is to be understood that any given elements of the disclosed embodiments of the invention may be embodied in a single structure, a single step, a single substance, or the like. Similarly, a given element of the disclosed embodiment may be embodied in multiple structures, steps, substances, or the like.

The foregoing description illustrates and describes the processes, machines, manufactures, compositions of matter, and other teachings of the present disclosure. Additionally, the disclosure shows and describes only certain embodiments of the processes, machines, manufactures, compositions of matter, and other teachings disclosed, but, as mentioned above, it is to be understood that the teachings of the present disclosure are capable of use in various other combinations, modifications, and environments and are capable of changes or modifications within the scope of the teachings as expressed herein, commensurate with the skill and/or knowledge of a person having ordinary skill in the relevant art. The embodiments described hereinabove are further intended to explain certain best modes known of practicing the processes, machines, manufactures, compositions of matter, and other teachings of the present disclosure and to enable others skilled in the art to utilize the teachings of the present disclosure in such, or other, embodiments and with the various modifications required by the particular applications or uses. Accordingly, the processes, machines, manufactures, compositions of matter, and other teachings of the present disclosure are not intended to limit the exact embodiments and examples disclosed herein. Any section headings herein are provided only for consistency with the suggestions of 37 C.F.R. § 1.77 or otherwise to provide organizational queues. These headings shall not limit or characterize the invention(s) set forth herein.

References

1. Huxley, H. E. 2004. Fifty years of muscle and the sliding filament hypothesis. Eur J Biochem 271 :1403-1415.

2. Squire, J. M., H. A. Al-Khayat, C. Knupp, and P. K. Luther. 2005. Molecular architecture in muscle contractile assemblies. Adv Protein Chem 71 :17-87.

3. Ebashi, S., M. Endo, and I. Otsuki. 1969. Control of muscle contraction. Q Rev Biophys 2:351-384.

4. Brown, J. H. and C. Cohen. 2005. Regulation of muscle contraction by tropomyosin and troponin: how structure illuminates function. Adv Protein Chem 71 :121-159.

5. Kobayashi, T. and R. J. Solaro. 2005. Calcium, thin filaments, and the integrative biology of cardiac contractility. Annu Rev Physiol 67:39-67.

6. Takeda, S., A. Yamashita, K. Maeda, and Y. Maeda. 2003. Structure of the core domain of human cardiac troponin in the Ca2+-saturated form. Nature 424:35-41.

7. Ohtsuki, 1. 1979. Molecular arrangement of troponin-T in the thin filament. J Biochem 86:491-497.

8. Cabral-Lilly, D., L. S. Tobacman, J. P. Mehegan, and C. Cohen. 1997. Molecular polarity in tropomyosin-troponin T co-crystals. Biophys J 73:1763-1770.

9. Tobacman, L. S., M. Nihli, C. Butters, M. Heller, V. Hatch, R. Craig, W. Lehman, and E. Homsher. 2002. The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity. J Biol Chem 277:27636-27642.

10. Bremel, R. D. and A. Weber. 1972. Cooperation within actin filament in vertebrate skeletal muscle. Nat New Biol 238:97-101.

11. Moss, R. L., M. Razumova, and D. P. Fitzsimons. 2004. Myosin crossbridge activation of cardiac thin filaments: implications for myocardial function in health and disease. Circ Res 94:1290-1300. 12. Robinson, J. M., W.-J. Dong, J. Xing, and H. C. Cheung. 2004. Switching of troponin I: Ca-and myosin-induced activation of heart muscle. J Mol Biol 340:295-305.

13. Li, K. L., D. Rieck, R. J. Solaro, and W. Dong. 2014. In situ time-resolved FRET reveals effects of sarcomere length on cardiac thin-filament activation. Biophys J 107:682-693. 14. Fusi, L, E. Brunello, I. R. Sevrieva, Y. B. Sun, and M. Irving. 2014. Structural dynamics of troponin during activation of skeletal muscle. Proc Natl Acad Sci U S A 111 :4626-4631.

15. Allen, D. G. and J. C. Kentish. 1985. The cellular basis of the length-tension relation in cardiac muscle. J Mol Cell Cardiol 17:821-840.

16. Hofmann, P. A. and F. Fuchs. 1987. Evidence for a force-dependent component of calcium binding to cardiac troponin C. Am J Physiol 253:C541-546.

17. Pan, B. S. and R. J. Solaro. 1987. Calcium-binding properties of troponin C in detergent-skinned heart muscle fibers. J Biol Chem 262:7839-7849.

18. Wang, Y. and W. G. L. Kerrick. 2002. The off rate of Ca2+ from troponin C is regulated by force- generating cross bridges in skeletal muscle. J Appl Phys 92:2409-2418.

