Kuhlman P A, Bagshaw C R
Department of Biochemistry, University of Leicester, UK.
J Muscle Res Cell Motil. 1998 Jun;19(5):491-504. doi: 10.1023/a:1005304408812.
Structural characterization of the mode of interaction of nucleotides bound to myosin has relied upon the crystal structure of the Dictyostelium discoideum myosin II motor domain. This fragment, denoted S1dC, lacks the regulatory domain and light chain subunits and may therefore fail to display the normal ATPase activity of the intact myosin molecule. Here we show that the elementary steps of the S1dC ATPase pathway and the effects of actin are similar to those of the complete myosin head fragment. This indicates that truncation at residue E759, with the removal of the light chain binding sites, is not crucial to catalytic activity. In particular, S1dC does not show the anomolous tight binding of ADP displayed by slightly shorter M754 construct reported elsewhere. We also show that the fluorescent analogue Cy3-EDA-ATP is a good substrate for S1dC and demonstrate the use of fluorescence correlation spectroscopy to determine the affinity of Cy3-EDA-ADP using microgram quantities of proteins.
与肌球蛋白结合的核苷酸相互作用模式的结构表征依赖于盘基网柄菌肌球蛋白II运动结构域的晶体结构。这个片段，记为S1dC，缺少调节结构域和轻链亚基，因此可能无法展现完整肌球蛋白分子正常的ATP酶活性。在这里我们表明，S1dC ATP酶途径的基本步骤以及肌动蛋白的作用与完整肌球蛋白头部片段的相似。这表明在E759位点截断，去除轻链结合位点，对催化活性并非至关重要。特别是，S1dC没有表现出其他地方报道的稍短的M754构建体所显示的ADP异常紧密结合。我们还表明，荧光类似物Cy3-EDA-ATP是S1dC的良好底物，并展示了使用荧光相关光谱法，利用微克量的蛋白质来确定Cy3-EDA-ADP的亲和力。
