Conformational transitions in the myosin head induced by temperature, nucleotide and actin

Abstract

Tryptic digestion patterns reveal a close similarity of the substructure of frog subfragment-1 (Sl) to that established for rabbit S1. The 97-kDa heavy chain of chymotryptic S1 of frog myosin is preferentially cleaved into three fragments with apparent molecular masses of 29 kDa, 49 kDa and 20 kDa. These fragments correspond to the 27-kDa, 50-kDa and 20-kDa fragments of rabbit S1, respectively; this is indicated by the sequence of their appearance during digestion, by the suppression by actin of the generation of the 49-kDa and 20-kDa peptides, and by a nucleotide-promoted cleavage of the 29-kDa peptide to a 24-kDa fragment and the 49-kDa peptide to a 44-kDa fragment, analogous to the nucleotide-promoted cleavage of the 27-kDa and 50-kDa fragments of rabbit S1 to the 22-kDa and 45-kDa peptides.The same changes in the digestion patterns as those produced by the presence of nucleotide (ATP or its P,yimido analog AdoPP[NH]P) at 25 "C were observed when the digestion was carried out at 0°C in the absence of nucleotide. The low-temperature-induced changes were particularly well seen in the preparations from frog myosin. The presence of ATP or AdoPP[NH]P at 0°C enhanced, whereas the complex formation with actin prevented, the low-temperature-induced changes. The results are consistent with there being two fundamental conformational states of the myosin head in an equilibrium that is dependent on the temperature, the nucleotide bound at the active site, and the presence or absence of actin.It is generally accepted that contraction of muscle results from a relative sliding movement of the myosin thick filaments past the actin thin filaments driven by a cyclic interaction of myosin heads or cross-bridges with actin, each cycle being coupled to hydrolysis of one molecule of ATP [l]. However, the detailed mechanism of conversion of the free energy of ATP hydrolysis into mechanical energy remains a subject of debate. A prevailing model suggested by H. E. Huxley [l] and by A. F. Huxley and Simmons [2] assumes that force is generated by an active change in the angle of attachment of myosin heads to actin filaments by rotation about a flexible joint between the head (myosin subfragment-1) and the neck region (subfragment-2) of the cross-bridge. An alternative proposal is the rotation of one portion or domain of the head relative to the other portion which remains rigidly attached to actin [3,4]. The latter possibility is in better agreement with the evidence from recent X-ray diffraction studies on muscle [5 -71 and with fluorescence polarization [8] and EPR studies [9] on glycerinated muscle fibers.The possible existence of domains within the myosin head has been inferred from limited proteolysis studies on myosin subfragment-1. The heavy chain of chymotryptic S1 of myosin from rabbit skeletal muscle is split by trypsin into three major fragments with molecular masses of 27 kDa, 50 kDa, and 20 kDa [lo, 111 which are aligned in this order relative to the NH2-terminus of the heavy chain [12]. Two heavy-chain segments (about ...

Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II

“…The presence of nucleotide has a strong modulating effect on the proteolytic cleavage pattern of muscle myosin. Additionally, modifications in the rates of cleavage of loop 1 and loop 2 are observed [12,59–61]. The most important change detected with nonmuscle myosin II, regardless of the nature of the nucleotide, is the protection of a cleavage site located 2 kDa from loop 2 inside the 70‐kDa NH 2 ‐terminal fragment.…”

Dimerization of Native Myosin LC2(RLC)-Free Subfragment 1 from Adult Rabbit Skeletal Muscle

“…Other experimental work has corroborated and extended their results (see ref 25 and references therein). By means of proteolysis, Redowicz et al (26) have shown that conformational transitions in the myosin heads are induced by temperatures between 0 and 25 °C. They digested S1 in a buffer of composition comparable to that of our dimer buffers, and we deduce that at 0 °C S1 is in the dimeric form, whereas it is in the monomeric form at 25 °C, leading possibly to different digestion products at the two temperatures.…”