Quenching of fluorescent nucleotides bound to myosin: a probe of the active-site conformation.
Quenching of fluorescent nucleotides bound to myosin: a probe of the active-site conformation.
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与肌球蛋白结合的荧光核苷酸的淬灭：活性位点构象的探针。
DOI：
10.1021/bi00208a025
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Cooke,R
中科院分区：
文献类型：
--
作者：
Franks-Skiba,K;Hwang,T;Cooke,R
1994年8月8日收到的修订版Mandarin pt ®摘要：通过测量结合的乙烯基吡咯烷酮eADP和eATP的溶剂可及性（稳态水解期间），探测肌原纤维中肌球蛋白亚片段1（SI）和肌动球蛋白的活性ATP酶位点的构象。通过测量由溶剂相猝灭剂丙烯酰胺（25-400 mM）产生的荧光猝灭来确定溶剂可及性。在存在和不存在5 mM ADP的情况下，在沉降后确定特异性结合至活性位点的核苷酸的分数。与以前的调查一致，eATP和eADP几乎完全保护猝灭剂结合时的活性位点的肌球蛋白。eADP和eATP的溶剂可及性随温度和离子强度而变化。核苷酸在较高的温度和较高的离子强度下变得更容易接近。在1 Mkc 1的淬灭曲线是双相的，表明肌球蛋白的核苷酸口袋可以存在于一个封闭的形式，允许很少的淬灭和更开放的形式，允许相当大的淬灭。然而，形式之间的过渡并没有强烈耦合到核苷酸的状态，在稳态循环过程中观察到类似的保护eADP和eATP。eADP与肌动蛋白-S1或肌原纤维中的肌动球蛋白结合，显示出与单独使用S1时相同的保护程度。在稳态水解期间获得类似的结果。因此，当肌球蛋白与肌动蛋白结合时，肌球蛋白口袋中的核苷酸不会变得更容易接近溶剂，无论是刚性ADP还是活性复合物。这些结果支持了核苷酸口袋可以以开放和封闭形式存在的假设，但它们表明这些形式之间的转换不涉及动力冲程。肌动蛋白和肌球蛋白头部的结构现在已经被确定为2.8 μ m的分辨率（Kabsch et al.，1990; Holmes埃塔尔，1990; Schutt埃塔尔，1993; Rayment埃塔尔，1993年a）。SI的结构是由一个大的球状区域连接到一个更细长的颈部。球状区域，被称为催化结构域，包含结合肌动蛋白和核苷酸的位点。细长的颈从球状区域延伸到与杆状物的连接处，进而延伸到粗丝。催化结构域包含与来自其它蛋白质的ATP结合序列具有同源性的区域。这部分结构由蛋白质结构中的浅凹陷组成，称为核苷酸口袋。该口袋大且开放，并且通过与光反应性核苷酸交联而鉴定的两个氨基酸位于相对侧，间隔15 μ m（Yount et al.，1992年）。许多酶核苷酸结合位点的构象现在是已知的，并且其中大多数以更封闭的形式存在，其中蛋白质结构包围核苷酸，将其与溶剂隔离（Schultz，1991）。在核苷酸水解之前从活性位点排除溶剂被认为是必要的，以便从反应中排除水。例如，腺苷酸激酶（一种其活性位点与肌球蛋白的活性位点显示出一定同源性的酶）在磷酸转移之前经历包围结合的核苷酸的结构域移位（Schultz等人，1991年）。这些结构表明，在周期中的某个点，肌球蛋白可能会发生结构域转移，关闭核苷酸口袋。如下文所述，这种畴运动可以在力的生成中发挥关键作用。
Revised Manuscript Received August 8, 1994® abstract: The conformation of the active ATPase site of myosin subfragment 1 (SI) and actomyosin in myofibrils was probed by measuring the solvent accessibility of the bound ethenonucleotides eADP and eATP (during steady-state hydrolysis). Solvent accessibility was determined by measuring the quenching of fluorescence produced by the solvent-phase quencher acrylamide, 25-400 mM. The fraction of the nucleotides that were specifically boundto the active site was determined following sedimentation in the presence and absence of 5 mM ADP. In agreement with previous investigations, both eATP and eADP were almost completely protected from the quencher when bound to the active site of myosin. The solvent accessibility of both eADP and eATP varied with both temperature and ionic strength. The nucleotides became more accessible at higher temperatures and higher ionic strength. At 1 Mkc1 the quenching curve was biphasic, indicating that the nucleotide pocket of myosin can exist in both a closed form that allows little quenching and a more open form that allows considerable quenching. However, the transition between forms was not strongly coupled to the state of the nucleotide, with a similar protection observed for both eADP and for eATP during steady-state cycling. eADP bound to acto-Sl or to actomyosin in myofibrils displayed the same degree of protection as seen with SI alone. A similar result is obtained during steadystate hydrolysis. Thus nucleotides in the myosin pocket do not become more accessible to the solvent when myosin binds to actin in either rigor-ADP or active complexes. These results support the hypothesis that the nucleotide pocket can exist in both open and closed forms, but they suggest that transitions between these forms are not involved in the powerstroke.The structures of both actin and the myosin head have now been determined to a resolution of 2.8 Á (Kabsch et al., 1990; Holmes etal., 1990; Schutt etal., 1993; Rayment etal., 1993a). The structure of SI is composed of a large globular region connected to a more slender neck. Theglobular region, known as the catalytic domain, contains sites for binding both actin and nucleotide. The long tapering neckextends from this globular region to the junction with the rod, which leads in turn to the thick filament. The catalytic domain contains a region which has homology to ATP binding sequences from other proteins. This portion of the structure consists of a shallow depression in the protein structure termed the nucleotide pocket. The pocket is large andopen, and two amino acids identified by cross-linking to photoreactive nucleotides reside on opposite sides, spaced 15 Á apart (Yount et al., 1992). The conformations of a number of enzyme nucleotide binding sites are now known, and most of these exist in a more closed form in which the protein structure encloses the nucleotide, isolating it from the solvent (Schultz, 1991). The exclusion of solvent from the active site prior to nucleotide hydrolysis is thought to be necessary in order to exclude water from the reaction. For instance, adenylate kinase, an enzyme whose active site displays some homology with that of myosin, undergoes a domain shift that encloses the bound nucleotides prior to the phosphate transfer (Schultz et al., 1991). These structures suggest that, at some point in the cycle, myosin may undergo a domain shift which closes the nucleotide pocket. As outlined below, such domain movement could play a crucial role in the generation of force.