Acrylamide fluorescence quenching studies on the actin-induced change in protein dynamics in the subfragment-1/subfragment-2 link region of cardiac myosin.
In order to obtain information about the actin-induced conformational change around the subfragment-1/subfragment-2 link region of myosin, measurements of the fluorescence quenching by acrylamide were made on cardiac myosin and its heavy meromyosin, in which the reactive lysyl residue located in the link region was labeled with an extrinsic fluorophore, the N-methyl-2-anilino-6-naphthalenesulfonyl group. The results with the model compound indicated the involvement of a collisional quenching mechanism for the fluorophore. The quenching rate constant calculated from measured quenching constants using available lifetime data was extremely low for the labeled myosin (0.59 X 10(8) M-1 . S-1), suggesting that the fluorophore bound to myosin is surrounded by segments of proteins. This value was independent of the solvent viscosity, indicating that the quenching reaction is limited by fluctuations in the protein matrix, which produce the inward movement of acrylamide. Chymotryptic digestion of the labeled myosin, which yielded the light chain-2-deficient heavy meromyosin, made the bound fluorophore slightly exposed. Addition of F-actin resulted in about 40% reduction in the quenching rate constants for the labeled myosin and heavy meromyosin. The actin effect was reversed by adding ATP. These results suggest that the binding of actin to myosin makes the protein matrix around the subfragment-1/subfragment-2 link region less mobile.
D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D000199:Actins / Q000494:pharmacology*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D009206:Myocardium / Q000378:metabolism*; D009218:Myosins / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000302:isolation & purification*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013552:Swine
D000178:Acrylamides; D000199:Actins; D000817:Anilino Naphthalenesulfonates; D010446:Peptide Fragments; C019057:2,6-mansyl chloride; D020106:Acrylamide; D000251:Adenosine Triphosphatases; D009218:Myosins