Conformational dynamics of the SH1-SH2 helix in the transition states of myosin subfragment-1.
Conformational dynamics of the SH1-SH2 helix in the transition states of myosin subfragment-1.
复制标题
肌球蛋白亚片段 1 过渡态 SH1-SH2 螺旋的构象动力学。
DOI：
10.1016/s0006-3495(02)75283-1
复制
发表时间：
2002
影响因子：
3.4
通讯作者：
Reisler,Emil
中科院分区：
文献类型：
--
作者：
Nitao,LisaK;Yeates,ToddO;Reisler,Emil
含有巯基SH 1（Cys-707）和SH 2（Cys-697）的α-螺旋被认为是负责肌球蛋白头部（亚片段-1（S1））构象变化转导的结构元件之一。以前的研究，使用分离和测量SH 1-SH 2交联步骤的速率的方法，表明该螺旋经历配体诱导的构象变化。然而，由于形成过渡态复合物（S1.ADP.BeFx、S1.ADP.AlF4−和S1.ADP.Vi）所需的孵育时间较长，因此该方法无法用于确定这些状态的交联速率常数。在这项研究中，从SH 1-SH 2交联反应的动力学数据进行了分析，通过计算方法提取速率常数的两步机制。对于S1.ADP.BeFx，所得结果与S1.ATPγS相似。对于涉及S1.ADP.AlF4−和S1.ADP.Vi的反应，第一步（SH 1修饰）是速率限制;因此，只能确定交联步骤速率常数的下限。然而，这些结果表明，与无核苷酸的S1相比，对于所有试剂，包括最短的试剂，过渡态复合物中的交联速率常数增加至少20倍。因此，SH 1-SH 2螺旋似乎在水解后状态下不稳定。
Theα-helix containing the thiols, SH1 (Cys-707) and SH2 (Cys-697), has been proposed to be one of the structural elements responsible for the transduction of conformational changes in the myosin head (subfragment-1 (S1)). Previous studies, using a method that isolated and measured the rate of the SH1-SH2 cross-linking step, showed that this helix undergoes ligand-induced conformational changes. However, because of long incubation times required for the formation of the transition state complexes (S1.ADP.BeFx, S1.ADP.AlF4−, and S1.ADP.Vi), this method could not be used to determine the cross-linking rate constants for such states. In this study, kinetic data from the SH1-SH2 cross-linking reaction were analyzed by computational methods to extract rate constants for the two-step mechanism. For S1.ADP.BeFx, the results obtained were similar to those for S1.ATPγS. For reactions involving S1.ADP.AlF4− and S1.ADP.Vi, the first step (SH1 modification) is rate limiting; consequently, only lower limits could be established for the rate constants of the cross-linking step. Nevertheless, these results show that the cross-linking rate constants in the transition state complexes are increased at least 20-fold for all the reagents, including the shortest one, compared with nucleotide-free S1. Thus, the SH1-SH2 helix appears to be destabilized in the post-hydrolysis state.