STRUCTURAL CONNECTIVITY IN ACTIN - EFFECT OF C-TERMINAL MODIFICATIONS ON THE PROPERTIES OF ACTIN
STRUCTURAL CONNECTIVITY IN ACTIN - EFFECT OF C-TERMINAL MODIFICATIONS ON THE PROPERTIES OF ACTIN
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DOI：
10.1016/s0006-3495(94)80678-2
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发表时间：
1994-11-01
影响因子：
3.4
通讯作者：
REISLER, E
中科院分区：
文献类型：
--
作者：
CROSBIE, RH;MILLER, C;REISLER, E
在这项研究中，我们使用荧光探针和蛋白水解消化来证明肌动蛋白远距离区域之间的结构耦合。我们表明，肌动蛋白C末端的半胱氨酸 - 374的修饰减缓了核苷酸裂隙中核苷酸交换的速率。在亚结构域II中，C末端和DNA酶I环之间的构象耦合在蛋白水解消化实验中被观察到，在该实验中，DNA酶I结合时会暴露一个新的C末端切割位点。C末端修饰的功能影响从S - 1 ATP酶活性以及用修饰的肌动蛋白进行的体外运动实验中可以明显看出。在半胱氨酸 - 374处标记的芘肌动蛋白激活S - 1 ATP酶活性的能力仅为对照肌动蛋白的一半。这种降低归因于较低的V - max值，因为芘肌动蛋白对S - 1的亲和力没有显著改变。芘肌动蛋白的体外滑动速度也降低了。然而，肌动蛋白的IAEDANS标记（同样在半胱氨酸 - 374处）使肌动蛋白 - S - 1 ATP酶活性的V - max以及体外滑动速度提高了约25%。我们根据肌动蛋白远距离区域之间的构象耦合以及肌动蛋白结合蛋白与肌动蛋白C末端相互作用的功能影响来讨论这些结果。
In this study, we use fluorescent probes and proteolytic digestions to demonstrate structural coupling between distant regions of actin. We show that modifications of Cys-374 in the C-terminus of actin slow the rate of nucleotide exchange in the nucleotide cleft. Conformational coupling between the C-terminus and the DNaseI loop in subdomain II is observed in proteolytic digestion experiments in which a new C-terminal cleavage site is exposed upon DNaseI binding. The functional consequences of C-terminal modification are evident from S-1 ATPase activity and the in vitro motility experiments with modified actins. Pyrene actin, labeled at Cys-374, activates S-1 ATPase activity only half as well as control actin. This reduction is attributed to a lower V-max value because the affinity of pyrene actin to S-1 is not significantly altered. The in vitro sliding velocity of pyrene actin is also decreased. However, IAEDANS labeling of actin (also at Cys-374) enhances the V-max of acto-S-1 ATPase activity and the in vitro sliding velocity by approximately 25%. These results are discussed in terms of conformational coupling between distant regions in actin and the functional implications of the interactions of actin-binding proteins with the C-terminus of actin.