Whole length myosin binding protein C stabilizes myosin S2 as measured by gravitational force spectroscopy

Accepted Manuscript Whole length myosin binding protein C stabilizes myosin S2 as measured by gravitational force spectroscopy Rohit R. Singh, James W. Dunn, Motamed M. Qadan, Nakiuda Hall, Kathy K. Wang, Douglas D. Root PII:

S0003-9861(17)30646-X

DOI:

10.1016/j.abb.2017.12.002

Reference:

YABBI 7603

To appear in:

Archives of Biochemistry and Biophysics

Received Date: 18 September 2017 Revised Date:

30 November 2017

Accepted Date: 1 December 2017

Please cite this article as: R.R. Singh, J.W. Dunn, M.M. Qadan, N. Hall, K.K. Wang, D.D. Root, Whole length myosin binding protein C stabilizes myosin S2 as measured by gravitational force spectroscopy, Archives of Biochemistry and Biophysics (2018), doi: 10.1016/j.abb.2017.12.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Whole length myosin binding protein C stabilizes myosin S2 as measured by gravitational

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force spectroscopy.

Rohit R. Singh, James W. Dunn, Motamed M. Qadan, Nakiuda Hall, Kathy K. Wang and

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Douglas D. Root*.

Department of Biological Sciences, Division of Biochemistry and Molecular Biology, University of North Texas, Denton, TX 76203

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*Corresponding author

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Douglas D. Root Life Sciences Complex, Building A Room # LS-A114 1511 West Sycamore Denton, Texas 76203 Phone: 940-565-2683 FAX: 940-565-4136 E-mail: [email protected]

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Abstract The mechanical stability of the myosin subfragment-2 (S2) was tested with simulated force spectroscopy (SFS) and gravitational force spectroscopy (GFS). Experiments examined

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unzipping S2, since it required less force than stretching parallel to the coiled coil. Both GFS and SFS demonstrated that the force required to destabilize the light meromyosin (LMM) was greater than the force required to destabilize the coiled coil at each of three different locations along S2.

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GFS data also conveyed that the mechanical stability of the S2 region is independent from its association with the myosin thick filament using cofilaments of myosin tail and a single intact

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myosin. The C-terminal end of myosin binding protein C (MyBPC) binds to LMM and the Nterminal end can bind either S2 or actin. The force required to destabilize the myosin coiled coil molecule was 3 times greater in the presence of MyBPC than in its absence. Furthermore, the in vitro motility assay with full length slow skeletal MyBPC slowed down the actin filament sliding

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over myosin thick filaments. This study demonstrates that skeletal MyBPC both enhanced the mechanical stability of the S2 coiled coil and reduced the sliding velocity of actin filaments over

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polymerized myosin filaments.

Graphical Abstract

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Highlights Myosin subfragment-2 has less mechanical stability than light meromyosin.

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The stability of myosin S2 is unaffected by polymerization of the myosin thick filament.

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MyBPC binding to myosin S2 increases the stability of the myosin S2 coiled coil.

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MyBPC stabilizes myosin S2 and reduces the motility of actin filament sliding.

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Keywords

Myosin subfragment-2, myosin binding protein C, gravitational force spectroscopy, molecular simulations, single molecule assays, in vitro motility assay.

Abbreviations

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Subfragment-1 (S1), Subfragment-2 (S2), Light meromyosin (LMM), Simulated force spectroscopy (SFS), Gravitational force spectroscopy (GFS), Myosin binding protein C (MyBPC), Familial Hypertrophic Cardiomyopathy (FHC), Phenylmethylsufonylfluoride (PMSF), Diethylenetriamine pentaacetate (DTPA), Worm Like Chain (WLC).

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Introduction Myosin subfragment-2 (S2) is a coiled coil linker between myosin subfragment-1 (S1) and myosin light meromyosin (LMM) that occurs in myosin heavy chain isoforms shared in

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striated muscles including cardiac and skeletal muscles. Myosin binding protein C (MyBPC) is known to bind to the coiled coil tail of striated muscle myosins, and derives from three genes, MyBPC1 (sometimes called the skeletal slow muscle isoform and expressed in skeletal muscle

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and at low levels in cardiac myocytes), MyBPC2 (sometimes called the skeletal fast muscle isoform and expressed only in skeletal muscle), and MyBPC3 (sometimes called the cardiac

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isoform and expressed only in cardiac myocytes) [1,2]. MyBPC binds LMM with high affinity to anchor it in a location that promotes lower affinity interactions with myosin S2 or F-actin in a phosphorylation dependent manner [3-15]. The impact of MyBPC binding on the structure of the myosin S2 coiled coil is not yet known, but a recent hypothesis is that it may promote binding of

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the myosin heads to S2 that regulates the number of heads available to bind thin filaments. The mechanical stability of myosin S2 could play a key role in the affinity of myosin heads for S2. Since the mechanical stability of a protein is the amount of force required to unfold the protein,

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this property can be measured by force spectroscopy to provide insight into the function of MyBPC and S2 interactions [16].

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It has long been known that smooth muscle myosins are regulated in part by forming a

bent conformation that restricts S1 mobility. Earlier work has shown that striated and smooth muscle myosins share certain structural properties such as the ability to achieve a bent conformation, as seen in electron micrographs of myosin S1 heads folding back on their long tail domain [17]. Velocity sedimentation measurments of both smooth and skeletal muscle myosins gave both a 6S and a 10S monomeric structure, where the 10S monomeric myosin had its head folding back onto its tail, while the 6S myosin monomer had its heads free and unbound to its tail 4

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[17,18]. Electron micrographs of a chimera created by skeletal myosin HMM with smooth muscle myosin light chain also showed the folding of myosin S1 heads onto the available myosin S2 region [19]. Anisotropy experiments with rabbit skeletal myosin gave two regions of dynamic

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wobbling, one being the myosin S1 head and the other being a 14 nm long region following the myosin S1-S2 hinge which is the proximal myosin S2 region [20]. Cryo-electron micrograph and atomic modelling studies of tarantula thick filaments have shown that myosin S1 heads have a

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switched off state where they bind to each other and then fold back and bind to the proximal myosin S2 region [21]. The transition of these myosin heads from the switched off to the

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switched on state is achieved by calcium-mediated phosphorylation of the myosin light chain or direct binding of calcium to the light chain in some invertebrate thick filaments [21]. While vertebrate striated muscle is predominantly regulated via the thin filament, there is evidence that myosin light chain phosphorylation can potentiate force generation, perhaps by a related

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mechanism [22].

Studies on the mechanical stability of myosin S2 in the myosin molecule are limited. The impact of MyBPC binding over myosin S2 stability and its resultant effect over the force

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produced through actomyosin interaction have not been previously determined. This study

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examines whether the stabilization of the myosin S2 coiled coil by myosin binding protein C may be a mechanism to reduce the amount of force produced through actomyosin interaction. Simulated force spectroscopy (SFS) and gravitation force spectroscopy (GFS) [23] test whether unraveling of the myosin coiled coil requires less force when pulled in a perpendicular direction to the thick filament axis than when pulled in a parallel direction to the axis. To facilitate the use of antibodies to separate the coiled coil strands at very low forces, GFS was utilized to determine the molecular length under varying loads. GFS tests whether the stability of the myosin S2 is 5

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independent from its association with the myosin thick filament by comparing the force-distance curve of the S2 coiled coil in a single myosin molecule to that of a myosin S2 in the myofilament. SFS and GFS test the mechanical stability of different sites in the proximal myosin

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S2 and myosin S1-S2 hinge and compare them to the LMM region that is expected to be more stable. The effects of whole length MyBPC on S2 stability in GFS and in vitro motility of actin filaments sliding over myosin thick filaments are further examined as possible modulatory

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functions of this myosin binding protein.

