REGULATION OF ACTOMYOSIN ATPASE
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Abstract
Myosin is a molecular motor that converts chemical energy to mechanical energy, thus generating force and movement in muscles and cells. Myosin interacts with two ligands, ATP (fuel) and actin filaments (motor’s tracks). Actomyosin interaction is regulated by the availability of ATP and by the proteins specifically binding actin or myosin. We would like to know how else myosin can be regulated. In this dissertation, we have examined actomyosin regulation by (a) metal ions, complexed with ATP, (b) crowding of actomyosin environment, and (c) ionic strength of actomyosin solution. We have used steady state myosin ATPase activity measurements, myosin intrinsic fluorescence, fluorescence of pyrene labeled actin and home-built transient time-resolved Förster resonance energy transfer to monitor myosin and actomyosin kinetics. We have perturbed myosin nucleotide binding site with magnesium-, manganese-, or calcium-nucleotide complexes, using metal cation as a probe to examine the pathways of myosin ATPase in the presence of actin. We found that actin activation of myosin ATPase does not depend on metal cation, regardless of the cation-specific kinetics of nucleotide binding and dissociation. The rate limiting step of myosin ATPase depends on the metal cation. The rate of the recovery stroke and the reverse recovery stroke is directly proportional to the ionic radius of the cation. The rate of nucleotide release from myosin and actomyosin, and ATP binding to actomyosin depends on the cation coordination number.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.The rate of actin and myosin rigor binding depends on the ionic strength of the solution, confirming that actomyosin interaction is electrostatically driven. The dependence of the rate of actin and myosin interaction on the ionic strength of the solution reflects the charge of the interface of the interacting molecules. We used recent data on the structure of actomyosin complex to determine the charge and charge distribution of the actin binding interface of myosin head. The experimental transient kinetics data and the computational analysis were used to compare cellular and muscle myosin in terms of similarities and differences of their actin binding interface.In summary, the results of this study suggest that cross-bridge kinetics, and therefore muscle performance, significantly depends on intracellular environments. What we have learned in this study is essential not only for revealing movement at the cellular and molecular levels but also for enhancing muscle power output from environments.