Photochemical cross-linking between rabbit skeletal troponin and alpha-tropomyosin. Attachment of the photoaffinity probe N-(4-azidobenzoyl-[2-3H]glycyl)-S-(2-thiopyridyl)-cysteine to cysteine 190 of alpha-tropomyosin.
The heterobifunctional photoaffinity probe N-(4-azidobenzoyl-[2-3H]glycyl)-S-(2-thiopyridyl)-cysteine was attached to cysteine 190 of alpha-tropomyosin to determine which component of rabbit skeletal troponin was in close proximity to cysteine 190. The troponin-tropomyosin complex was photolyzed in the presence and absence of Ca2+, and the radiolabeled troponin, (CM-Tn-AGC) was isolated by hydroxylapatite chromatography. CM-Tn-AGC was separated into its individual components of DEAE-Sephadex chromatography in 8 M urea, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and dithiothreitol. Radioactive measurements indicated that only troponin T (TnT) was radiolabeled. A limited 15-min chymotryptic digest of CM-Tn-AGC isolated from the CM-Tn-AGC-tropomyosin complex formed in the presence or absence of Ca2+ resulted in the isolation of T2-AGC (residues 156-259 of TnT) which bound to a tropomyosin-Sepharose affinity column. More proteolysis of CM-Tn-AGC allowed the isolation of T2'-AGC (residues 156-227). When the tropomyosin-troponin complex was photolyzed in the absence of Ca2+, as compared to the presence of Ca2+, there was a 1.7-fold increase in the cross-linking yield to troponin. This result suggests that there is a Ca2+-sensitive conformational change in the binding region of TnT around cysteine 190. A tightening of the complex around cysteine 190 in the absence of Ca2+ could explain the decrease in reaction of the arylnitrene with solvent. Proteolysis of T2 and sequence analysis of the radiolabeled peptides is now in progress.
D000818:Animals; D001386:Azides / Q000378:metabolism*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism*; D010777:Photochemistry; D011817:Rabbits; D014335:Tropomyosin / Q000378:metabolism*; D014336:Troponin / Q000378:metabolism*
D001386:Azides; D003432:Cross-Linking Reagents; D009124:Muscle Proteins; D014335:Tropomyosin; D014336:Troponin; C029966:N-(4-azidobenzoylglycyl)-S-(2-thiopyridyl)cysteine; D003545:Cysteine