19. Robinson, J. M., W.-J. Dong, and H. C. Cheung. 2008. The cardiac Ca-sensitive regulatory switch, a system in dynamic equilibrium. Biophys J 95:4772-4789.

20. Robinson, J. M. 2008. Physical Limits on Computation by Assemblies of Allosteric Proteins. Phys Rev Lett 101 :178104.178101 -178104.178104.

21. Rieck, D. C, K.-L. Li, Y. Ouyang, R. J. Solaro, and W.-J. Dong. 2013. Structural basis for the in situ Ca2+ sensitization of cardiac troponin C by positive feedback from force-generating myosin cross- bridges. Archives of Biochemistry and Biophysics 537:198-209.

22. Kalinin, S., S. Felekyan, A. Valeri, and C. A. M. Seidel. 2008. Characterizing Multiple Molecular States in Single-Molecule Multiparameter Fluorescence Detection by Probability Distribution Analysis. The Journal of Physical Chemistry B 112:8361 -8374.

23. Rothwell, P. J., S. Berger, O. Kensch, S. Felekyan, M. Antonik, B. M. Wohrl, T. Restle, R. S. Goody, and C. A. M. Seidel. 2003. Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes. Proc Natl Acad Sci U S A 100:1655-1660.

24. Nir, E., X. Michalet, K. M. Hamadani, T. A. Laurence, D. Neuhauser, Y. Kovchegov, and S.

Weiss. 2006. Shot-Noise Limited Single-Molecule FRET Histograms: Comparison between Theory and Experiments!. The Journal of Physical Chemistry B 110:22103-22124.

25. Kalinin, S., A. Valeri, M. Antonik, S. Felekyan, and C. A. Seidel. 2010. Detection of structural dynamics by FRET: a photon distribution and fluorescence lifetime analysis of systems with multiple states. J Phys Chem B 114:7983-7995.

26. Chakraborty, A., D. Wang, Y. W. Ebright, Y. Korlann, E. Kortkhonjia, T. Kim, S. Chowdhury, S. Wigneshweraraj, H. Irschik, R. Jansen, B. T. Nixon, J. Knight, S. Weiss, and R. H. Ebright. 2012. Opening and closing of the bacterial RNA polymerase clamp. Science 337:591-595.

27. Fredriksen, D. W., Cunningham, L.W. 1982. Purification of muscle actin. 28. Fredriksen, D. W., Cunningham, L.W. 1982. Purification of tropomyosin from acetone powder.

29. Margossian, S. S. and S. Lowey. 1982. Preparation of myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol 85 Pt B:55-71.

30. Dong, W., S. S. Rosenfeld, C. K. Wang, A. M. Gordon, and H. C. Cheung. 1996. Kinetic studies of calcium binding to the regulatory site of troponin C from cardiac muscle. J Biol Chem 271 :688~694.

31. Dong, W. J., J. Xing, J. M. Robinson, and H. C. Cheung. 2001. Ca induces an extended conformation of the inhibitory region of troponin I in cardiac muscle troponin. J Mol Biol 314:51-61.

32. Sumandea, M. P., W. G. Pyle, T. Kobayashi, P. P. de Tombe, and R. J. Solaro. 2003.

Identification of a functionally critical protein kinase C phosphorylation residue of cardiac troponin T. J Biol Chem 278:35135-35144.

33. MATLAB. R2014b. The MathWorks, Natick, MA.

34. Kuhnemuth, R. and C. A. Seidel. 2001. Principles of single molecule multiparameter fluorescence spectroscopy. Single Molecules 2:251-254.

35. Kapanidis, A. N., T. A. Laurence, N. K. Lee, E. Margeat, X. Kong, and S. Weiss. 2005.

Alternating-laser excitation of single molecules. Acc Chem Res 38:523-533.

36. Mijller, B. K., E. Zaychikov, C. Brauchle, and D. C. Lamb. 2005. Pulsed Interleaved Excitation. Biophysical Journal 89:3508-3522.

37. Andrieu, C, N. de Freitas, A. Doucet, and M. Jordan. 2003. An Introduction to MCMC for Machine Learning. Machine Learning 50:5-43.

38. Robertson, S. P., J. D. Johnson, and J. D. Potter. 1981. The time-course of Ca2+ exchange with calmodulin, troponin, parvalbumin, and myosin in response to transient increases in Ca2+. Biophys J 34:559-569.

39. Holroyde, M. J., S. P. Robertson, J. D. Johnson, R. J. Solaro, and J. D. Potter. 1980. The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase. J Biol Chem 255:11688-11693.

