Substructure of skeletal myosin subfragment 1. Preferential destabilization of a domain by methanol and its effect on catalytic activity.
Evidence is presented that in increasing concentrations of methanol the structure of the subfragment 1 is perturbed in such a way that the Mr = 50,000 central portion of the associated heavy chain is preferentially unfolded. This unfolding is accompanied by the loss in ATPase function where the rate of inactivation can be correlated with the loss in the amount of the Mr = 50,000 fragment generated under standard tryptic digestion conditions. The residual protein appears to be a soluble aggregate of a complex of the Mr = 27,000, 21,000, and light chain with no intact Mr = 50,000 fragment. Tryptic digestions in the presence of MgATP are restricted to the usual linker regions and the Mr = 50,000 fragment is completely protected from attack. Binding of actin to subfragment 1 also results in the protection of the Mr = 50,000 segment and of the Mr = 50,000/21,000 junction from tryptic attack. The data suggest that, in terms of methanolic perturbation, the subfragment 1 appears to be comprised of two domains with differential stability. One domain appears to be the central Mr = 50,000 segment of the heavy chain which is preferentially unfolded by methanol and requires the presence of MgATP or of actin for stabilization. The other domain is more stable and appears to consist of the interacting Mr = 27,000, 21,000, and light chain. The results also suggest that the integrity of the Mr = 50,000 segment is essential for the ATPase function of the protein.
D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D000432:Methanol / Q000494:pharmacology*; D008970:Molecular Weight; D015879:Myosin Subfragments; D009218:Myosins / Q000032:analysis*; D009391:Nephelometry and Turbidimetry; D010446:Peptide Fragments / Q000032:analysis*; D011817:Rabbits; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
D015879:Myosin Subfragments; D010446:Peptide Fragments; D014357:Trypsin; D009218:Myosins; D000432:Methanol