Direct evidence for the calcium-induced change in the quaternary structure of troponin in situ. Millisecond cross-linking of troponin components by a photosensitive heterobifunctional reagent.
Flash irradiation of the reconstituted troponin of thin filament complex in which one of their components, troponin C, was modified with a heterobifunctional photosensitive reagent before reconstitution of the troponin complex resulted in the formation of cross-links between troponin C and other components in contact with it. Quantitative analysis of the cross-linked products by gel electrophoresis has revealed interesting features of the quaternary structure of troponin. When the reconstituted troponin was photo-cross-linked with a xenon flash, an appreciable amount of cross-linking was detected between troponin C and troponin I and also between troponin C and troponin T. No effect of calcium on the cross-linking could be detected. This arrangement of components was found to change when troponin was complexed with F-actin-tropomyosin. The arrangement of troponin components in the thin filament complex was sensitive to calcium and magnesium; maximum cross-linking of troponin C and troponin I was observed when the thin filament was cross-linked in the presence of calcium and magnesium, while an appreciable decrease in the extent of the cross-linking was detected when calcium alone or calcium and magnesium were removed from the cross-linking medium. The cross-linking of troponin C and troponin T remained marginal irrespective of the concentration of calcium and magnesium.
D002118:Calcium; D003374:Coumarins; D003432:Cross-Linking Reagents; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium; D008301:Maleimides; D009124:Muscle Proteins; D010777:Photochemistry; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014336:Troponin
D003374:Coumarins; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D008301:Maleimides; D009124:Muscle Proteins; D014336:Troponin; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D008274:Magnesium; D002118:Calcium