40. Solaro, R. J., P. Rosevear, and T. Kobayashi. 2008. The unique functions of cardiac troponin I in the control of cardiac muscle contraction and relaxation. Biochem Biophys Res Commun 369:82-87.

41. Yang, S., L. Barbu-Tudoran, M. Orzechowski, R. Craig, J. Trinick, H. White, and W. Lehman. 2014. Three-dimensional organization of troponin on cardiac muscle thin filaments in the relaxed state. Biophys J 106:855-864.

42. Li, M. X., L. Spyracopoulos, and B. D. Sykes. 1999. Binding of cardiac troponin-1147-163 induces a structural opening in human cardiac troponin-C. Biochemistry 38:8289-8298. 43. Dong, W. J., J. Xing, M. Villain, M. Hellinger, J. M. Robinson, M. Chandra, R. J. Solaro, P. K. Umeda, and H. C. Cheung. 1999. Conformation of the regulatory domain of cardiac muscle troponin C in its complex with cardiac troponin I. J Biol Chem 274:31382-31390.

44. Erickson, H. P. 2009. Size and Shape of Protein Molecules at the Nanometer Level Determined by Sedimentation, Gel Filtration, and Electron Microscopy. Biological Procedures Online 11 :32-51.

45. Eggeling, C, S. Berger, L. Brand, J. R. Fries, J. Schaffer, A. Volkmer, and C. A. Seidel. 2001. Data registration and selective single-molecule analysis using multi-parameter fluorescence detection. J Biotechnol 86:163-180.

46. Stryer, L. and R. P. Haugland. 1967. Energy transfer: a spectroscopic ruler. Proceedings of the National Academy of Sciences of the United States of America 58:719.

47. Gopich, I. V. and A. Szabo. 2012. Theory of the energy transfer efficiency and fluorescence lifetime distribution in single-molecule FRET. Proceedings of the National Academy of Sciences 109:7747-7752.

48. Margittai, M., J. Widengren, E. Schweinberger, G. F. Schroder, S. Felekyan, E. Haustein, M. Konig, D. Fasshauer, H. Grubmuller, R. Jahn, and C. A. Seidel. 2003. Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1. Proc Natl Acad Sci U S A 100:15516-15521.

49. Thomas, D. D., E. Prochniewicz, and O. Roopnarine. 2002. Changes in actin and myosin structural dynamics due to their weak and strong interactions. Results and problems in cell differentiation 36:7-19.

50. Rosenfeld, S. S. and E. W. Taylor. 1985. Kinetic studies of calcium binding to regulatory complexes from skeletal muscle. J Biol Chem 260:252-261.

51. Dong, W. J., C. K. Wang, A. M. Gordon, S. S. Rosenfeld, and H. C. Cheung. 1997. A kinetic model for the binding of Ca2+ to the regulatory site of troponin from cardiac muscle. J Biol Chem 272:19229-19235.

52. Brenner, B. and J. M. Chalovich. 1999. Kinetics of thin filament activation probed by fluorescence of N-((2-(lodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1 , 3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle: implications for regulation of muscle contraction. Biophys J 77:2692-2708.

53. Solzin, J., B. lorga, E. Sierakowski, D. P. Gomez Alcazar, D. F. Ruess, T. Kubacki, S. Zittrich, N. Blaudeck, G. Pfitzer, and R. Stehle. 2007. Kinetic Mechanism of the Ca2+-Dependent Switch-On and Switch-Off of Cardiac Troponin in Myofibrils. Biophys J 93:3917-3931. 54. Davis, J. P., C. Norman, T. Kobayashi, R. J. Solaro, D. R. Swartz, and S. B. Tikunova. 2007. Effects of thin and thick filament proteins on calcium binding and exchange with cardiac troponin C. Biophys J 92:3195-3206.

55. Criddle, A. H., M. A. Geeves, and T. Jeffries. 1985. The use of actin labelled with N-(1- pyrenyl)iodoacetamide to study the interaction of actin with myosin subfragments and

troponin/tropomyosin. Biochem J 232:343-349.

56. Guth, K. and J. D. Potter. 1987. Effect of rigor and cycling cross-bridges on the structure of troponin C and on the Ca2+ affinity of the Ca2+-specific regulatory sites in skinned rabbit psoas fibers. J Biol Chem 262:13627-13635.

57. Ishii, Y. and S. S. Lehrer. 1990. Excimer fluorescence of pyrenyliodoacetamide-labeled tropomyosin: a probe of the state of tropomyosin in reconstituted muscle thin filaments. Biochemistry 29:1160-1166.