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Materials and Methods

Materials

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Proteins Rabbit skeletal myosin was purified by the method described by Godfrey and Harrington, 1970 [24]. Rabbit skeletal actin was purified by the method described by Spudich and Watt, 1971

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[25]. Rabbit skeletal back muscle MyBPC was isolated by the method described by Furst et al., 2011 [26] and sequentially chromatographed by hydroxyapatite, gel filtration, anion exchange,

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and chromatofocusing yielding a predominant isoform (75%) that corresponded to rabbit skeletal slow MyBPC based on electrophoretic mobility and pI (Supplementary Data). Monoclonal antibodies to myosin S2 (MF30) and LMM (MF20) were obtained from the Developmental Studies Hybridoma Bank and purified by Sephadex G-75 FPLC [27]. A site-specific polyclonal

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antibody to the glutamate-rich region in human β-cardiac myosin S2 (924-942) was created by conjugating the myosin S2 peptide with albumin and injecting it into a guinea pig. The peptide was created synthetically and the polyclonal antibody was supplied by Bio-synthesis Inc.,

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Lewisville, Texas, United States. The specificity of polyclonal antibody to myosin S2 was determined by competitive enzyme linked immunosorbent assay (cELISA) with the synthetic

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myosin S2 peptide and whole rabbit skeletal myosin (supplementary data). The protein concentrations were determined by utilizing extinction coefficients at 280 nm of 0.55 (mg/ml)-1 cm-1, 0.20 (mg/ml)-1 cm-1 and 1.09 (mg/ml)-1 cm-1 for myosin, rod and MyBPC, respectively, and 0.63 (mg/ml)-1 cm-1 at 290 nm for actin [28,29]. Fragmentation and purification of myosin rods were performed on the method described by Gundapaneni et al., 2005 [30]. Fragmentation of myosin into myosin rod was achieved by

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adding 0.3 M EDTA in 0.32 M Tris, pH 7 to the dialyzed supernatant to obtain a final concentration of 2.0 mM EDTA and warmed to room temperature. A fresh stock of 5 mg/mL αchymotrypsin was added to the myosin for a final concentration of 0.05 mg/mL. After incubation

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at 22 °C with shaking for 15 minutes in α-chymotrypsin, the proteolysis was stopped by adding 30 µL/mL of a stock of 100 mM phenylmethylsufonylfluoride (PMSF). The proteolyzed myosin products were dialyzed overnight at 10 °C in 1 L of 0.04 M KCl, 10 mM imidiazole, pH 7.0. A

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150 µL portion was saved for gel electrophoresis. To separate the fragments, the proteolyzed products were centrifuged at 47,000 RPM in a TLA 100.3 rotor for 1 hour at 4 °C. The

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supernatant containing the S1 myosin subfragment was collected and saved for electrophoresis. The pellet containing leftover undigested myosin and rod fragments was re-suspended in low salt buffer (100 mM KCl, 25 mM K2HPO4, 2 mM MgCl2, pH 7.0). To affinity purify the myosin rod, actin was added in a 9:1 molar ratio to the re-suspended pellet of undigested myosin and rod

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fragments. Then the mixture was incubated for an hour in 0.3 M KCl. After incubation, the mixture was centrifuged at 40,000 RPM for 1 hour at 4 °C. The supernatant containing the purified rod fragments were transferred into a separated microcentrifuge tube for further use

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while the pellet containing the undigested myosin bound to actin was re-suspended in low salt

DNA

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buffer and saved for electrophoresis.

Lambda Phage DNA (N3110S, New England Biolabs, Inc.) was used as a control to test

the free fall mode of GFS. Lambda phage DNA was extended at its overhanging ends for an hour at 30 0C in presence of Taq DNA polymerase (D1806, Millipore Sigma) and dATP, dGTP, dTTP (Thermo Fisher Scientific) with diethylenetriamine pentaacetate (DTPA) tagged dCTP to crosslink the DNA to edge of the aminosilanated glass coverslip and glass beads using 1-ethyl-3-(38

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dimethyaminopropyl) carbodiimide in a manner similar to the methods for cross-linking proteins described in the GFS method section.

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Methods Simulated Force Spectroscopy

X-ray crystallography structures of myosin were imported as atomic models from the

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Protein Data Bank. Proximal regions of the human cardiac myosin S2 (2FXM) [31] and LMM (4XA3) [32] after removing nonmyosin end segments were used as starting points for the

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simulations.

Molecular mechanics simulations were performed with the Macromodel 9.7 and Maestro 9.0 software (Schrödinger, Inc). For conformational searches and energy minimizations, the OPLS-2005 force field and generalized-born surface area effective solvation model (simulation

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of aqueous environment) were applied. Energy minimizations were run to convergence at 0.05, or 10000 iterations. Global energy conformational searches were run to convergence at 1.00, or 10000 iterations using the mixed torsional/large scale low mode sampling method and the

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structures were saved at an energy window of 21 kJ/mol. SFS was performed as previously described [33]. The atomic models were pulled in opposite directions from 4 different positions.

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The first position was the N-terminus for the prehinge position. The second was E875 to simulate where MF30 antibody binds on the S1-S2 hinge. The third was E927 to simulate where the sitespecific polyclonal antibody binds on S2, which is considered a posthinge position. The last was K1392 in the LMM region. The same amino acids were picked on both helices to initiate the pulling. The distance between the amino acids was measured first, then incremented 1 angstrom at a time until right before the helix separated. Calculation of the force required to extend the

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coiled coil myosin molecules was based on the assumption that the coiled-coil structures behave as springs in molecular mechanics simulations. Force was calculated by the derivation of the Hooke’s law;






= √2

(1) (2)

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Substituting x in above equation with




=    =  

(3)

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where, k is the force constant, x is the length of extended molecule at a given force and E is the

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given constraint energy. Ramachandran plots were generated for each structure, and the α-helix content was determined throughout the separation procedure. Note that SFS produces forces larger than experimental values because the experimental time scale is much longer than the simulated time scale, so the structures can relax more. As previously published, a similar shape of force-distance curve with lower forces approaching that of experimental values is possible

current experiments [33].

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with extraordinarily long simulations times that are not possible with the larger molecules in the

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Gravitational Force Spectroscopy

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GFS assays were performed as described by Dunn and Root, 2011, with modifications to the protocol [23]. The cofilament assays used only the rotational mode with a two bead assay as previously to facilitate statistical analysis. All other assays used free fall mode and edge assays as described hereafter: The edge of a coverslip was used instead of the large bead as the immobile component in the free fall assays, to simplify the molecular length calculation and to enable assays of multiple single molecules simultaneously. The free fall mode of GFS allows a graded force to be implemented on a single molecule. The entire GFS can be dropped on a spring 10

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supported platform with the gravitational force acting on to the molecule tethered between edge and bead. The acceleration trace of GFS combined with the mass of the bead yields the force distance curve for the molecule in the free fall state (Fig. 1). For the GFS assay, a myosin

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molecule was tethered, at myosin S1-S2 hinge, posthinge, and at LMM position with the antibodies MF30, polyclonal anti-S2 and MF20, respectively (Fig. 2). The antibodies were conjugated to cut edges of glass coverslips and silica beads with the method described earlier.

