Abstract
Actomyosin kinetics is usually studied in dilute solutions, which do not reflect conditions in the cytoplasm. In cells, myosin and actin work in a dense macromolecular environment. High concentrations of macromolecules dramatically reduce the amount of free space available for all solutes, which results in an effective increase of the solutes' chemical potential and protein stabilization. Moreover, in a crowded solution, the chemical potential depends on the size of the solute, with larger molecules experiencing a larger excluded volume than smaller ones. Therefore, since myosin interacts with two ligands of different sizes (actin and ATP), macromolecular crowding can modulate the kinetics of individual steps of the actomyosin ATPase cycle. To emulate the effect of crowding in cells, we studied actomyosin cycle reactions in the presence of a high-molecular-weight polymer, Ficoll70. We observed an increase in the maximum velocity of the actomyosin ATPase cycle, and our transient-kinetics experiments showed that virtually all individual steps of the actomyosin cycle were affected by the addition of Ficoll70. The observed effects of macromolecular crowding on the myosin-ligand interaction cannot be explained by the increase of a solute's chemical potential. A time-resolved Förster resonance energy transfer experiment confirmed that the myosin head assumes a more compact conformation in the presence of Ficoll70 than in a dilute solution. We conclude that the crowding-induced myosin conformational change plays a major role in the changed kinetics of actomyosin ATPase.