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1 advances.sciencemag.org/cgi/content/full/1/9/e /dc1 Supplementary Materials for Contractility parameters of human -cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function Suman Nag, Ruth F. Sommese, Zoltan Ujfalusi, Ariana Combs, Stephen Langer, Shirley Sutton, Leslie A. Leinwand, Michael A. Geeves, Kathleen M. Ruppel, James A. Spudich The PDF file includes: Published 9 October 2015, Sci. Adv. 1, e (2015) DOI: /sciadv Fig. S1. Schematic of the different constructs of the human -cardiac ss1 used in our experiments and relative purity of the wild type and R403Q protein preparations. Fig. S2. Representative experimental traces of wild-type and R403Q ss1 unloaded motility. Fig. S3. Single-molecule stroke size measurements of wild type and R403Q human -cardiac ss1. Table S1. Rate constants and equilibrium constants for the various kinetic steps of wild type and R403Q human -cardiac ss1. Reaction schemes Scheme 1. Kinetic schemes for interaction of myosin only with nucleotide. Scheme 2. Kinetic schemes for interaction of myosin with actin and nucleotide. Scheme 3. A combined equilibrium scheme for myosin, actin, and ADP binding to each other. Reference (70)

2 WT-eGFP R403Q-eGFP WT-affinity clamp R403Q-affinity clamp Supplementary materials Supplementary Figures A B GSG egfp ss1 100 kd Human b-cardiac ss1 50 kd ss1 GSG RGSIDTWV 25 kd Human ELC Figure S1. Expression of recombinant human β-cardiac ss1. (A) A schematic of two different C- terminally-tagged constructs of human β-cardiac short Subfragment 1 (ss1, residues) used in our experiments. For all ATPase and single molecule trap experiments a C-terminal egfp tag on the ss1 was used (top). For all other experiments, except stopped-flow experiments, a C-terminal eight residue peptide (RGSIDTWV) was used (bottom), which specifically binds to the SNAP-PDZ18 system (70). We call this an 'affinity clamp' construct. For both of these constructs, a flexible GSG linker separates the N-terminal portion of the ss1 from the C-terminal tag. Both constructs have an N-terminal FLAGtagged (DYKDDDDK) human ventricular essential light chain (ELC) containing a TEV-precision protease site for affinity purification of the ss1. For most of the stopped flow experiments, we used ss1 with no C-terminal heavy chain tag, but with a 6X-His tag on the ELC; we duplicated the ADP-release kinetic experiments with the C-terminal egfp tagged version and found no differences compared to the untagged version (Table S1). (B) An overloaded SDS/PAGE of purified recombinant human β-cardiac ss1. Lane 1 contains the Invitrogen BenchMark Ladder. Three different molecular weights are marked for convenience. Lanes 2, 3, 4 and 5 contain wild-type (WT ss1-egfp), R403Q ss1-egfp, wild type (WT ss1-affinity clamp) and R403Q ss1-affinity clamp, respectively. The C-terminal egfp constructs migrate around 110 kda and the affinity clamp construct migrates around 90 kda. Both ss1 fragments co-purify with the human ventricular ELC, which migrates around 24 kda.

