Interaction of myosin with monomeric actins

肌球蛋白横桥与F-肌动蛋白的循环相互作用代表了肌肉收缩期间的力产生步骤。这种相互作用的动力学和伴随的ATP水解已进行了详细分析，通过使用水溶性肌球蛋白亚片段1和F-肌动蛋白。这些研究表明，S1优先与丝状（F-）肌动蛋白相互作用。不太好的特点是它与单体肌动蛋白的相互作用。该项目的目的是对不同的S1分子与单体形式的肌动蛋白的相互作用进行动力学分析，单体形式的肌动蛋白可以通过生化修饰（ADP-核糖基化）或与螯合肌动蛋白结合蛋白（如DNA酶I、胸腺素β 4、维生素D结合蛋白或凝溶胶蛋白片段1）复合后获得。S1对这些单体肌动蛋白的亲和力将通过动力学方法测定，并与F-肌动蛋白进行比较。不同的S1分子将测试，特别强调突变的重组网骨藻S1，据报道，表现出更高的肌动蛋白结合亲和力。条件将被定义下，可以产生稳定的复合物S1与单体肌动蛋白，这将被用于结晶尝试。

The cyclic interaction of the myosin cross-bridge with F-actin represents the force generating step during muscle contraction. The kinetics of this interaction and the accompanying ATP-hydrolysis have been analysed in great detail by using the water soluble myosin subfragment 1 and F-actin. These studies have shown that S1 preferentially interacts with filamentous (F-) actin. Less well characterised is its interaction with monomeric actin. The aim of this project is the kinetic analysis of the interaction of different S1 molecules with monomeric forms of actin which can be obtained by biochemical modification (ADP-ribosylation) or after complexation to sequestering actin binding proteins such as DNase I, thymosin ß4, Vitamin D binding protein, or gelsolin segment 1. The affinity of S1 to these monomeric actins will be determined by kinetic procedures and compared to F-actin. Different S1 molecules will tested with particular emphasis on mutated recombinant Dictyostelium S1s reportedly exhibiting a higher actin binding affinity. Conditions will be defined under which stable complexes of S1s with monomeric actin can be generated which will be employed in crystallisation attempts.