Dynamics at Lys-553 of the acto-myosin interface in the weakly and strongly bound states.
Dynamics at Lys-553 of the acto-myosin interface in the weakly and strongly bound states.
复制标题
弱结合态和强结合态肌动球蛋白界面 Lys-553 处的动力学。
DOI：
10.1016/s0006-3495(00)76697-5
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发表时间：
2000
影响因子：
3.4
通讯作者：
Berger,CL
中科院分区：
文献类型：
--
作者：
MacLean,JJ;Chrin,LR;Berger,CL
骨骼肌肌球蛋白亚片段 1 (S1) 的 Lys-553 用荧光探针 FHS（6-[荧光素-5（和 6）-甲酰氨基]己酸琥珀酰亚胺酯）特异性标记，并进行荧光猝灭实验以确定该探针在 MgATP 酶循环的强和弱肌动蛋白结合状态下在 Lys-553 上的可及性。使用不同电荷的溶剂猝灭剂 [硝基甲烷、(2,2,6,6-四甲基-1-哌啶氧基) (TEMPO)、碘化物 (I−) 和铊 (Tl+)] 来评估 FHS 探针在 Lys-553 处的空间和静电可及性。在强结合严格状态（无核苷酸）和 MgADP 状态下，肌动蛋白无法防止硝基甲烷、TEMPO 或铊对 FHS 进行溶剂猝灭，但当使用碘化物作为猝灭剂时，确实将 Stern-Volmer 常数降低了几乎两倍。添加 150mM KCl 后，对碘化物猝灭的保护几乎完全逆转，表明这种效应本质上是离子效应而不是空间效应。相反，在稳态 ATP 水解过程中，即使有相当一部分肌球蛋白头与肌动蛋白结合，肌动蛋白也无法在低离子强度下提供碘猝灭保护。因此，包含 Lys-553 的肌球蛋白的较低 50 kD 亚结构域似乎在弱结合状态和强结合状态下与肌动蛋白相互作用不同。
Lys-553 of skeletal muscle myosin subfragment 1 (S1) was specifically labeled with the fluorescent probe FHS (6-[fluorescein-5(and 6)-carboxamido]hexanoic acid succinimidyl ester) and fluorescence quenching experiments were carried out to determine the accessibility of this probe at Lys-553 in both the strongly and weakly actin-bound states of the MgATPase cycle. Solvent quenchers of varying charge [nitromethane, (2,2,6,6-tetramethyl-1-piperinyloxy) (TEMPO), iodide (I−), and thallium (Tl+)] were used to assess both the steric and electrostatic accessibilities of the FHS probe at Lys-553. In the strongly bound rigor (nucleotide-free) and MgADP states, actin offered no protection from solvent quenching of FHS by nitromethane, TEMPO, or thallium, but did decrease the Stern-Volmer constant by almost a factor of two when iodide was used as the quencher. The protection from iodide quenching was almost fully reversed with the addition of 150mM KCl, suggesting this effect is ionic in nature rather than steric. Conversely, actin offered no protection from iodide quenching at low ionic strength during steady-state ATP hydrolysis, even with a significant fraction of the myosin heads bound to actin. Thus, the lower 50 kD subdomain of myosin containing Lys-553 appears to interact differently with actin in the weakly and strongly bound states.