Resolution of conformational states of Dictyostelium myosin II motor domain using tryptophan (W501) mutants:: Implications for the open-closed transition identified by crystallography
Resolution of conformational states of Dictyostelium myosin II motor domain using tryptophan (W501) mutants:: Implications for the open-closed transition identified by crystallography
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DOI：
10.1021/bi001125j
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发表时间：
2000-12-26
期刊：
影响因子：
2.9
通讯作者：
Bagshaw, CR
中科院分区：
文献类型：
--
作者：
Málnási-Csizmadia, A;Woolley, RJ;Bagshaw, CR
当肌球蛋白与ATP相互作用时，色氨酸的荧光有一个特征的增强，这在动力学研究中得到了广泛的利用。利用Dictyostoma运动区突变体，我们发现W501位于转换区附近的继电螺旋末端，对两个独立的核苷酸结合构象事件做出反应。首先，快速异构化产生小的荧光猝灭，然后控制水解率的较慢的可逆步骤(对应于由结晶学识别的开放-闭合转变)产生较大的增强。缺失W501的突变体没有表现出ATP诱导的荧光增强，但猝灭流动测量表明，与野生型一样，ATP被迅速水解以产生产物复合体。含有色氨酸的单一结构W501+的核苷酸残基、开放和闭合状态显示出明显的荧光光谱和对丙烯酰胺猝灭的敏感性，这表明W501在闭合状态下内化。开放-闭合转变本身不需要水解，并且可以由不可水解的类似物诱导。在20℃时，平衡可能有利于开放状态，但以三磷酸腺苷为底物，随后的水解步骤将平衡拉向关闭状态，从而使仅含有W501的色氨酸突变体产生总体80%的提高。这些研究使得基于溶液的分析可以用肌球蛋白马达结构域的晶体结构合理化，并表明在继电器和转换器区域的界面上可以区分三种不同的状态。
When myosin interacts with ATP there is a characteristic enhancement in tryptophan fluorescence which has been widely exploited in kinetic studies. Using Dictyostelium motor domain mutants, we show that W501, located at the end of the relay helix close to the converter region, responds to two independent conformational events on nucleotide binding. First, a rapid isomerization gives a small fluorescence quench and then a slower reversible step which controls the hydrolysis rate (and corresponds to the open-closed transition identified by crystallography) gives a large enhancement. A mutant lacking W501 shows no ATP-induced enhancement in the fluorescence, yet quenched-flow measurements demonstrate that ATP is rapidly hydrolyzed to give a products complex as in the wild-type. The nucleotidefree, open and closed states of a single tryptophan-containing construct, W501+, show distinct fluorescence spectra and susceptibilities to acrylamide quenching which indicate that W501 becomes internalized in the closed state. The open-closed transition does not require hydrolysis per se and can be induced by a nonhydrolyzable analogue. At 20 degrees C, the equilibrium may favor the open state, but with ATP as substrate, the subsequent hydrolysis step pulls the equilibrium toward the closed state such that a tryptophan mutant containing only W501 yields an overall 80% enhancement. These studies allow solution-based assays to be rationalized with the crystal structures of the myosin motor domain and show that three different states can be distinguished at the interface of the relay and converter regions.