Rapid helix--coil transitions in the S-2 region of myosin.

Abstract

Temperature-jump studies on the long S-2 fragment (100,000 daltons) isolated from myosin show that this structure can undergo a-helix-random coil transitions in a time range approximating the cycle time of a crossbridge. Two relaxation times are observed after temperature jumps of 50C over the range 35-55°C, one in the submillisecond (Tf) and the other in the millisecond (r,) time ranges. Both processes exhibit maxima near the midpoint of the helix-coil transition (ttm = 45 + 2°C) as determined by optical rotation melt experiments. Similar results were observed for the low temperature transition (t. = 45°C) of the myosin rod. Viscosity studies reveal that the S-2 particle has significant flexibility at physiological temperature. Results are considered in terms of the Huxley-Simmons and helix-coil transition models for force generation in muscle. The subfragment 2 (S-2) region of the myosin molecule plays a major role in most current theories of muscle contraction. In the model of H. E. Huxley (1) for contraction, this segment, which forms the coiled-coil a-helical tail of heavy meromyosin, swings away from the thick filament surface during each contractile cycle of a crossbridge, thus allowing the myosin heads (S-1 subunits) to attach to neighboring thin filaments with the same stereospecific orientation over a wide range of interfilament spacing. S-2 is considered to be a rigid, inextensible rod which is fastened to the thick filament backbone by a hinge at the junction of light meromyosin (LMM) and heavy meromyosin (HMM) and is joined to the myosin head by a second hinge-the S-1/S-2 linkage. This arrangement provides the freedom required to accommodate the variable interfilament spacing as the sarcomere shortens. After attachment of the S-1 subunit to the thin filament, it rotates relative to this structure, generating a relative sliding force between the thick and thin filaments. During each cycle of a crossbridge, ATP is hydrolyzed. In the A. F. Huxley-Simmons model (2-4), S-2 serves as an elastic element which, by spring-like extension, acts to exert a part of the tensile force developed when a crossbridge interacts with the thin filament. The force-generating event is again considered to be rotation of the myosin head on the thin filament with the S-2 elastic element stretching instantaneously as the S-1 subunit alters its angle of attachment through a series of stable positions with progressively lower potential energy.In the Harrington model (5) for contraction, the force-generating event is considered to be an a helix-random coil transition within the S-2 element during each cycle of a crossbridge. In this model the myosin head remains fixed in orientation during its transient attachment to the thin filament and the melting of part of the double a helix near the LMM-HMM junction is effected by the ATP cleavage reaction occurring on a neighboring myosin head.Our crosslinking studies of synthetic myosin thick filaments and glycerinated rabbit muscle fibers in rigor indicate that the The publicatio...

1H NMR study of long and short myosin S2 fragments

“…The larger proportion of sharp signals in the long $2 spectrum compared with the short $2 spectrum seen at 40°C and 50°C, but not at 20°C and 30°C, thus indicates the existence of a region or regions in long $2 which have a lower thermal stability than short $2 and which melt at a lower temperature, which is in qualitative agreement with other melting studies [7,14,15]. If one assumes that the part of the molecule common to both fragments shows the same melting behaviour in both, then this region of lower thermal stability must be in the -225 residues present in long $2 but not in short $2.…”

“…Close inspection also indicated that the resonances at 2.2, 2.35 and 3.3 ppm were sharper in the long $2 spectrum at these temperatures. At these temperatures the structure of the molecules will be partially melted [7,14,15]. Protein unfolding is commonly observed to be a slow process on the NMR time scale [8] and the absence of any progressive shifts of the resonances with temperature is consistent with this.…”

“…At 60°C the spectra of long and short $2 were again virtually identical since at this temperature both molecules had been completely denatured [7,14,15] and their amino acid compositions are very similar [6]. However at 40°C and 50°C the spectrum from long $2 contained a larger proportion of sharp signals than that from short $2.…”

Force generation by muscle fibers in rigor: a laser temperature-jump study.

“…The high cooperativity and the kinetic form of the T-jump data are typical of a helix-coil transition in a linear polymer. Conventional T-jump experiments on isolated segments of myosin a-helical coiledcoil structures reveal a typical biexponential response in which the reciprocal relaxation times decline with increasing temperature toward the midpoint of the transition (28,29). In fiber experiments, the reciprocal relaxation time of the slow process 1/; and probably the fast 1/rf also decrease in magnitude as the temperature is elevated.…”

Effect of the integrity of the myofibrillar structure on the tryptic accessibility of a hinge region of the myosin rod

“…One of the main differences was that the cleavage at the HMM/LMM hinge in the rod segment of myosin was reduced in myofibrils relative to isolated myosin. It seemed to us of interest to study this question in greater detail, in particular because of the significance of the HMM~LMM hinge region in the molecular mechanism of contraction, as assumed by Harrington and his co-workers [ 12,22,23]. Here, we studied the effects of actin, of the regulatory proteins of the thin filament (tropomyosin and troponin), as well as those of the myofibrillar structure on the tryptic cleavage of the HMM/LMM hinge region.…”