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The myosin molecule was tethered at its prehinge position by rigor binding of myosin S1 to actin. Actin was cross-linked to the aminosilanated glass coverslips and silica beads with the

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help of 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (0.6M). N-hydroxysuccinamide was added to 60 mM to assist the cross-linking of actin to glass coverslips and silica beads. The unreacted groups were blocked by adding the glycine quenching solution (1 M glycine in water with pH 7.0) to stop the reaction. After quenching, the actin coupled glass coverslips and silica

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beads underwent several washes with low salt buffer (0.1 M potassium chloride, 0.02 M imidazole, 0.5 mg/ml bovine serum albumin, 5 mM magnesium chloride, with pH 7.0) and were later stored in this low salt buffer for force spectroscopy purposes. A vacuum grease sealed four

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walled chamber was created on a glass slide containing actin or antibody-conjugated mobile beads and the molecule of interest, with one of the walls being the edge of the coverslip

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conjugated with actin or antibody. The slide was scanned along the immobile edge for a molecule tethered between the edge of the coverslip and mobile bead. The GFS measurements were performed with images captured on a digital video camera (Sony XCD-V60). The images were analyzed in Image J (NIH) to calculate the length of the molecule using the force distance equation as previously described [23]. The carbodiimide and N-hydroxysuccinamide coupled cross-linking reaction was also utilized to tether lambda DNA amplified with DTPA tagged

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dCTP between the edge of the coverslip and glass bead to test the GFS. DNA measurements were made at room temperature, in buffer containing 10 mM Tris hydrochloride, 50 mM potassium chloride, and 2 mM magnesium chloride at pH 8.3. Note that the unique experimental

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ability of GFS to determine absolute molecular lengths means that in all GFS force-distance

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curves presented, the distance is the molecular length and not just a relative amount of extension.

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Figure 1. Free fall assay of GFS. (A) Schematic of a GFS with microscope on an alt-azimuthal telescopic mount suspended on springs, capable of doing rotational mode [23] (green arrow) and a free fall mode (red arrow) against the gravity vector (orange arrow). (B) Sketch of a molecule

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of interest (red) tethered between edge of the coverslip and silica or glass bead (grey sphere) by antibody (blue) raised against the molecule of interest. The molecular length before free fall is calculated as dmax-dmin. The force in pN imparted by the microsphere, including the subtraction

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of buoyancy, is a(4/3)(ρ-w)π [(dmax-dmin)/(1/cos(αmin)-1)]3 in which a is the acceleration due to gravity (0.0098 km/s2) multiplied by the unit conversion correction, ρ is the density of the

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microsphere in g/cm3, w is the density of water = 1 g/cm3, αmin is the angle between the gravity vectors for dmax and dmin at the angle of its first occurrence, and dmax and dmin are in microns. (C) Acceleration trace for GFS when dropped freely against the spring constant under gravitational force. The acceleration is in the frame of reference of the mobile bead, so it is initially zero

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during free fall. Force is calculated with Newton’s second law of motion, F=ma, and the buoyancy of the microsphere is subtracted. (D) The trace of the molecular length for the

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molecule undergoing the free fall mode of GFS.

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Figure 2. Schematic of points for force applied perpendicular to coiled coil axis, from right to left. Prehinge point where actin (blue sphere) and myosin subfragment-1 interaction were used as the source of suspending a single molecule of myosin for the force assay. Hinge point where

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MF30 (green arrow), a monoclonal antibody raised against myosin S1-S2 hinge and confirmed to be within 5 nm of the regulatory light chain [28], was used as the source of suspension. Posthinge point where polyclonal anti-S2 antibody (red arrow) was used as the source of suspension. LMM

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point where MF20 (orange arrow), a monoclonal antibody raised against LMM, was used as a

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source of suspension for force assay.

Outlier Removal in Cofilament Assays and Other Statistical Methods To ensure that measurements were made only within a single subunit of a cofilament,

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intact myosin was copolymerized with affinity purified myosin rod at a ratio of 1:100, and statistical filtering of outliers was performed. To test the method, higher ratios than 1:100 of myosin to rod were used in separate experiments to yield multiple intact myosin subunits per

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cofilament, yet no significant difference in the force-distance curves were detected at these higher ratios. Outliers were removed from force distance graphs using the fourth spread method

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[34]. Since the data followed a normal distribution in the probability of occurrence, the outliers were assumed to lie on the far end of the data. The fourth spread method was used to quantify the ranges in which the bulk of the data lies [34]. The extremes were defined by boundaries calculated within the data set that approximated data within one standard deviation of the data on the lower and the upper ends.

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The data sets were normalized by calculating the ratio of distance to force. For each experimental data set of sample size n, each observed ratio xi, was listed in order from least to greatest, x1 ≤ x2 ≤… ≤ xn. From the ordered list of observed ratios the upper quartile (FU) and

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lower quartiles (FL) were established. The upper quartile (FU) was calculated by finding the median of the upper half of the observed ratios: FU = x([3n+1]/4). The lower quartile was calculated by finding the median of the lower half of the observed ratios: FL = x(0.5[

(n + 3)/2]).

A set of

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resistant rules were applied to the data sets to establish the upper (IFU) and lower (IFL) inner fences of the data. Outliers were removed if greater than the inner upper fence boundary: IFU =

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FU + 1.5 (FU – FL) or lower than the inner lower fence boundary: IFL= FL – 1.5 (FU – FL). For free fall GFS and corresponding SFS, molecular lengths were binned into 5 nm intervals to enable averaging between groups of individual single molecules. The 5 nm bin range was chosen to correspond to the standard deviation of molecular lengths of S2 measured by GFS

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on a group of 8 individual single molecules. Unless otherwise indicated, all error bars represent

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the standard error of the mean (SEM).

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In vitro motility assay

The in vitro motility assay of actin filaments sliding over myosin filaments on a 2%

dimethyldichlorosilane-coated coverslip was performed in 1 mM ATP diluted in buffer containing 4 mM magnesium chloride, 10 mM imidazole, 10 mM dithioerythritol, 25 mM potassium chloride, 0.5 mg/ml bovine serum albumin, 0.5% methylcellulose, 1% glucose, 45 µg/ml catalase and 25 µg/ml glucose oxidase at 30 0C. Actin filaments were labeled with rhodamine phalloidin and excited with a green LED light source on an epifluorescent microscope 15

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and imaged with an ICCD 350F (Videoscope Int., Sterling, VA). The microscope was equipped with a stage heater to regulate the temperature at 30 0C as measured with a thermocouple. The motility of actin was recorded on a SVHS video recorder (Panasonic) before and after adding an

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equimolar concentration of MyBPC to the concentration of myosin used to treat the coverslip. Images were grabbed from SVHS tape using the frame grabber and later analyzed by Image J (NIH) software. The movements of actin filaments were tracked using the manual tracking

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Image J plugin.

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Results

Myosin S2 coiled coil is mechanically unstable in a direction perpendicular to the thick filament axis

SFS and GFS tested the mechanical instability of the myosin S2 coiled coil by pulling the

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coiled coil parallel or perpendicular to the thick filament axis. The crystal structure of β-cardiac myosin S2 coiled coil (2FXM) was uncoiled through molecular simulation. The atomic model was pulled from the N-terminus on one end and the C-terminus on the other side of the coiled

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coil to simulate parallel uncoiling, and the N-terminal ends of both the helices to simulate perpendicular uncoiling. SFS results showed that perpendicular uncoiling provided less

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resistance compared to parallel uncoiling. The force required to uncoil the myosin S2 coiled coil in the perpendicular direction was typically less than 500 pN, while force required to uncoil in the parallel direction was up to nearly 3500 pN (Fig. 3A).

