Fluorescence resonance energy transfer within the complex formed by actin and myosin subfragment 1. Comparison between weakly and strongly attached states.
Fluorescence resonance energy transfer within the complex formed by actin and myosin subfragment 1. Comparison between weakly and strongly attached states.
复制标题
肌动蛋白和肌球蛋白亚片段形成的复合物内的荧光共振能量转移 1. 弱附着态和强附着态的比较。
作者：
H. Trayer;I. Trayer
荧光共振能量转移测量Cys-374对肌动蛋白和Cys-177对肌球蛋白亚片段1（S1）的碱性轻链使用几对供体-受体发色团。将标记的轻链交换成亚片段1，并通过SP-Trisacryl上的离子交换色谱分离所得荧光标记的亚片段1。能量转移的效率通过acto-S1的强结合复合物中的稳态荧光来测量，并发现代表两个探针之间5.6-6.3 nm的空间分离。然后用以两种方式产生的弱结合acto-S1复合物进行相同的测量。首先，将肌动蛋白与对亚苯基二马来酰亚胺-S1复合，所述对亚苯基二马来酰亚胺-S1是S1-腺苷5 ′-三磷酸（ATP）的稳定类似物，其通过使亚片段1的SH 1和SH 2重链硫醇交联而获得[格林，L. E、Chalovich，J. M.，& Eisenberg，E.（1986）Biochemistry 25，704-709]。转移效率的大幅增加表明，两个探针已经移近了约3 nm。第二，在调节蛋白肌钙蛋白和原肌球蛋白的存在下，不存在钙，并且存在ATP时，在亚片段1和肌动蛋白之间形成弱结合复合物[Chalovich，J.M.，& Eisenberg，E.（1982）J.Biol.Chem.257，2432-2437]。测量的能量转移效率再次表明，两个标记位点之间的距离已经移动近了约3 nm。这些数据支持的想法，有一个相当大的差异之间的弱结合态和强结合态的acto-S1复合物的结构。
Fluorescence resonance energy transfer measurements have been made between Cys-374 on actin and Cys-177 on the alkali light chain of myosin subfragment 1 (S1) using several pairs of donor-acceptor chromophores. The labeled light chain was exchanged into subfragment 1 and the resulting fluorescently labeled subfragment 1 isolated by ion-exchange chromatography on SP-Trisacryl. The efficiency of energy transfer was measured by steady-state fluorescence in a strong binding complex of acto-S1 and found to represent a spatial separation between the two probes of 5.6-6.3 nm. The same measurements were then made with weak binding acto-S1 complexes generated in two ways. First, actin was complexed with p-phenylenedimaleimide-S1, a stable analogue of S1-adenosine 5'-triphosphate (ATP), obtained by cross-linking the SH1 and SH2 heavy-chain thiols of subfragment 1 [Greene, L. E., Chalovich, J. M., & Eisenberg, E. (1986) Biochemistry 25, 704-709]. Large increases in transfer efficiency indicated that the two probes had moved closer together by some 3 nm. Second, weak binding complexes were formed between subfragment 1 and actin in the presence of the regulatory proteins troponin and tropomyosin, the absence of calcium, and the presence of ATP [Chalovich, J. M., & Eisenberg, E. (1982) J. Biol. Chem. 257, 2432-2437]. The measured efficiency of energy transfer again indicated that the distance between the two labeled sites had moved closer by about 3 nm. These data support the idea that there is a considerable difference in the structure of the acto-S1 complex between the weakly and strongly bound states.
通过使用硫醇交联试剂捕获金属核苷酸的活性位点对肌球蛋白进行化学修饰。
影响因子：
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作者：
Wells,JA;Yount,RG
通讯作者：
Yount,RG