Actin-Myosin Kinetics and the Effect of Myosin Binding on Cooperative Thin Filament Activation
Biochemistry & Molecular Biology
Biochem and Molecular Biology
StatisticsView Usage Statistics
Activation of the acto-myosin complex is controlled by the regulatory proteins tropomyosin (Tm) and troponin (Tn) which is made up of the C, I and T subunits. TnC undergoes a conformational change upon calcium binding triggering the release of actin by TnI thereby allowing the TmTn complex to move away from myosin binding sites. This process is known as regulation. Regulation allows for what is known as cooperativity, or the ability of a single initial event to make a subsequent event more or less likely. As this relates to muscle mechanics cooperativity is defined in two ways, these being calcium sensitive and myosin dependent respectively. With regard to myosin dependency cooperativity is a measure of the ability of one myosin head binding the thin filament to increase the chances of another myosin head binding to the thin filament and is therefore related to the number of myosin heads bound to the thin filament (????). Cooperativity is related to calcium sensitivity in that the regulatory complex must move away from the actin binding sites for a myosin head to bind. In addition to myosin dependent this becomes calcium dependent with the binding of calcium to the TnC subunit of Tn. In vitro motility assays were used to study the cooperative nature of thin filament activation measuring the velocity of the actin+TmTn complex. Velocities of the thin filament were measured while varying myosin density, pCa, and at constant myosin density varying pCa with results showing that with increasing myosin concentration there is a decrease in cooperativity based on the Hill function cooperative coefficient. In addition this shows an increase in ??????50, or the concentration of calcium at which we attain half maximal velocity of the thin filament. With a decrease in myosin density there is an increase in cooperativity and a subsequent decrease in ??????50 . This ??????50 value is known to be affected by the duty ratio, whereas cooperativity is again a function of Nb. In addition the effects of phosphate on the binding pathway of unregulated myosin were studied again using an in vitro motility assay varying the concentration of myosin density and the presence of phosphate. Experiments were performed using rabbit striated muscle. Additional studies of the binding pathways of actinmyosin interactions were done using N,N’p-phenylenedimaleimide (pPDM) which is a structural analogue of myosin subfragment-1 thought to force actin-myosin interaction towards a weakly bound state (14).