3 Number of events Number of events A B Filtered Unfiltered Filtered Unfiltered Actin filament length (mm) % Tolerance Actin filament length (mm) % Tolerance Figure S2. A representative unloaded motility output of human β-cardiac ss1. Actin filaments were tracked for at least 30 consecutive frames, actin velocities and actin filament lengths were averaged over 5 consecutive frames, a 20%-tolerance filter was applied to eliminate intermittently moving filaments with velocities fluctuating higher than 20 percent of the mean velocity, and stuck filaments were excluded from the scatter plot. Scatter plot of actin filament length and velocities for (A) wild-type and (B) R403Q β-cardiac ss1. Top left panel of (A) and (B): Maximum velocity of each filament is in triangles (black for wild-type and red for R403Q) and lower velocities are in gray circles. The solid line is the fit to a single exponential decay function to the maximum velocities to obtain the PLATEAU velocity. The dashed line marks the mean of the top 5% velocities (TOP5%). Top center panel of (A) and (B): A 20%-tolerance-filtered filament velocity distribution excluding stuck filaments. The solid line denotes the mean of the velocity distribution (MVEL20). Top right panel of (A) and (B): The unfiltered filament velocity distribution including stuck filaments. The solid line marks the mean of the distribution (MVEL). Filaments with a mean velocity less than 1 pixel length per second (80 nm/s) were classified as stuck. %STUCK represents the time-weighted fraction of stuck filaments. Bottom left panel of (A) and (B): Actin filament length distribution. The solid line marks the mean of the distribution (<FIL-LENGTH>). Bottom right panel of (A) and (B): MVEL (solid line) and TOP5% velocity (dashed line) from the data subjected to different levels of tolerance filtering. For more details see Aksel et al. (37).

4 Fraction of events Fraction of events Step size Step (nm) size (nm) Figure S3. Single-molecule stroke size measurements. Combined histogram of stroke size distribution from several single-molecules of wild-type (grey bars) and R403Q (red bars) human β-cardiac ss1 at 0.5 µm ATP. A Gaussian function using the least square method was used to fit the histogram (solid black line for wild-type and red for R403Q) to give a mean stroke size value.

5 Supplementary Table R403Q ss1 wild-type ss1 ATP-binding to 20 o C, Scheme 1 ATP binding rate constant K 1k +2 (µm -1 s -1 ) 5.4 ± ± 0.4 Rate constant of conformational change upon ATP binding k +2 (s -1 ) ± ± 1.8 Equilibrium dissociation constant 1/K 1 (µm) 20.8 ± ± 1.4 ADP-binding to 20 o C, Scheme 1,3 Equilibrium dissociation constant K D =K 6K 7 (µm) 0.49 ± ± 0.07 Rate constant for ADP release k +6 (s -1 ) 0.6 ± ± 0.03 ATP-binding to actin 20 o C, Scheme 2 ATP induced actin ss1dissociation rate constant K' 1k' +2 (µm -1 s -1 ) 4.9 ± ± 0.2 Equilibrium dissociation constant 1/K' 1 (µm) n.d n.d Rate constant of strong-to-weak binding transition of actin ss1 k' +2 (s -1 ) n.d n.d Nucleotide pocket isomerization equilibrium constant K α1 n.d n.d Nucleotide pocket isomerization forward rate constant k +α1 (s -1 ) n.d n.d ATP-binding to actin 10 o C, Scheme 2 ATP induced actin ss1dissociation rate constant K' 1k' +2 (µm -1 s -1 ) 2.5 ± ± 0.07 Equilibrium dissociation constant 1/K' 1 (µm) 276 ± 12 * ± 15 Rate constant of strong-to-weak binding transition of actin ss1 k' +2 (s -1 ) 677 ± 12 *** 991 ± 17 Nucleotide pocket isomerization equilibrium constant K α1 3.4 ± ± 0.1 Nucleotide pocket isomerization forward rate constant k +α1 (s -1 ) 53.3 ± 1.8 *** 78.9 ± 3 ADP-binding for actin 20 o C, Scheme 2,3 Equilibrium dissociation constant K AD =K' 6K' 7(µM) 7.6 ± ± 0.3 ADP release rate constant k' +6 (s -1 ) 60.0 ± ± 1.7 ADP release rate 23 o C k' +6 (s -1 ) 80.0 ± ± 10 Rate constant of ADP binding K' 7k' -6 (µm -1 s -1 ) Nucleotide pocket isomerization equilibrium constant in the presence of ADP K αd 16.4 ± 5 No second phase Nucleotide pocket isomerization forward rate constant in the presence of ADP k +αd (s -1 ) 4.2 ± 0.5 No second phase Coupling constant K' 6K' 7 / K 6K ss1-affinity for 20 o C, Scheme 3 Dissociation constant (nm) K A (nm) 19.7 ± 6.2 * 10 ± 1.8 ss1-affinity for actin at 100 mm 20 o C, Scheme 3 Dissociation constant (nm) K A (nm) 63.2 ± 16.7 * 23.4 ± 2.5 ss1-affinity for actin in the presence of 20 o C, Scheme 3 Dissociation constant (nm) K DA (nm) ± ± 24.1 Table S1: Rate constants and equilibrium constants for the various kinetic steps of wild-type and R403Q human β-cardiac ss1. All kinetic measurements for wild-type human β-cardiac ss1 (n = 3-4 exp, 3 preps) and R403Q ss1 (n = 3-4 exp, 2 preps) were done according to methodologies published in Deacon et al. (34) and Bloemink et al. (36). The buffer used contained 20 mm MOPS, 25 mm KCl, 5 mm MgCl2 and 1 mm DTT at 20 C, unless otherwise stated in the table. Errors reported are SEM. The values for R403Q that are shaded in orange are the ones which had statistical differences from wild-type

