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

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Abstract
Previous experiments demonstrated that two thiols of skeletal myosin subfragment 1 (SF1) could be oxidized to a disulfide bone by treatment with a 2-fold excess of 5,5''-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of MgADP [Wells, J. A., and 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., and 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., and Collins, J. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4281-4284; M. Elzinga, personal communication]. Parallel experiments with [14C]pPDM and unmodified SF1 similar to those above gave an identical SH1, SH2 tryptic peptide, verifying earlier labeling results [Burke, M., and Knight, P. (1980) J. Biol. Chem. 255, 8385-8387]. These combined results demonstrate that SH1 and SH2 cross-linked by pPDM (12-13 .ANG., S to S) or by oxidation with DTNB (2 .ANG., S to S) can move a minimum of 10 .ANG. under the influence of nucleotide binding. Because these residues are separated by only nine amino acids in the primary sequence, this small section of the heavy chain must possess extraordinary flexibility.