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Figure 3. Uncoiling of myosin S2 in parallel and perpendicular direction of myosin thick filament axis. (A) Uncoiling of myosin S2 in both parallel and perpendicular directions to the thick filament axis by SFS. (B) Uncoiling of myosin S2 in a parallel (red circles with dashed lines for WLC fit between MF20 and MF30) (n=5) and perpendicular (dashed orange line, logarithmic fit from Fig. 8D, and dashed green line, logarithmic fit from Fig. 8B) direction by GFS. Inset figure (dashed black line) indicates the pulling of myosin molecule (red) with MF30 17

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antibody (green arrow and circles) and MF20 antibody (orange arrow and circles). (C) Stretching of DNA in a direction parallel to the axis of DNA length using GFS. The composite trace of force distance curves for DNA with red circles and WLC fit with dashed lines for one DNA molecule subjected to forces less than 60 pN, and blue circles and fit with dashed lines for

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another DNA molecule subjected to forces higher than 60 pN are shown. Inset figure illustrates the DNA (blue) with DTPA-dCTP (blue circle) on opposite strands with a black arrow indicating the direction of stretching. Video for the GFS measurement of DNA molecule is available online

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in supplementary data (Supplementary Video 1).

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For GFS, the myosin molecule was tethered by the MF30 antibody from the myosin S1S2 hinge on one end and the MF20 antibody from myosin LMM on the other end, thus encompassing the myosin S2 between both ends. The myosin molecule was pulled against the gravity vector, thus uncoiling the myosin molecule in a direction parallel to the long axis of the coiled coil. The force-distance curve (Fig. 3B) demonstrated the three typical phases: rise phase,

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plateau phase and extension phase in which both the rise phase and the extension phase could be separately fit by the worm like chain model (WLC) [33]. In contrast, the myosin molecule

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tethered at its myosin S1-S2 hinge by MF30 antibody on both the ends and by MF20 antibody at its LMM position, and pulled against the gravity vector, allowed uncoiling of the myosin

phase.

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molecule in a direction perpendicular to the myosin thick filament axis without an extension

The force-distance curve of the myosin coiled coil pulled parallel to its long axis is

readily fit by a WLC model [33] (Fig. 3B), yielding the contour length of 131 ± 16 nm at force less than 25 pN and 249 ± 6 nm at forces higher than 25 pN with 1.9 times extension of myosin molecule at higher forces:

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=

  







 / −  +  

(4)

in which F is the force at extension x,  is Boltzmann’s constant,

is the absolute

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temperature, ! is the persistence length, and " is the contour length. Analogously, the forcedistance curve of DNA pulled parallel to its long axis is also fit well by the WLC model with a B to Z DNA transition at nearly 60 pN [35,36]. The force distance curve for parallel stretching of

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DNA by GFS fitted the WLC model (Fig. 3C) and yielded a contour length 1.8 times higher for the DNA molecule at high force load (Z DNA) compared to one at low force (B DNA). The

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altered DNA extension trace at lower forces may be due to the partial single stranded form of DNA at lower ionic condition as observed by Smith et al (1996) [36]. While the force-extension curve of DNA stretched parallel to its long axis is well fit by WLC, when DNA is unzipped perpendicular to its long axis, the resulting force-distance curve is best fit by a model other than

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the WLC [37,38]. Similarly, when the myosin coiled coil is unzipped perpendicular to its long axis, a different model better fits the data. The apparent logarithmic fit of these data from unzipping myosin suggest that the force is entropically driven:

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∫F⋅dx = ∫dW = ∫dG = ∫dH - ∫T dS,

(5)

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in which F is force, W is work whether reversible or irreversible according to the Jarzynski equality [39], G is free energy, H is enthalpy, T is temperature, S is entropy, and x is the extension. F ∫-dx = ∫dH -  T ln Ω = ∫dH -  T Σ pi ln pi ≈ ∫dH - v  T x ln x,

(6)

Dividing by ∫-dx and integrating yields, F = v  T ln x - v  T ln xo - ∫dH/∫dx = v  T ln (x/xo) - ∫dH/∫dx

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(7)

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where - v  T ln xo is the integration constant in which the reference extension, xo, may be set to 1 for the chosen unit system to simplify to, F = v  T ln x - ∫dH/∫dx

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(8)

in which Ω is the number of microstates from the third law of thermodynamics,  is Boltzmann’s constant, pi is the probability that a microstate will be occupied, and v is a

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proportionality constant (in units of distance-1) that is related to the reduction in entropy as the polypeptide length is extended or more specifically the relation between extension and the

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probabilities that various microstates will be occupied. If the coiled coil unzips linearly with extension into an extended polypeptide as depicted in the images of the dynamics simulations in Figure 4, then the probabilities should be changed proportionally, since the fully extended peptide has very low entropy. The sign of ∫-dx is negative because the force vector of the

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molecule resisting stretch is in an opposite direction to the externally applied force and extension. In this model, the force-distance relationship is logarithmic if the change in enthalpy with extension is relatively constant.

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Prehinge extension

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Posthinge extension

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LMM Extension

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Hinge extension

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Figure 4. Images from SFS and plots of broken hydrogen bonds of the myosin S2 coiled coil uncoiled at different positions along the length of the myosin rod. (A) and (E) Myosin S2 uncoiled at pre-myosin S1-S2 hinge position. The plot of broken hydrogen bonds per extension of myosin S2 molecule at prehinge position is linear with a correlation coefficient of 0.971. (B) and (F) Myosin S2 uncoiled at hinge position. The plot of broken hydrogen bonds per extension of myosin S2 molecule at hinge position is linear with a correlation coefficient of 0.991. (C) and (G) Myosin S2 uncoiled at post-myosin S1-S2 hinge position. The plot of broken hydrogen bonds per extension of myosin S2 molecule at posthinge position is linear with a correlation 21

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coefficient of 0.982. (D) and (H) Myosin S2 uncoiled at LMM position. The plot of broken hydrogen bonds per extension of myosin S2 molecule at LMM position is linear with a correlation coefficient of 0.961. Videos of the simulations are available in the online supporting

In the equation used to fit the unzipping data,

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F = a ln (x + b) + c

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information [Supplemenatry Video 2].

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the floated parameters are a, b, and c. The parameter a = v  T. The term (x + b) replaces x in the derived equation, because b represents the length of the linker elements such as antibodies. The parameter c = -∫dH/∫dx is assumed to be nearly constant due to the mostly uniform breaking of hydrogen bonds and other noncovalent bonds of the coiled coil during extension as illustrated in the SFS simulations in Fig. 4E-H. (Note that c may also contain the integration constant from

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equation (7) if the reference extension, xo, is chosen to be other than 1.) The force-distance curve fit yielded correlation coefficients of 0.942 and 0.969 for hinge

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and LMM positional uncoiling, respectively (Table 2). The force-distance relationship for uncoiling of the myosin molecule at the myosin S2 region showed that the myosin S2 instability

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is in the direction perpendicular to the thick filament axis rather than the parallel direction. All subsequent SFS and GFS experiments were performed to uncoil the myosin molecule in a direction perpendicular to thick filament axis to test the instability of myosin S2.

