Molecular movements promoted by metal nucleotides in the heavy-chain regions of myosin heads from skeletal muscle.

Molecular movements generated in the heavy-chain regions (27-50-20(X 10(3)) Mr) of myosin S1 on interaction with nucleotides ATP, AMPPNP, ADP and PPi were investigated by limited proteolysis of several enzyme-metal nucleotide complexes in the absence and presence of reversibly bound and crosslinked F-actin. The rate and extent of the nucleotide-promoted conversion of the NH2-terminal 27 X 10(3) Mr and 50 X 10(3) Mr segments into products of 22 X 10(3) Mr and 45 X 10(3) Mr, respectively, were estimated to determine the amplitude of the molecular movements. The 22 X 10(3) Mr peptide was identified by amino acid sequence studies as being derived from cleavage of the peptide bond between Arg and Ile (at position 23 to 24). The 45 X 10(3) Mr peptide, previously shown to represent the NH2-terminal part of the 50 X 10(3) Mr region, would be connected to the adjacent C-terminal 20 X 10(3) Mr region by a pre-existing loop segment of about 5 X 10(3) Mr; the proteolytic sensitivity of the latter region is increased particularly by nucleotide binding. The tryptic reaction proved to be a sensitive indicator of the conformational state of the liganded heavy chain as the rate of peptide bond cleavage in the two regions is dependent on the nature of the bound ligand; it decreases in the order: ATP greater than AMPPNP greater than ADP greater than PPi. It depends also on the nature of the metal present, Mg2+ and Ca2+ being much more effective than K+. Binding of F-actin to the S1-MgAMPPNP complex affords significant protection against breakdown of 27 X 10(3) Mr and 50 X 10(3) Mr peptides, but with concomitant hydrolysis of the 50 X 10(3) Mr-20 X 10(3) Mr junction. Additionally, interaction of MgATP with HMM modulates the tryptic fission of the S1-S2 region. The overall data provide a molecular support for the two-state model of the myosin head and emphasize the involvement of the 50 X 10(3) Mr unit in the mechanism of coupling between the actin and nucleotide binding sites.