Flexibility of the myosin heavy chain: direct evidence that the region containing SH1 and SH2 can move 10 A under the influence of nucleotide binding

Biochemistry. 1988 Dec 13;27(25):8945-52. doi: 10.1021/bi00425a011.

Abstract

Previous experiments demonstrated that two thiols of skeletal myosin subfragment 1 (SF1) could be oxidized to a disulfide bond by treatment with a 2-fold excess of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of MgADP [Wells, J. A., & Yount, R. G. (1980) Biochemistry 19, 1711-1717]. The resulting characteristic changes in the ATPase activities of SF1 and the fact that MgADP was stably trapped at the active site [Wells, J. A., & Yount, R. G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970] suggested that the two thiols cross-linked were SH1 (Cys-707) and SH2 (Cys-697) from the myosin heavy chain. To verify this suggestion, SF1, after DTNB treatment as above, was treated with an excess of N-ethylmaleimide to block all accessible thiols. The single protein disulfide produced by DTNB oxidation was reduced with dithioerythritol and the modified SF1 internally cross-linked with equimolar [14C]p-phenylenedimaleimide (pPDM) in the presence of MgADP. After extensive trypsinization, the major 14C-labeled peptide was isolated, characterized, and shown to be Cys-Asn-Gly-Val-Leu-Gly-Ile-Arg-Ile-Cys-Arg, in which the two cysteines were cross-linked by pPDM. This peptide is known to contain SH2 and SH1 in this order and to come from residues 697-708 in the rabbit skeletal myosin heavy chain [Elzinga, M., & Collins, J. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4281-4284; M. Elzinga, personal communication].(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Chemical Phenomena
  • Chemistry
  • Chromatography, High Pressure Liquid
  • Cross-Linking Reagents
  • Disulfides
  • Dithionitrobenzoic Acid / pharmacology
  • Ethylmaleimide / pharmacology
  • Maleimides
  • Molecular Sequence Data
  • Myosins / metabolism*
  • Nucleotides / metabolism*
  • Oxidation-Reduction
  • Peptide Fragments
  • Protein Conformation
  • Rabbits
  • Sulfhydryl Compounds*
  • Trypsin

Substances

  • Cross-Linking Reagents
  • Disulfides
  • Maleimides
  • Nucleotides
  • Peptide Fragments
  • Sulfhydryl Compounds
  • Dithionitrobenzoic Acid
  • N,N'-4-phenylenedimaleimide
  • Trypsin
  • Myosins
  • Ethylmaleimide