Thick filament interactions of myosin do not alter the instability of myosin S2

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Figure 5. Individual single molecules of myosin (red circles and dashed line) have a similar

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force-distance curve when compared to those of a single myosin (blue triangles and dotted line) or multiple myosin (green squares and line) dimers in a myofilament when pulled from the prehinge position. Higher ratios of myosin to rod in the cofilament (green squares) do not alter the conclusion that S2 stability is not affected significantly by myosin polymerization. Each

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plotted point is a different single molecule measured by GFS without free fall. Myosin molecules are arranged into thick filaments in the muscle sarcomere by the association of myosin rods and anchored at the M-line of the sarcomere [40,41]. It is possible

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that the myosin S2 coiled coil could be stabilized by these interactions of myosin molecules in the thick filament. GFS was performed to uncoil the myosin dimer as a single molecule, a

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cofilament of a myosin rod and a single intact myosin, or a cofilament of a myosin rod and multiple intact myosins. This test compares the instability of the myosin coiled coil as a single molecule with the coiled coil in a thick filament assembly. Testing the instability of the myosin coiled coil was also performed on a myosin dimer with multiple myosin dimers present on the same thick filament to assess whether the presence of other myosin dimers affects the forcedistance curve. The results indicate similar force-distance curves for a single molecule of myosin

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dimer, or a single myosin dimer in a cofilament with the myosin rod, or multiple myosin dimers in a cofilament with the myosin rod (Fig. 5). The force required to uncoil the myosin molecule at the prehinge position to a length of 100 nm was similar (Table 1), and Student’s t-tests on each

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compared pair of the curve fitted parameters did not detect a statistically significant difference (p>0.05). Therefore, the mechanical stability of myosin S2 is independent of its association with

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the thick filament, and the presence of other myosin dimers do not affect this stability.

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Myosin coiled coil is unstable at its S2 region compared to its LMM region The myosin coiled coil was uncoiled at four different positions to test its instability at various points along its length. The four positions tested were the N-terminal start of the coiled coil (prehinge), the MF30 binding site (hinge), the site-specific polyclonal binding site

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(posthinge), and the MF20 binding site (myosin LMM) (Fig. 2). For GFS, actin was bound to the myosin S1 heads in order to uncoil the myosin molecule at the prehinge position. Similarly, the MF30 monoclonal antibody against myosin S1-S2 at the hinge position, polyclonal anti-S2

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antibody at the posthinge position, and MF20 monoclonal antibody against LMM at the LMM position were utilized (Fig. 2). The myosin coiled coil was uncoiled at similar positions in the

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perpendicular direction for SFS (Fig. 6). Since the data for SFS in Figure 6 are based on atomic models with no extra linker (e.g. such as an antibody), there is an additional measured point below 2.5 nm at zero applied force. The data have been binned to the same degree as the GFS data, but because the available atomic models are short, there are a limited number of points (5-7 points per curve depending on the available atomic model and the position of the applied force) The correlation coefficients for the fits range from 0.928 to 0.987. The plots with fewer data

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points yielded lower correlation coefficients, but a correlation coefficient of even 0.928 from the LMM atomic model is not inconsistent with the fitted model; although, the larger S2 atomic

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model produced more convincing fits and correlation coefficients.

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Figure 6. Simulated force spectroscopy of the myosin molecule uncoiled from pre-myosin S1-S2 hinge (blue diamond), myosin S1-S2 hinge (green square), post-myosin S1-S2 hinge position

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(red triangle), and LMM (orange circle).

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E

Figure 7. Force-distance curve for uncoiling of myosin molecule at different positions along the

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myosin coiled coil. (A) Force-distance curve for uncoiling of myosin molecule at prehinge position (n = 7). (B) Force-distance curve for uncoiling of myosin molecule at hinge position (n= 3). (C) Force-distance curve for uncoiling of myosin molecule at posthinge position (n = 7). (D) Force distance curve for uncoiling of myosin molecule at LMM position (n = 3). (E) Force distance curve for uncoiling of myosin molecule with bound MyBPC at posthinge position (n =10). For cartoons (dashed square): Myosin molecule (red), Prehinge position with actin (blue

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arrow), Hinge position with MF30 antibody (green arrow), Posthinge position with polyclonal anti-S2 antibody (red arrow), LMM position with MF20 antibody (orange arrow), and MyBPC (purple line).

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Force-distance curves from GFS and SFS indicated that the myosin molecule is more unstable at its myosin S2 region compared to its LMM (Fig. 6 and Fig. 7, Table 2). SFS and GFS

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results with myosin uncoiling at the prehinge, hinge, and posthinge positions gave similar trends. The uncoiling at the prehinge and hinge positions were less resistant to force compared to that of the posthinge position (Fig. 6 and Fig. 7, Table 2). Differences between prehinge and hinge force-distance curves are most apparent at the start of the SFS curve, when the pulling at the hinge must initially unravel two coiled coils per strand, while the prehinge unravels only one coiled coil per strand. In GFS, the MF30 antibody binding may largely unravel the N-terminus of

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the coiled coil before force is applied, so the resulting force-distance curve is more similar to the GFS of the prehinge position.

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Such impacts of the antibodies as linkers are accommodated in the fitted equation by parameter b. Since ln (0) is not a defined function, the y intercept does not necessarily occur at all and does not have a specific meaning. Parameters a, b, and c, all influence the approach to the

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the y axis, and each contribute to all points along the curve. The logarithm of a small number can be very negative, but the experimental force is never a negative number, and Brownian motion

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will become dominant at very small forces. For simplicity, the fitting equation does not include Brownian motion terms for such low forces, since the experiment did not acquire significant data in that region.

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MyBPC stabilizes the myosin S2 coiled coil

MyBPC binds to myosin, with its C-terminal end binding to myosin LMM and its Nterminal end binding to myosin S2 [8]. This binding appears to stabilize the myosin S2 coiled

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coil. GFS was performed with myosin treated with full length MyBPC at an equimolar

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concentration and uncoiled at the posthinge position. The force required to uncoil the myosin molecule in the presence of MyBPC at the posthinge position was 3 times greater compared to that in the absence of MyBPC (Fig. 7E, Table 2 values in bold). The deviation in the force fit for the myosin molecule with MyBPC was expected, since the binding of MyBPC to the myosin S2 coiled coil would alter the enthalpy change in parameter c of the logarithmic force fit. This result indicates that the myosin S2 coiled coil is stabilized upon binding of MyBPC. MyBPC bound myosin reduces the motility of actin thin filaments 28

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Stabilization of the myosin S2 coiled coil by MyBPC could decrease the number of myosin S1 heads to binding actin. Thus, the in vitro motility of Rh-Ph labeled actin thin filaments over myosin thick filament in the presence of MyBPC was assessed. Since MyBPC

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requires the LMM along with myosin S2 to bind to the myosin thick filament, the use of myosin HMM alone would be insufficient to bind full-length MyBPC; hence, myosin thick filaments were used. For the assay, the motility of actin filaments in the absence of MyBPC and after the

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addition of an equimolar concentration of whole length skeletal MyBPC to the same slide

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enabled the comparison of actin thin filament motility in the presence and absence of MyBPC.

Figure 8. Histogram of in vitro motility assay with MyBPC. (A) Histogram plot showing distribution of in vitro actin filament motility over myosin thick filaments in the absence (blue) (n=33) and presence (orange) of full-length skeletal MyBPC (n=44). (B) Histogram showing 29

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average velocity of actin thin filaments in absence (blue) and presence (orange) of full-length skeletal MyBPC. Histogram distribution plots of the motility of actin filaments in absence and presence of

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MyBPC showed reduced motility of actin sliding over the myosin thick filament treated with MyBPC (Fig. 8A). Sliding velocities of actin filaments in the absence of MyBPC were on average 6.64 um/sec ± 0.211, compared to 5.12 um/sec ± 0.153 in the presence of MyBPC (Fig.

