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.
D000818:Animals; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology*; D001952:Bridged-Ring Compounds / Q000494:pharmacology*; D002918:Chymotrypsin; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003488:Cyanogen Bromide; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005456:Fluorescent Dyes; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism*; D011817:Rabbits
D001643:Bridged Bicyclo Compounds; D001952:Bridged-Ring Compounds; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D010446:Peptide Fragments; D008623:Mercaptoethanol; C037094:dibromobimane; D004228:Dithionitrobenzoic Acid; D002918:Chymotrypsin; D009218:Myosins; D003488:Cyanogen Bromide; C028381:monobromobimane