6 ss1. * P<0.05 and *** P< were determined by Student's t tests. For some of the measurements under 'ATP-binding to actin 20 C ' the rates were too fast and therefore unreliable, and hence are not determined (n.d). These measurements were repeated at 10 C (see 'ATP-binding to actin 10 C '). ki denotes a forward rate constant and k-i denotes a backward rate constants of steps (i) shown in scheme 1 and scheme 2 below. Ki (= ki/k-i) denotes equilibrium rate constants for a particular step. All rate constants and equilibrium constants without a prime sign denote kinetic steps involving only the ss1 and nucleotide (scheme 1), whereas those with the prime sign (') are for steps involving the ss1, actin and nucleotide (scheme 2). KA is the equilibrium dissociation constant of actin interaction with ss1 in the rigor state (scheme 3), KAD is the equilibrium dissociation constant of ADP affinity with the actin.ss1 complex (scheme 3), KDA is the equilibrium dissociation constant of actin affinity with the ADP.sS1 complex (scheme 3) and KD is the equilibrium dissociation constant of ss1 affinity with ADP (scheme 3).

7 Reaction Schemes Kinetic reaction scheme 1 shows the reaction mechanism of ss1 (M) with either ATP (T) or ADP (D) (33, 35). Phosphate is denoted as P. * and ** indicates different level of tryptophan fluorescence and represents different conformational states of ss1. Kinetic reaction scheme 2 shows the interaction of ss1 (M) with actin (A), ATP (T) and ADP (D). Actin complexed with ss1 exists in two conformations (A.M and A.M') in equilibrium. A M' (closed configuration) is unable to bind to nucleotide and must isomerize to A M (open configuration) before ATP can bind. A similar pair of conformations exist in the presence of bound ADP, where A M' D must isomerize to A M D before ADP can dissociate. * indicates different level of tryptophan fluorescence and represents different conformational states of ss1. Scheme 3 is a simplified combined scheme showing the affinity of ss1 with actin in the rigor state (the equilibrium dissociation constant denoted by KA), the affinity of actin ss1 complex to ADP (the equilibrium dissociation constant denoted by KAD=K'6K'7), the affinity of ss1 ADP complex to actin (the equilibrium dissociation constant denoted by KDA) and the affinity of ss1 to ADP (the equilibrium dissociation constant denoted by KD=K6K7). Scheme 1: Kinetic schemes for interaction of myosin only with nucleotide k 1 k 2 * k 3 ** k 4 * k 5 * k 6 k 7 M T M T M T M D P M D P P M D M D M D k 1 k 2 k 3 k 4 k 5 k 6 k 7 Scheme 2: Kinetic schemes for interaction of myosin with actin and nucleotide k D k 6 * k 7 T k 1 k 2 A M ' D A M D A M D D A M A M T A M T k D k 6 k 7 k 1 k 2 closed open open open open closed k 1 k 1 Scheme 3: A combined equilibrium scheme for myosin, actin and ADP binding to each other A M closed K AD K A K DA KD