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8B). Thus, stabilization of myosin S2 by MyBPC is accompanied by a reduction in the motility

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of actin filaments. Discussion

The myosin S2 has long been viewed as an elastic element in the myofilaments of muscle. While its exact role has been debated, many observations suggest that it plays a significant role in the function of muscle myosin. In vitro motility assays exhibit similar actin

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sliding velocities for HMM and myosin, which both contain S2. Yet S1, which lacks S2, produces greatly reduced velocities, suggesting at a minimum that the instability of the S2 coiled coil imparted freedom of movement to the S1 heads to facilitate motility. Polyclonal antibodies

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against the folded S2 coiled coil structure, which likely increase S2 rigidity, inhibit force

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generation in skinned fibers [29]. Furthermore, temperature jump experiments that preferentially melt S2 accelerate actin sliding in in vitro motility assays and also enhance fiber contraction [42,43]. The medical importance of S2 is underlined by the observation that mutations causing hypertrophic cardiomyopathy are found at a high frequency in S2, near the N-terminal side (supplementary data) [44-50] and that a subset of these mutations are among the most lethal, with sudden cardiac arrest rates of around 50% for family members who inherit them [51,52].

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Force spectroscopy has previously been applied to the myosin coiled coil, which is one of the longest coiled coils in nature. Schwaiger et al. (2002) [52] identified the characteristic shape of the force-distance curve of myosin when pulled parallel to the long axis of the filament, while

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Taniguchi et al. (2010) [54] analyzed contributions of a free-energy barrier to the uncoiling. Root et al. (2006) [33] examined the S2 proteolytic subfragment and identified specific uncoiling events that occurred at approximately 40 pN of force and suggested the existence of unfolded

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segments in S2 prior to force-dependent unfolding. Given that 40 pN is greater than the forces generated by single myosin heads, the intact coiled coil is expected to remain stable when pulled

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parallel to the axis of the thick filament; however, Gundapaneni et al. (2005) [30] found that the myosin coiled coil spontaneously unwinds perpendicular to the thick filament axis even under zero load. In line with this observation, (Fig. 3) illustrates that GFS requires much less force to unravel the coiled coil when applied perpendicular to the filament axis than when applied

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parallel to the filament axis, and SFS yields similar results. Thus, it is quite likely that S2 partially unravels and refolds when not under tension, which may impart more degrees of freedom for myosin heads to find a productive actin binding orientation. To clarify which parts

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of the myosin rod are most susceptible to unraveling upon the application of force perpendicular to the myosin filament axis, several sites were probed by GFS and SFS. The site in the LMM

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proved to be the most resistant to unraveling compared to sites in the S2 (Fig. 7D). This result correlates well with previously reported measurements of proteolytic susceptibility of S2, which can yield long or short S2, while LMM preparations are more consistent in size, indicating greater stability [55]. Similarly, Highsmith et al. (1977) [56] found through spectroscopic measurements that LMM behaved like a rigid cylinder while S2 was more flexible. In addition,

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Walker et al. (1985) [57] observed more bending in the S2 than the LMM by electron microscopy.

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The instability of S2 relative to LMM may not be consistent along its entire length, so three sites were compared by both GFS and SFS (Fig. 7A-D, Table 2). While each site in S2 was more readily unraveled than LMM, the prehinge and hinge sites required less application of force

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to uncoil S2 than the posthinge site. From the atomic modeling results, it is apparent that pulling on the coiled coil from an internal position requires the extension of twice as many strands

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compared to pulling the coiled coil from its N-terminal end, and the relative increase in force required for extending twice as many strands is similar in both SFS and GFS measurements. It has been reported that scallop myosin S2 is particularly unstable at its N-terminal end, indicating that this region carries out a unique function [58]. Indeed, some nonmuscle myosins are thought to increase their step size by uncoiling the N-terminal coiled coil [59]. The high variability of

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myosin head positions seen in electron microscopy of vertebrate striated muscle myosin may be related to this N-terminal coiled coil instability [57].

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The stability of S2 may be modulated by its interaction with other proteins. For this reason, the effect of myosin filament formation on GFS of the prehinge region was investigated

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using cofilaments of myosin rod and a low ratio of intact myosin. Our results did not show a significant difference in the force-distance curves between the myosin in the cofilament and unpolymerized myosin (Fig. 5). Consistent with this finding, the rate of tryptic digestion of the S1/S2 hinge is not inhibited by myosin filament formation, even though actin binding strongly inhibits this proteolysis, perhaps by a steric effect [60].

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Myosin binding protein C (MyBPC) is another potential modulator of S2 stability. MyBPC binds with high affinity to LMM (Kd less than 500 nM) [8], but its affinities for actin (Kd less than 4300 nM) [12], and S2 (Kd = 5000 nM) [7] are much weaker. Thus, LMM likely

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acts as a docking site for MyBPC while its N-terminal region interacts with other sites due to increased effective concentration. The data in (Fig. 7E) indicates that MyBPC increases the stability of S2 by 3-fold (Table 2), while not competing with the site-specific polyclonal

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antibody for binding to the posthinge region. It is unlikely that such an increase in stability can be conferred by binding of MyBPC to LMM alone, so MyBPC is interacting with S2 at

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concentrations well below its Kd to S2 due to its higher effective concentration. MyBPC’s interaction with S2 has been reported to be abolished by phosphorylation [15]. The purified MyBPC is typically dephosphorylated [13]. Furthermore, the peak measured pI of the purified MyBPC by chromatofocusing of 5.6 matched the calculated pI of 5.6 for the

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dephosphorylated protein. If the MyBPC were phosphorylated, the pI should be lower than 5.6. Therefore, MyBPC is expected to retain its affinity for S2 in these experiments.

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The addition of MyBPC to an in vitro motility assay immediately slowed actin sliding over myosin filaments (Fig. 8), which may be caused by the stabilizing effect of MyBPC on the

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S2, if this stabilization reduces the ability of myosin heads to bind productively to actin. Such an inhibition could be caused by facilitating binding of the myosin heads to S2 as occurs in unphosphorylated smooth muscle myosin, certain nonmuscle myosins, and especially the thick filaments of invertebrate striated muscle myosins [21, 61-63]. It is possible that a stable coiled coil structure is necessary for myosin heads to dock on S2, and an unstable S2 might therefore reduce docking and increase myosin activity.

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An alternative explanation for the inhibition of actin sliding in the in vitro motility assay by MyBPC is the potential for interaction between MyBPC and actin [64]. While MyBPC phosphorylation does not affect the Kd of MyBPC for actin, phosphorylation has been reported to

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reduce the Bmax [12,14,65]. In a nonphosphorylatable MyBPC mouse model, contractile functions are compromised compared to wild-type, which suggests that phosphorylation is required for optimum crossbridge attachment rates [66,67]. Such a response is more consistent

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with the phosphorylation reducing MyBPC binding to S2 and thereby increasing myosin head availability to bind actin. Nag et al. (2017) [68] has also shown that myosin S1 forms a complex

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with myosin S2 and MyBPC while these interactions are dampened by the phosphorylation of myosin S1 or MyBPC. However, it is also possible that the phosphorylated MyBPC that is released from binding S2 could then bind actin and displace tropomyosin to help activate the thin filament and possibly reduce actin sliding velocity as a consequence of the tether between thick

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and thin filaments [69]. Such a switch from binding to S2 to binding actin could correspond with a need for higher isometric force rather than more rapid shortening velocity, since few myosin heads are required for high shortening velocities against a low load. When near isometric

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conditions are present, MyBPC could be phosphorylated and undock from S2 which releases more force generating myosin heads to bind to actin. Its subsequent binding to actin could aid

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force generation by increasing thin filament activation, and the slowing of shortening velocity caused by tethering the thin and thick filaments would have minimal impact during isometric conditions.

