Cysteine mutants of light chain-2 form disulfide bonds in skeletal muscle myosin.
Multiple disulfide bonds form in recombinant myosin light chain-2 mutants that contain an engineered cysteine at positions 2, 73, or 94, in addition to the endogenous cysteines at residues 126 and 155 (Saraswat, L.D., and Lowey, S. (1991) J. Biol. Chem. 266, 1977). By replacing one of the native cysteines with an alanine, mutants with a single pair of thiols were created: Cys2/Cys155, Cys2/Cys126, Cys73/Cys155 and Cys94/Cys155. Oxidation of these mutants resulted in a fast migrating band on nonreducing SDS gels, which was attributed to an intramolecular disulfide bond. To determine if disulfide formation could also occur when the light chains (LC) are bound to the myosin heavy chains, LCs were added to myosin which had been depleted of its native LC2 by an immunoadsorbent. When the reconstituted myosin was reacted with 5,5'-dithiobis(2-nitrobenzoic acid) in the absence of divalent cations, intramolecular disulfide bonds formed in the mutant and wild type LCs, but the LCs did not remain bound to the myosin heavy chains. Addition of magnesium ions prevented LC dissociation, but intramolecular disulfide bonds no longer formed. Instead, mutants containing cysteines in the NH2-terminal domain formed intermolecular disulfide bonds between the two heads of myosin. The ability to cross-link the heads demonstrates the existence of close head/head interactions in the myosin molecule, a feature that may be essential for regulation.
Wolff-Long
V L
VL
Rosenstiel Basic Medical Sciences Research Center, Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
Saraswat
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LD
Lowey
S
S
D000818:Animals; D001483:Base Sequence; D002413:Cations, Divalent; D002645:Chickens; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000737:chemistry* / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship
D002413:Cations, Divalent; D004220:Disulfides; D009838:Oligodeoxyribonucleotides; D004228:Dithionitrobenzoic Acid; D009218:Myosins; D003545:Cysteine