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.
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