Electrostatic interaction of loop 1 and backbone of human cardiac myosin regulates the rate of ATP induced actomyosin dissociation.
Electrostatic interaction of loop 1 and backbone of human cardiac myosin regulates the rate of ATP induced actomyosin dissociation.
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DOI：
10.1007/s10974-021-09611-z
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发表时间：
2022-03
影响因子：
2.7
通讯作者：
Nesmelov YE
中科院分区：
文献类型：
--
作者：
Gargey A;Nesmelov YE
在人心肌肌球蛋白的β同种型中引入双突变D208 Q：K450 L以去除连接环1和肌球蛋白头部内的七链β折叠的盐桥D208：K450。β异构体特异性盐桥D208：K450，限制环1的灵活性，先前在分子动力学模拟中发现。早些时候，有人提出，1号环调节核苷酸亲合力肌动球蛋白，我们假设1号环和肌球蛋白头骨架之间的静电相互作用调节ATP结合和ADP解离肌动球蛋白，因此，时间的强肌动球蛋白结合。为了检验这一假设，我们表达了野生型和突变体的肌球蛋白头部结构（1-843个氨基酸残基）在分化的C2 C12细胞，ATP诱导的肌动球蛋白解离和ADP释放的动力学特征，使用停流荧光分光光度法。这两种结构都表现出快速的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的强结合状态的时间。我们观察到一个更快的速率ATP诱导肌动球蛋白解离的突变体，相比野生型肌动球蛋白。ADP从肌动球蛋白释放的速率对于突变体和野生型肌动球蛋白保持相同。我们的结论是环1的灵活性是一个因素，影响ATP结合肌动球蛋白和肌动球蛋白解离率。环1的柔性不影响ADP从人心脏肌动球蛋白的释放速率。
Double mutation D208Q:K450L was introduced in the beta isoform of human cardiac myosin to remove the salt bridge D208:K450 connecting loop 1 and the seven-stranded beta sheet within the myosin head. Beta isoform-specific salt bridge D208:K450, restricting the flexibility of loop 1, was previously discovered in molecular dynamics simulations. Earlier it was proposed that loop 1 modulates nucleotide affinity to actomyosin and we hypothesized that the electrostatic interactions between loop 1 and myosin head backbone regulate ATP binding to and ADP dissociation from actomyosin, and therefore, the time of the strong actomyosin binding. To examine the hypothesis we expressed the wild type and mutant of the myosin head construct (1–843 amino acid residues) in differentiated C2C12 cells, and the kinetics of ATP-induced actomyosin dissociation and ADP release were characterized using stopped-flow spectrofluorometry. Both constructs exhibit 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. We observed a faster rate of ATP-induced actomyosin dissociation with the mutant, compared to the wild type actomyosin. The rate of ADP release from actomyosin remains the same for the mutant and the wild type actomyosin. We conclude that the flexibility of loop 1 is a factor affecting the rate of ATP binding to actomyosin and actomyosin dissociation. The flexibility of loop 1 does not affect the rate of ADP release from human cardiac actomyosin.
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影响因子：
2.7
作者：
Gargey A;Iragavarapu SB;Grdzelishvili AV;Nesmelov YE
通讯作者：
Nesmelov YE
DOI：
10.1016/j.bbrc.2019.01.045
发表时间：
2019-02-19
影响因子：
3.1
作者：
Gargey, Akhil;Ge, Jinghua;Nesmelov, Yuri E.
通讯作者：
Nesmelov, Yuri E.
影响因子：
2.9
作者：
Murphy, CT;Spudich, JA
通讯作者：
Spudich, JA
影响因子：
4.8
作者：
WALLER, GS;OUYANG, G;LOWEY, S
通讯作者：
LOWEY, S
影响因子：
2.9
作者：
Furch, M;Geeves, MA;Manstein, DJ
通讯作者：
Manstein, DJ