Cross-linking study on tropomyosin.
The cross-linking reaction of alpha-tropomyosin with dimethyl adipimidate yielded a dimer of the alpha-subunit of tropomyosin as a major product, which was isolated by gel filtration on Sephadex G-150 in the presence of urea. Amino acid analyses revealed that the cross-linked alpha-tropomyosin contained about two adipimidate cross-links per molecule. Selective cleavage of the cross-linked molecule at the cysteinyl residue, Cys 190 (a single cysteinyl residue in the alpha-subunmit), gave a new band at a position corresponding to a molecular weight of 48,000 on SDS-gel electrophoresis, suggesting that the cross-links were incorporated in the N-terminal fragment. When the cross-linked molecule was cleaved with CNBr, two large fragments from residue 11 to 127, and from 142 to 281, were obtained, as in the case of the intact molecule. Therefore, it is inferred that the location  of the intersubunit cross-links is in the region from residue 2 to 8 and/or from 128 to 141. These results indicate that the arrangement of alpha-subunits of tropomyosin in solution must be in parallel and in register. Although the exact positions of the reactive sites could not be determined in the present study, stereochemical examination of the coiled-coil model suggests that the most probable sites of cross-linking are Lys 5 of one subunit and Lys 7 of the other.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D003545:Cysteine; D004119:Dimethyl Adipimidate; D007096:Imidoesters; D008970:Molecular Weight; D009132:Muscles; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D011817:Rabbits; D014335:Tropomyosin
D000596:Amino Acids; D003432:Cross-Linking Reagents; D007096:Imidoesters; D010446:Peptide Fragments; D014335:Tropomyosin; D004119:Dimethyl Adipimidate; D003545:Cysteine; D003488:Cyanogen Bromide