Probing the mechano-chemical transduction mechanism of skeletal muscle myosin II using a feedback enhanced optical trap

Skeletal muscle uses more energy when it is shortening rapidly and less energy when it is maintaining a static load, a phenomenon discovered in 1923 by W. O. Fenn and A. V. Hill. This physiological feedback, termed the "Fenn effect", was one of the key observations leading to the actomyosin cross-bridge theory of muscle contraction by A. F. Huxley in 1957. The efficient control of energy liberation in isometric vs. shortening skeletal muscle implies that one or more steps in the actomyosin ATPase cycle are controlled by mechanical load borne by the molecular motor, myosin. The objective of this study is to obtain a precise correlation between the mechanical and biochemical aspects of actomyosin interactions at the single molecule level to understand the molecular mechanism of this load adaptive molecular motor.; In this work, a novel feedback enhanced infrared laser-based optical gradient trap, the "isometric clamp", was constructed. The isometric clamp was used to study the mechanics of individual skeletal muscle myosin in order to detect the reaction steps that depend on the dynamic properties of the external load. The method also enabled reliable and quantitative measurement of the mechanical properties such as the isometric force, power stroke displacement and stiffness, produced by single actomyosin interactions.; The results obtained from this work indicate that single myosin molecules transduce energy as efficiently as whole muscle and that a component of the Fenn effect is reversal of the force-generating actomyosin transition under high load without net utilization of ATP. Our results, together with the earlier studies of muscle contraction suggest that the actomyosin enzymatic cycle has multiple strain dependent steps, and that two factors contribute toward the Fenn effect: at low shortening velocities, the economical reversal of the force-generating actomyosin transition saves ATP and at high shortening velocities strain-dependent acceleration of ADP release increases ATP turnover. Both of these effects probably occur in muscle contraction and thus contribute to its efficient control of energy utilization.