Probing the conformational states of the SH1-SH2 helix in myosin: a cross-linking approach.
Probing the conformational states of the SH1-SH2 helix in myosin: a cross-linking approach.
复制标题
探索肌球蛋白中 SH1-SH2 螺旋的构象状态：一种交联方法。
DOI：
10.1021/bi9817212
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发表时间：
1998
期刊：
影响因子：
2.9
通讯作者：
Reisler,E
中科院分区：
文献类型：
--
作者：
Nitao,LK;Reisler,E
先前的生化研究表明，兔骨骼肌肌球蛋白亚片段1（S1）上的SH 1（Cys 707）和SH 2（Cys 697）基团可以通过使用不同交联长度的试剂进行交联。在核苷酸的存在下，这种交联被加速。在S1的晶体结构中，SH 1和SH 2残基位于α-螺旋上，相距19个碱基。因此，交联结果可以指示在核苷酸存在下的螺旋解链或增加的柔性。在这项研究中，通过监测S1上的SH 1和SH 2与跨度为5至15 μ m的双马来酰亚胺试剂的交联，检查了该区域中核苷酸诱导的变化。设计了一种直接测量核苷酸对交联反应速率的动力学影响的方法。在没有核苷酸的情况下，SH 1 − SH 2交联的缓慢和试剂不敏感的速率表明，SH 1 − SH 2螺旋在不同SH 1 − SH 2分离状态之间的平均分配很少发生。在存在MgADP、MgATP和MgATPγS的情况下，对于最短的试剂（5−8 μ m），SH 1和SH 2交联的速率增加了1.2 −7倍。对于较长的试剂（9 - 15 μ m），速率加速要大得多：MgADP为40 - 50倍，MgATP为25 - 40倍，MgATPγS为80 - 270倍。为了解释试剂对SH 2的反应性的任何核苷酸依赖性差异，还测量了每种试剂在SH 1标记的S1上的单官能SH 2修饰率。这些实验表明，核苷酸诱导的SH 2修饰速率的增加对于所有试剂都是相似的。因此，观察到的交联速率的变化不仅是由于活性位点中结合的核苷酸的类型，而且是由于交联剂的跨度。这些发现被解释为核苷酸诱导的SH 1 − SH 2螺旋构象状态平衡的变化。
Previous biochemical studies have shown that the SH1 (Cys707) and SH2 (Cys697) groups on rabbit skeletal myosin subfragment 1 (S1) can be cross-linked by using reagents of different cross-linking lengths. In the presence of nucleotide, this cross-linking is accelerated. In the crystal structure of S1, the SH1 and SH2 residues are located on an α-helix, 19 Å apart. Thus, the cross-linking results could be indicative of helix melting or increased flexibility in the presence of nucleotides. Nucleotide-induced changes in this region were examined in this study by monitoring the cross-linking of SH1 and SH2 on S1 with dimaleimide reagents of spans ranging from 5 to 15 Å. A method was devised to directly measure the kinetic effects of nucleotides on the rates of cross-linking reactions. The slow and reagent-insensitive rates of the SH1−SH2 cross-linking in the absence of nucleotides reveal that the equipartitioning of the SH1−SH2 helix among states with different SH1−SH2 separations occurs infrequently. In the presence of MgADP, MgATP, and MgATPγS, the rates of SH1 and SH2 cross-linking were increased ∼2−7-fold for the shortest reagent (5−8 Å). Rate accelerations were much greater for the longer reagents (9−15 Å): 40−50-fold for MgADP, 25−40-fold for MgATP, and 80−270-fold for MgATPγS. To account for any nucleotide-dependent differences in the reactivities of the reagents toward SH2, the rates of monofunctional SH2 modification on SH1-labeled S1 were also measured for each reagent. These experiments showed that the nucleotide-induced increases in the rates of SH2 modification were similar for all of the reagents. Thus, the changes observed in the cross-linking rates are due not only to the type of nucleotide bound in the active site but also to the span of the cross-linking reagent. These findings are interpreted in terms of nucleotide-induced shifts in the equilibria among conformational states of the SH1−SH2 helix.