The results presented here indicate that the stability of S2 can play a significant role in muscle contraction. S2 is intrinsically unstable relative to LMM based on GFS when force is applied perpendicular to the myosin filament axis. The stability of S2 is not increased by 34

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polymerization of myosin into filaments; however, MyBPC has a profound impact on increasing the stability of S2. This increase in S2 stability caused by MyBPC binding can decrease actin sliding velocities during in vitro motility assays. MyBPC’s stabilizing role is consistent with

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observed impacts of MyBPC phosphorylation in mouse models reported in the literature. S2 stability appears to modulate contractile responses, which can have important implications for

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disease-causing mutations localized to this region.

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Acknowledgment The authors thank Dr. Kuan Wang for his discussions on the implementation of the

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free fall gravitational force spectroscopy and Dr. David M. Warshaw for his insightful comments on the manuscript. Funding from NSF #0842736 ARRA to D.D.R. supported

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this research.

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44. Tesson, F., Richard, P., Charron, P., Mathieu, B., Cruaud, C., Carrier, L., Dubourg, O., Lautié, N., Desnos, M., Millaire, A., Isnard, R., Hagege, A.A., Bouhour, J.B., Bennaceur, M., Hainque, B., Guicheney, P., Schwartz, K., and Komajda, M. (1998) Genotypephenotype analysis in four families with mutations in beta-myosin heavy chain gene responsible for familial hypertrophic cardiomyopathy. Hum Mutat. 12, 385-392. 45. Richard, P., Charron, P., Carrier, L., Ledeuil, C., Cheav, T., Pichereau, C., Benaiche, A., Isnard, R., Dubourg, O., Burban, M., Gueffet, J.P., Millaire, A., Desnos, M., Schwartz, K., Hainque, B., and Komajda, M. (2003) Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation 107, 2227-2232.

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46. Erdmann, J., Daehmlow, S., Wischke, S., Senyuva, M., Werner, U., Raible, J., Tanis, N., Dyachenko, S., Hummel, M., Hetzer, R., Regitz-Zagrosek, V. (2003) Mutation spectrum in a large cohort of unrelated consecutive patients with hypertrophic cardiomyopathy. Clin. Genet. 64, 339-349.

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48. Cho, K.W., Lee, J., and Kim, Y. (2016) Genetic variations leading to familial dilated cardiomyopathy. Mol Cells. 39, 722-727. 49. McNally, E.M., Barefield, D.Y., and Puckelwartz, M.J. (2015) The genetic landscape of cardiomyopathy and its role in heart failure. Cell Metab. 21, 174-82. 50. Kooij, V., Holewinski, R.J., Murphy, A.M., Van Eyk, J.E. (2013) Characterization of the cardiac myosin binding protein-C phosphoproteome in healthy and failing human hearts. J. Mol. Cell. Cardiol. 60, 116-120. 51. Tesson, F., Richard, P., Charron, P., Mathieu, B., Cruaud, C., Carrier, L., Dubourg, O., Lautié, N., Desnos, M., Millaire, A., Isnard, R., Hagege, A.A., Bouhour, J.B., Bennaceur, M., Hainque, B., Guicheney, P., Schwartz, K., and Komajda, M. (1998) Genotype40

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phenotype analysis in four families with mutations in beta-myosin heavy chain gene responsible for familial hypertrophic cardiomyopathy. Hum Mutat. 12, 385-392.

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52. Waldmüller, S., Sakthivel, S., Saadi, A.V., Selignow, C., Rakesh, P.G., Golubenko, M., Joseph, P.K., Padmakumar, R., Richard, P., Schwartz, K., Tharakan, J.M., Rajamanickam, C., and Vosberg, H.P. (2003) Novel deletions in MYH7 and MYBPC3 identified in Indian families with familial hypertrophic cardiomyopathy. J Mol Cell Cardiol. 35, 623-636. 53. Schwaiger, I., Sattler, C., Hostetter, D.R., and Rief, M. (2002) The myosin coiled-coil is a truly elastic protein structure. Nat Mater. 1, 232-235.

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54. Taniguchi, Y., Khatri, B.S., Brockwell, D.J., Paci, E., and Kawakami, M. (2010) Dynamics of the coiled-coil unfolding transition of myosin rod probed by dissipation force spectrum. Biophys J. 99, 257-262.

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58. Li, Y., Brown, J.H., Reshetnikova, L., Blazsek, A., Farkas, L., Nyitray, L., and Cohen, C. (2003) Visualization of an unstable coiled coil from the scallop myosin rod. Nature 424, 341-345.

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60. Duong, A.M., and Reisler, E. (1987) The binding of myosin heads on heavy meromyosin and assembled myosin to actin in the presence of nucleotides. Measurements by the proteolytic rates method. J Biol Chem. 262, 4129-4133. 61. Zoghbi M.E., Woodhead, J.L., Moss, R.L., and Craig, R. (2008) Three-dimensional structure of vertebrate cardiac muscle myosin filaments. Proc Natl Acad Sci U S A. 105, 2386-90 62. Kensler R.W., and Harris, S.P. (2008) The structure of isolated cardiac Myosin thick filaments from cardiac Myosin binding protein-C knockout mice. Biophys J. 94, 1707-18 63. Colson, B.A., Bekyarova, T., Fitzsimons, D.P., Irving, T.C., and Moss, R.L. (2007) Radial displacement of myosin cross-bridges in mouse myocardium due to ablation of myosin binding protein-C. J Mol Biol. 367, 36-41

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64. Walcott, S., Docken, S., and Harris, S.P. (2015) Effects of cardiac Myosin binding protein-C on actin motility are explained with a drag-activation-competition model. Biophys J. 108, 10-13.

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65. Weith, A., Sadayappan, S., Gulick, J., Previs, M.J., VanBuren, P., Robbins, J., and Warshaw, D.M. (2012) Unique single molecule binding of cardiac myosin binding protein C to actin and phosphorylation-dependent inhibition of actomyosin motility requires 17 amino acids of the motif domain. J Mol Cell Cardiol. 52, 219-227. 66. Mamidi, R., Gresham, K.S., Verma, S., and Stelzer, J.E. (2016) Cardiac myosin binding protein-C phosphorylation modulates myofilament length-dependent activation. Front Physiol. 7, 38.

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67. Colson, B.A., Bekyarova, T., Locher, M.R., Fitzsimons, D.P., Irving, T.C., and Moss, R.L. (2008) Protein kinase A-mediated phosphorylation of cMyBP-C increases proximity of myosin heads to actin in resting myocardium. Circ Res. 103 244–251.

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68. Nag, S., Trivedi, D.V., Sarkar, S.S., Adhikari, A.S., Sunitha, M.S., Sutton, S., Ruppel, K.M., and Spudich, J.A. (2017) The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations. Nat Struct Mol Biol. 24, 525-533.

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69. Mun, J.Y., Previs, M.J., Yu, H.Y., Gulick, J., Tobacman, L.S., Beck Previs, S., Robbins, J., Warshaw, D.M., and Craig, R. (2014) Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism. Proc Natl Acad Sci U S A. 111, 2170-2175.

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TABLES

GFS experiment

Length (nm)

Force (pN)

Single myosin molecule

100

6.5

Single myosin

100

6.9

100

7.0

a

dimer/thick filamenta Multiple myosin

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dimers/thick filamenta

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Table 1: Length of uncoiled myosin dimer length measured with GFS.

a

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The correlation coefficient (R) calculated with simultaneous fit using a common linker distance was 0.866. The fitted parameters of the three fits had a standard deviation of 24% and the range of the fitted parameters for the single myosin with or without the cofilament was 3%.

