Interaction of myosin with monomeric actins

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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.