Kinetics of interaction of myosin-nucleotide complex and actin.

Abstract

In the actomyosin cycle myosin hydrolyses ATP and binds actin, initially weakly and then strongly. The timing of myosin ATP hydrolysis and binding to actin remains elusive. We performed double-mixing transient experiments to characterize the kinetics of actomyosin interaction shortly after ATP binding to myosin. In the experiment we first rapidly pre-mixed rabbit skeletal myosin S1 and stoichiometric ATP and after a controlled delay rapidly added unregulated F-actin to the mixture. The first mixing produces mainly complexes of myosin.ATP and myosin.ADP.Pi, we expected to have small quantity of unreacted myosin and complex of myosin.ADP in the mixture. In the second mixing myosin binds actin. We detected transient fluorescence of pyrene labeled actin and changes in the light scattering after the second mixing. It is accepted that the change of fluorescence of pyrene labeled actin reflects strong actomyosin binding due to the conformational change of actin induced by bound myosin. One can suggest that light scattering reflects the formation of weakly bound actomyosin, since light scattering is sensitive to the size of the protein complex, and not the strength of binding. In our experiments we varied protein concentrations and the time of the delay between the first and the second mixing, which is the myosin-nucleotide incubation time. We fitted obtained transients to the solution of the system of differential equations, describing the evolution of this composite protein system. We discuss models of actomyosin interaction that correspond to the observed transient kinetics and the possible cooperativity of myosin interaction with unregulated actin.