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.
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)
Huston
E E
EE
Institute of Biological Chemistry, Washington State University, Pullman 99164.
Grammer
J C
JC
Yount
R G
RG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004220:Disulfides; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D008301:Maleimides; D008969:Molecular Sequence Data; D009218:Myosins / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D010084:Oxidation-Reduction; D010446:Peptide Fragments; D011487:Protein Conformation; D011817:Rabbits; D013438:Sulfhydryl Compounds; D014357:Trypsin
D003432:Cross-Linking Reagents; D004220:Disulfides; D008301:Maleimides; D009711:Nucleotides; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; C011328:N,N'-4-phenylenedimaleimide; D014357:Trypsin; D009218:Myosins; D005033:Ethylmaleimide