Temperature and Ligand Dependence of Conformation and Helical Order in Myosin Filaments

Abstract

Mammalian myosin filaments are helically ordered only at higher temperatures (>20 degrees C) and become progressively more disordered as the temperature is decreased. It had previously been suggested that this was a consequence of the dependence of the hydrolytic step of myosin ATPase on temperature and the requirement that hydrolysis products (e.g., ADP.P(i)) 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 nonhydrolyzable ligands. The muscle fibers were overstretched to nonoverlap such that myosin affinity for nucleotides was not influenced by the interaction of myosin with the thin filament. We show that with bound ADP.vanadate, which mimics the transition state between ATP and hydrolysis products, or with the ATP analogues AMP-PNP or ADP.BeF(x)() the myosin filaments are substantially ordered at higher temperatures but are reversibly disordered by cooling. These results reinforce recent studies in solution showing that temperature as well as ligand influence the equilibrium between multiple myosin conformations [Málnási-Csizmadia, A., Pearson, D. S., Kovács, M., Woolley, R. J., Geeves, M. A., and Bagshaw, C. R. (2001) Biochemistry 40, 12727-12737; Málnási-Csizmadia, A., Woolley, R. J., and Bagshaw, C. R. (2000) Biochemistry 39, 16135-16146; Urbanke, C., and Wray, J. (2001) Biochem. J. 358, 165-173] and indicate that helical order requires the myosin heads to be in the closed conformation. Our results suggest that most of the heads in the 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 closed conformation in relaxed muscle. Analysis of the dependence on temperature of helical order and myosin conformation shows that in the presence of these analogues one ordered (closed) conformation and two disordered conformations with distinct thermodynamic properties coexist. Low temperatures favor one disordered conformation, while high temperatures favor the ordered (closed) conformation together with a second disordered conformation.

Myosin complexed with ADP and blebbistatin reversibly adopts a conformation resembling the start point of the working stroke

“…The ordered state of myosin heads was previously associated with the prepowerstroke state (24). In another x-ray diffraction study, Yu and coworkers also observed that, in overstretched muscle fibers, blebbistatin causes ordering of the myosin heads in the nonoverlapping (myosin-only) region (15).…”

Fiber type and temperature dependence of inorganic phosphate: implications for fatigue

“…In addition, it is possible that elevated fiber force with increasing temperature is in part due to an increase in the force per cross bridge. Xu et al (51) observed temperature to increase the helical order of the myosin head and hypothesized that this would reduce the internal drag, producing a faster filament sliding velocity. While the increased filament order might contribute to the temperature effect on fiber velocity and thus peak power, whether it exerts any effect on force is not known.…”

Myosin regulatory light chain phosphorylation inhibits shortening velocities of skeletal muscle fibers in the presence of the myosin inhibitor blebbistatin

“…If the array is stable then velocity does not tend to be inhibited as for dephosphorylated fibers in vanadate. Vanadate stabilizes the array to a greater extent than BeF 3 (Wray 1987;Xu et al 2003), and shortening velocities of dephosphorylated fibers are inhibited in BeF 3, but not in vanadate. At lower temperatures, the array is unstable, and all of the phosphate analogs inhibit both tension and velocity.…”

“…The array itself was first visualized in resting frog muscles by electron microscopy and X-ray diffraction (reviewed in (Huxley and Faruqi 1983) and has since been shown in a variety of muscle types (Levine 1993). The array is labile, and an example of disruption to the array can be found in mammalian muscles, where order is only seen at higher temperatures (Wray 1987;Xu et al 2003). In normal physiological conditions such as that in relaxed fibers, and probably also in fibers with large populations of non-force generating heads, these heads can be sequestered by binding to the thick filament where they are sterically unable to reach and interact with actin filaments.…”