58. Liao, R., C. K. Wang, and H. C. Cheung. 1994. Coupling of calcium to the interaction of troponin I with troponin C from cardiac muscle. Biochemistry 33:12729-12734.

59. Kitao, A. and N. Go. 1999. Investigating protein dynamics in collective coordinate space. Curr. Opin. Struct. Biol. 9:164-169.

60. Bahar, I., T. R. Lezon, A. Bakan, and I. H. Shrivastava. 2010. Normal mode analysis of biomolecular structures: functional mechanisms of membrane proteins. Chem Rev 110:1463-1497.

61. Frauenfelder, H., S. G. Sligar, and P. G. Wolynes. 1991. The energy landscapes and motions of proteins. Science 254:1598-1603.

62. Hilser, V. J., D. Dowdy, T. G. Oas, and E. Freire. 1998. The structural distribution of cooperative interactions in proteins: analysis of the native state ensemble. Proc Natl Acad Sci U S A 95:9903-9908.

63. Latt, S. A., H. T. Cheung, and E. R. Blout. 1965. Energy Transfer. A System with Relatively Fixed Donor-Acceptor Separation. J Am Chem Soc 87:995-1003.

64. Cheung, H. C, C. K. Wang, I. Gryczynski, W. Wiczk, G. Laczko, M. L. Johnson, and J. R.

Lakowicz. 1991. Distance distributions and anisotropy decays of troponin C and its complex with troponin I. Biochemistry 30:5238-5247.

65. Beechem, J. M. and E. Haas. 1989. Simultaneous determination of intramolecular distance distributions and conformational dynamics by global analysis of energy transfer measurements.

Biophys J 55:1225-1236.

66. Deniz, A. A., M. Dahan, J. R. Grunwell, T. Ha, A. E. Faulhaber, D. S. Chemla, S. Weiss, and P. G. Schultz. 1999. Single-pair fluorescence resonance energy transfer on freely diffusing molecules: Observation of Forster distance dependence and subpopulations. Proc Natl Acad Sci U S A 96:3670- 3675. 67. Deniz, A. A., S. Mukhopadhyay, and E. A. Lemke. 2008. Single-molecule biophysics: at the interface of biology, physics and chemistry. J R Soc Interface 5:15-45.

68. Tao, T., B. Gong, and P. Leavis. 1990. Calcium-induced movement of troponin-l relative to actin in skeletal muscle thin filaments. Science 247:1339-1341.

69. Xing, J. and H. C. Cheung. 1995. Internal movement in myosin subfragment 1 detected by fluorescence resonance energy transfer. Biochemistry 34:6475-6487.

70. Xing, J., M. Chinnaraj, Z. Zhang, H. C. Cheung, and W.-J. Dong. 2008. Structural studies of interactions between cardiac troponin I and actin in regulated thin filament using Forster resonance energy transfer. Biochemistry 47:13383-13393.

71. Miki, M., S. Makimura, Y. Sugahara, R. Yamada, M. Bunya, T. Saitoh, and H. Tobita. 2012. A three-dimensional FRET analysis to construct an atomic model of the actin-tropomyosin-troponin core domain complex on a muscle thin filament. J Mol Biol 420:40-55.

72. Miki, M., H. Hai, K. Saeki, Y. Shitaka, K. Sano, Y. Maeda, and T. Wakabayashi. 2004.

Fluorescence resonance energy transfer between points on actin and the C-terminal region of tropomyosin in skeletal muscle thin filaments. J Biochem 136:39-47.

73. Kimura-Sakiyama, C, Y. Ueno, K. Wakabayashi, and M. Miki. 2008. Fluorescence resonance energy transfer between residues on troponin and tropomyosin in the reconstituted thin filament: modeling the troponin-tropomyosin complex. J Mol Biol 376:80-91.

74. Miki, M. 1990. Resonance energy transfer between points in a reconstituted skeletal muscle thin filament. A conformational change of the thin filament in response to a change in Ca2+ concentration.

Eur J Biochem 187:155-162.

75. Wang, H., J. M. Chalovich, and G. Marriott. 2012. Structural dynamics of troponin I during Ca2+- activation of cardiac thin filaments: a multi-site Forster resonance energy transfer study. PLoS ONE 7:e50420.

76. Robinson, J. M., W.-J. Dong, and H. C. Cheung. 2003. Can Forster resonance energy transfer measurements uniquely position troponin residues on the actin filament? A case study in multiple- acceptor FRET. J Mol Biol 329:371-380.

77. Kozuka, J., H. Yokota, Y. Arai, Y. Ishii, and T. Yanagida. 2006. Dynamic polymorphism of single actin molecules in the actin filament. Nat Chem Biol 2:83-86.