Dynamics of β-cardiac myosin between the super-relaxed and disordered-relaxed states

Figures

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  Figure 1. Single- versus double-exponential equations fitted to mantATP nucleotide turnover data.A, reaction scheme of SRX-DRX transition followed by release of nucleotide as MantADP. A fast k1 relative to kDRX results in single phase kinetics for nucleotide release rate limited by kDRX; if k1 is substantially slower than kDRX, the fluorescence decay would be biphasic, with the slow phase rate limited by k1. B, single-nucleotide turnover of mantATP from the active site of WT-cHMM (blue) with double-exponential fit, along with a trace of mantATP in the absence of myosin (gray) demonstrating a significant amplitude and rate of mant fluorescence decrease (photobleaching) during kinetic acquisition. Inset: schematic of double-mixing stopped-flow apparatus for experimental conditions employed throughout this paper. C, WT-cHMM single-nucleotide turnover after correction for mant photobleaching. Left: double-exponential fit with residuals. Right: single-exponential fit with residuals. D, WT-cardiac subfragment-1 (-cS1) single-nucleotide turnover, corrected for mant photobleaching, with single-exponential fit and residuals. cHMM, cardiac heavy meromyosin; DE, double exponential; mantATP,…

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  Figure 2. Digestion of WT-cHMM to produce S1 and S2 speeds nucleotide release.A, coomassie stained SDS-PAGE gel of WT-cHMM demonstrates digestion by papain protease into S1, S2, and other fragments. B, undigested control samples incubated without the protease have single-turnover kinetics that are well fitted by a single-exponential function, similar to untreated WT-cHMM. C, upon papain digestion for 5 min, the single-turnover rate increases significantly to that of WT-cS1, as expected for myosin heads incapable of forming head–tail interactions. cHMM, cardiac heavy meromyosin.

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  Figure 3. Mutant-cHMM molecules increase single-turnover rate upon proteolytic digestion with papain.A and B, turnover rate for S532P-cHMM increases from 0.0035 ± 0.0005 s−1 to 0.011 ± 0.002 s−1 upon papain digestion, resulting in a KEQ of 0.32 ± 0.07, similar to WT. C and D, turnover rate for R712L-cHMM increases from 0.016 ± 0.004 s−1 to 0.05 ± 0.02 s−1 upon papain digestion, resulting in a KEQ of 0.3 ± 0.1, similar to WT. Inset: turnover of papain-digested R712L on a faster time-base to show single-exponential fit for early time points. E and F, turnover rate for E497D-cHMM increases from 0.032 ± 0.003 s−1 to 0.067 ± 0.003 s−1 upon papain digestion, resulting in a KEQ of 0.49 ± 0.04, significantly higher than WT. Inset: turnover of papain-digested E497D on a faster time-base to show single-exponential fit on early time points. Data are reported as mean ± SD. cHMM, cardiac heavy meromyosin.

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  Figure 4. Treatment with mavacamten slows nucleotide turnover by an equal percentage for WT-cHMM and WT-cS1.A and B, WT-cHMM treated with DMSO (A) or 10 μM mava (B) showing a 4-fold slowing of nucleotide release in the presence of mava. C and D, WT-cS1 treated with DMSO (C) or 10 μM mava (D), also showing a 4-fold slowing of nucleotide release in the presence of mava. The KEQ for the SRX/DRX equilibrium in the presence of mavacamten is 0.38 ± 0.07, statistically indistinguishable from untreated samples. Data are reported as mean ± SD. cHMM, cardiac heavy meromyosin.