Differences in local conformation around cysteine residues in alpha alpha, alpha beta, and beta beta rabbit skeletal tropomyosin.
The ability of 5,5'-dithiobis-2-nitrobenzoate (Nbs2) to form a disulfide crosslink between the Cys-190s of the alpha alpha and alpha beta molecular components of rabbit skeletal tropomyosin (Tm) and the Cys-36s and Cys-190s of purified beta beta was studied in separate experiments, as a function of urea concentration in 0.5 M NaCl, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 15 degrees C. In the absence of urea, complete reaction of the Cys-190s of Tm with Nbs2 as well as with 2- and 4-pyridine disulfide quantitatively produced two crosslinked species, alpha-alpha and alpha-beta, in a 60/40 ratio, respectively, visualized as bands on sodium dodecyl sulfate-polyacrylamide gels; similar reactions of beta beta produced both doubly (at Cys-36 and Cys-190) and singly crosslinked species (at Cys-190 as identified by amino acid analysis of separated tryptic peptides). In the presence of 4 M urea where the chains were unfolded and separated, only Nbs-blocked uncrosslinked species were obtained after complete reaction with Nbs2. The loss of Nbs2-crosslinking at increasing [urea] showed that the relative stability of the Cys-containing regions of the three species of Tm, alpha alpha, alpha beta, and beta beta increases in the order Cys-36 of beta beta, Cys-190 of alpha beta, Cys-190 of alpha alpha.
D000818:Animals; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000032:analysis*; D004220:Disulfides / Q000032:analysis; D004228:Dithionitrobenzoic Acid; D006868:Hydrolysis; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D011817:Rabbits; D014335:Tropomyosin; D014357:Trypsin; D014508:Urea
D003432:Cross-Linking Reagents; D004220:Disulfides; D010446:Peptide Fragments; D014335:Tropomyosin; D014508:Urea; D004228:Dithionitrobenzoic Acid; D014357:Trypsin; D003545:Cysteine