| Myosin subfragment-1 (SF(,1)) was treated with the bifunctional cross-linking reagent ('14)C -N-N'-p-phenylene dimaleimide (pPDM). In the presence of MgADP, both K('+)-EDTA- and Ca('2+)-ATPase activities were inactivated concurrent with the incorporation of 0.95 mole of ('14)C -pPDM per mole of SF(,1). The modified enzyme was extensively trypsinized, and the major radiolabeled peptide isolated. NH(,2)-terminal analysis revealed the presence of two peptides; amino acid composition showed the peptides spanned the region containing the reactive cysteines, SH(,1) and SH(,2). Detection of the modified amino acid S-succinylcysteine showed that the two tryptic peptides were covalently attached by the cross-linker, which has a 12-13 (ANGSTROM) sulfur to sulfur span.;SF(,1) was oxidized with dithio-bis-(2-nitrobenzoic acid) (DTNB) in the presence of MgADP, a treatment known to promote formation of a single protein disulfide. After all other accessible thiols were blocked, the disulfide was reduced and the newly exposed thiol groups reacted with ('14)C -pPDM. Trypsinization and purification of the major radiolabeled peptide showed the presence of an SH(,1) containing peptide cross-linked to an SH(,2) containing peptide. Formation of a disulfide results in a sulfur to sulfur distance of 2 (ANGSTROM).;The bifunctional reagent trans 4,4'-dimaleimidylstilbene-2,2'-disulfonate (DMSDS) becomes fluorescent upon formation of the thiol adduct. Addition to SF(,1) of trans DMSDS resulted in MgADP stimulated inactivation of both K('+)-EDTA- and Ca('2+)-ATPase activities. Inactivation, trapping of ADP, loss of thiol groups, and preliminary localization of DMSDS labeling by partial proteolysis were all consistent with DMSDS cross-linking of SH(,1) and SH(,2). This was confirmed by isolation of DMSDS labeled peptides after extensive trypsinization. The sulfur to sulfur span of trans DMSDS is 18-19 (ANGSTROM).;SH(,1) and SH(,2), separated by only nine amino acids in the sequence, can be cross-linked by rigid bifunctional agents at distances of 2 (ANGSTROM), 12-13 (ANGSTROM), and 18-19 (ANGSTROM). This region of the myosin head is thus demonstrated to be extraordinarily flexible. |
