Effects of interchain disulfide cross-links on the trypsin cleavage pattern and conformation of myosin subfragment 2.
The ability of 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) to produce interchain disulfide cross-links in both the long and short forms of myosin subfragment 2 (S2) and the conformational effects of these cross-links have been investigated. Short S2 (residues 3-287) contains two pairs of Cys residues at positions 66 and 108, and long S2 (residues 1-440) contains an additional pair at position 410. The reaction kinetics of each form of S2 with Nbs2 was biphasic. During the fast kinetic phase the reaction resulted in un-cross-linked species having Nbs-blocked Cys. During the slow phase disulfide-cross-linked species were formed via interchain S-Nbs/SH exchange. For short S2, Cys-66 appeared to react without forming disulfide cross-links, and the Cys- 108 pair reacted with partial cross-linking. For long S2, the Cys-66 pair appeared to react with partial cross-linking, and the Cys pairs at 108 and 410 reacted with complete cross-linking. Mild tryptic digestion of disulfide-cross-linked long S2, under conditions that resulted in partial production of short S2 from un-cross-linked LS2, produced peptides T1a and T1b (residues 1 to approximately 360), with one and two disulfide cross-links, respectively. Further digestion of cross-linked long S2 or cross-linked short S2 resulted in the same shorter fragment, T2, with an NH2-terminus beginning at 103 consistent with a sequence of residues 103-287. Circular dichroism studies on long S2 indicated that the presence of disulfide cross-links changed the thermal unfolding profile of the helix.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D014357:Trypsin / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004220:Disulfides; D015879:Myosin Subfragments; D010446:Peptide Fragments; D014357:Trypsin; D009218:Myosins; D003545:Cysteine