Table 2: Summarized length of uncoiled myosin dimer measured with SFS and GFS. Length (nm) ± range 12 ± 2

Force (pN) a ± SEM 521 ± 27b

Correlation c coefficient (R) 0.987

12 ± 2

636 ± 23 b

0.973

Posthinge

12 ± 2

868 ± 26 b

0.967

LMM

12 ± 2

965 ± 28 b

0.928

Prehinge

100 ± 10

5.25 ± 0.13 b

0.980

Hinge

100 ± 10

4.098 ± 0.44 b

0.942

Posthinge

100 ± 10

6.15 ± 0.07 b

0.983

LMM

100 ± 10

9.36 ± 0.32 b

0.969

Posthinge with MyBPC

100 ± 10

18.40 ± 0.32 b

0.975

Position on Myosin Prehinge

SFS

Hinge

a

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GFS

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Force Spectroscopy

The forces for SFS are the mean ± standard error for all force measurements with distances of 10 nm to 14 nm. The forces for GFS are the mean ± standard error for all force measurements with distances from 90 nm to 110 nm. Force values in bold indicate results ± MyBPC. b

These values are statistically significantly different from all other values using Student’s t-test with p<0.02 in each pair. Six passed and three failed a Shapiro-Wilk normality test, so a nonparametric Mann-Whitney test was also performed that demonstrated that the values are statistically significantly different from all other values in each pair with p<0.02.

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FIGURE LEGENDS (See separate PDF file for high resolution figures.)

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Figure 1. Free fall assay of GFS. (A) Schematic of a GFS with microscope on an alt-azimuthal telescopic mount suspended on springs, capable of doing rotational mode [23] (green arrow) and a free fall mode (red arrow) against the gravity vector (orange arrow). (B) Sketch of a molecule of interest (red) tethered between edge of the coverslip and silica or glass bead (grey sphere) by

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antibody (blue) raised against the molecule of interest. The molecular length before free fall is

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calculated as dmax-dmin. The force in pN imparted by the microsphere, including the subtraction of buoyancy, is a(4/3)(ρ-w)π [(dmax-dmin)/(1/cos(αmin)-1)]3 in which a is the acceleration due to gravity (0.0098 km/s2) multiplied by the unit conversion correction, ρ is the density of the microsphere in g/cm3, w is the density of water = 1 g/cm3, αmin is the angle between the gravity vectors for dmax and dmin at the angle of its first occurrence, and dmax and dmin are in microns. (C)

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Acceleration trace for GFS when dropped freely against the spring constant under gravitational force. The acceleration is in the frame of reference of the mobile bead, so it is initially zero

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during free fall. Force is calculated with Newton’s second law of motion, F=ma, and the buoyancy of the microsphere is subtracted. (D) The trace of the molecular length for the

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molecule undergoing the free fall mode of GFS. Figure 2. Schematic of points for force applied perpendicular to coiled coil axis, from right to left. Prehinge point where actin (blue sphere) and myosin subfragment-1 interaction were used as the source of suspending a single molecule of myosin for the force assay. Hinge point where MF30 (green arrow), a monoclonal antibody raised against myosin S1-S2 hinge and confirmed to be within 5 nm of the regulatory light chain [28], was used as the source of suspension. Posthinge point where polyclonal anti-S2 antibody (red arrow) was used as the source of suspension. LMM 44

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point where MF20 (orange arrow), a monoclonal antibody raised against LMM, was used as a source of suspension for force assay.

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Figure 3. Uncoiling of myosin S2 in parallel and perpendicular direction of myosin thick filament axis. (A) Uncoiling of myosin S2 in both parallel and perpendicular directions to the thick filament axis by SFS. (B) Uncoiling of myosin S2 in a parallel (red circles with dashed

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lines for WLC fit between MF20 and MF30) (n=5) and perpendicular (dashed orange line, logarithmic fit from Fig. 8D, and dashed green line, logarithmic fit from Fig. 8B) direction by

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GFS. Inset figure (dashed black line) indicates the pulling of myosin molecule (red) with MF30 antibody (green arrow and circles) and MF20 antibody (orange arrow and circles). (C) Stretching of DNA in a direction parallel to the axis of DNA length using GFS. The composite trace of force distance curves for DNA with red circles and WLC fit with dashed lines for one DNA molecule subjected to forces less than 60 pN, and blue circles and fit with dashed lines for

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another DNA molecule subjected to forces higher than 60 pN are shown. Inset figure illustrates the DNA (blue) with DTPA-dCTP (blue circle) on opposite strands with a black arrow indicating the direction of stretching. Video for the GFS measurement of DNA molecule is available online

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in supplementary data (Supplementary Video 1).

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Figure 4. Images from SFS and plots of broken hydrogen bonds of the myosin S2 coiled coil uncoiled at different positions along the length of the myosin rod. (A) and (E) Myosin S2 uncoiled at pre-myosin S1-S2 hinge position. The plot of broken hydrogen bonds per extension of myosin S2 molecule at prehinge position is linear with a correlation coefficient of 0.971. (B) and (F) Myosin S2 uncoiled at hinge position. The plot of broken hydrogen bonds per extension of myosin S2 molecule at hinge position is linear with a correlation coefficient of 0.991. (C) and (G) Myosin S2 uncoiled at post-myosin S1-S2 hinge position. The plot of broken hydrogen 45

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bonds per extension of myosin S2 molecule at posthinge position is linear with a correlation coefficient of 0.982. (D) and (H) Myosin S2 uncoiled at LMM position. The plot of broken hydrogen bonds per extension of myosin S2 molecule at LMM position is linear with a

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correlation coefficient of 0.961. Videos of the simulations are available in the online supporting information [Supplemenatry Video 2].

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Figure 5. Individual single molecules of myosin (red circles and dashed line) have a similar force-distance curve when compared to those of a single myosin (blue triangles and dotted line)

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or multiple myosin (green squares and line) dimers in a myofilament when pulled from the prehinge position. Higher ratios of myosin to rod in the cofilament (green squares) do not alter the conclusion that S2 stability is not affected significantly by myosin polymerization. Each plotted point is a different single molecule measured by GFS without free fall.

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Figure 6. Simulated force spectroscopy of the myosin molecule uncoiled from pre-myosin S1-S2 hinge (blue diamond), myosin S1-S2 hinge (green square), post-myosin S1-S2 hinge position (red triangle), and LMM (orange circle).

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Figure 7. Force-distance curve for uncoiling of myosin molecule at different positions along the myosin coiled coil. (A) Force-distance curve for uncoiling of myosin molecule at prehinge

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position (n = 7). (B) Force-distance curve for uncoiling of myosin molecule at hinge position (n= 3). (C) Force-distance curve for uncoiling of myosin molecule at posthinge position (n = 7). (D) Force distance curve for uncoiling of myosin molecule at LMM position (n = 3). (E) Force distance curve for uncoiling of myosin molecule with bound MyBPC at posthinge position (n =10). For cartoons (dashed square): Myosin molecule (red), Prehinge position with actin (blue arrow), Hinge position with MF30 antibody (green arrow), Posthinge position with polyclonal

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anti-S2 antibody (red arrow), LMM position with MF20 antibody (orange arrow), and MyBPC (purple line).

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Figure 8. Histogram of in vitro motility assay with MyBPC. (A) Histogram plot showing distribution of in vitro actin filament motility over myosin thick filaments in the absence (blue) (n=33) and presence (orange) of full-length skeletal MyBPC (n=44). (B) Histogram showing

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average velocity of actin thin filaments in absence (blue) and presence (orange) of full-length

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skeletal MyBPC.

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