Conformation and Helical Order of the Myosin Filaments in Skeletal Muscle Cells

Leepo C. Yu 
National Institute of Arthritis, Musculoskeletal and Skin Diseases
National Institutes of Health, Bethesda, MD. USA

The basic processes of muscle contraction are well understood: it is a result of cyclic interactions between myosin and actin, driven by the energy of actomyosin ATP hydrolysis. Muscle shortening involves the relative sliding of two sets of filaments: the thick, myosin containing filaments and the thin, actin containing filaments. Force is generated by myosin heads (cross-bridges) interacting cyclically with specific sites along the actin filaments. Since the availability of the crystal structures of the contractile proteins, and with the advent of single molecule assays, the field has made great strides in understanding the underlying processes. However, the details of the mechanism of transduction of chemical to mechanical energy still remains largely unresolved. One of the obstacles is that most of the studies at the molecular level are based on isolated, in vitro systems. The link between the information obtained from the in vitro systems and the actual processes occurring in intact muscle is still largely missing. The aim of our efforts is to provide such a link.
             Mammalian myosin filaments are helically ordered only at higher temperatures (>20°C) and become progressively more disordered as the temperature is lowered. It had previously been suggested that this was a consequence of the dependence on temperature of the hydrolytic step of myosin ATPase and the requirement that hydrolysis products (eg ADP.Pi) be bound at the active site. An alternative hypothesis is that temperature directly affects the conformation of the myosin heads and that they need to be in a particular conformation for helical order in the filament. To discriminate between these two hypotheses, we have studied the effect of temperature on the helical order of myosin heads in rabbit psoas muscle in the presence of non-hydrolysable ligands. We show that with bound ADP.vanadate, which mimics the transition state between ATP and hydrolysis products, or with the ATP analogs AMPPNP or ADP.BeFx, the myosin filaments are substantially ordered at higher temperatures but are reversibly disordered by cooling. These results, taken together with recent studies in solution on the effect of temperature on the equilibrium between myosin conformations, indicate that helical order requires the myosin heads to be in the switch-II closed conformation. Our results suggest that most of the heads in the switch-II closed conformation are ordered, and that order is not produced in a separate step. Hence helical order can be used as a signature of the switch-II closed conformation in relaxed muscle. Analysis of the dependence on temperature of helical order and myosin conformation suggests that in the presence of these analogs one ordered conformation and two disordered conformations with distinct thermodynamic properties coexist. Although the ordered state is identified with the switch-II closed conformation, the nature of the two disordered conformations is not clear. Low temperature favors one disordered conformation, while high temperature favors one ordered conformation together with a second disordered conformation.