Two single mutations, R694N and E45Q, were introduced in the beta isoform of human cardiac myosin to remove permanent salt bridges E45:R694 and E98:R694 in the SH1-SH2 helix of the myosin head. Beta isoform-specific bridges E45:R694 and E98:R694 were discovered in the molecular dynamics simulations of the alpha and beta myosin isoforms. Alpha and beta isoforms exhibit different kinetics, ADP dissociates slower from actomyosin containing beta myosin isoform, therefore, beta myosin stays strongly bound to actin longer. We hypothesize that the electrostatic interactions in the SH1-SH2 helix modulate the affinity of ADP to actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutants of the myosin head construct (1–843 amino acid residues) were expressed in differentiated C₂C₁₂ cells, and the duration of the strongly bound state of actomyosin was characterized using transient kinetics spectrophotometry. All myosin constructs exhibited a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. The mutant R694N showed a faster rate of ADP release from actomyosin, compared to the wild type and the E45Q mutant, thus indicating that electrostatic interactions within the SH1-SH2 helix region of human cardiac myosin modulate ADP release and thus, the duration of the strongly bound state of actomyosin.

将两个单一突变 R694N 和 E45Q 引入人类心肌肌球蛋白的 β 同种型，以去除肌球蛋白头部 SH1-SH2 螺旋中的永久盐桥 E45:R694 和 E98:R694。β 亚型特异性桥 E45:R694 和 E98:R694 在 α 和 β 肌球蛋白亚型的分子动力学模拟中被发现。α 和 β 亚型表现出不同的动力学，ADP 从含有 β 肌球蛋白亚型的肌动球蛋白解离较慢，因此，β 肌球蛋白与肌动蛋白的牢固结合时间更长。我们假设 SH1-SH2 螺旋中的静电相互作用调节 ADP 对肌动球蛋白的亲和力，因此调节强肌动球蛋白结合的时间。野生型和肌球蛋白头部构建体的突变体（1-843 个氨基酸残基）在分化的 C ₂ C中表达_12个细胞，并使用瞬态动力学分光光度法表征肌动球蛋白的强结合状态的持续时间。所有肌球蛋白构建体都表现出快速的 ATP 结合肌动球蛋白和缓慢的 ADP 解离速率，表明 ADP 释放限制了肌动球蛋白强结合状态的时间。与野生型和 E45Q 突变体相比，突变体 R694N 从肌动球蛋白释放 ADP 的速率更快，因此表明人心肌肌球蛋白 SH1-SH2 螺旋区域内的静电相互作用调节 ADP 释放，因此，强烈的持续时间肌动球蛋白的结合状态。