Intramolecular cross-linking of myosin subfragment 1 with bimane

We previously showed that the fluorescent inter-thiol cross-linker dibromobimane (DBB) [Kosower, N. S., Kosower, E. M., Newton, G. L., & Ranney, H. M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3382-3386] cross-links two [50 and 20 kilodaltons (kDa)] of the three major fragments of myosin subfragment 1 (S-1); on intact S-1, DBB quenches tryptophans and inhibits all ATPases [Mornet, D., Ue, K., & Morales, M. F. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1658-1662]. Here we characterize the modification chemically: DBB cross-links Cys-522 (50 kDa) with Cys-707 (20 kDa), thereby sealing a large preexisting heavy-chain loop containing important functionalities. Cross-linking rate is insensitive to nucleotides, but apparently sterically, either monobromobimane or DBB reduces Ca2+-ATPase to low, nonzero levels.