Two conformational states of didansylcystine-labeled rabbit cardiac tropomyosin.
The fluorescence properties of rabbit cardiac tropomyosin specifically labeled at Cys190 with didansylcystine has been correlated with its unfolding transitions. Circular dichroism studies at 222 nm showed that for both didansylcystine-labeled tropomyosin and reduced tropomyosin under physiological conditions, a small thermal pretransition near 30 degrees C was present before the main unfolding transition at 48 degrees C. In the absence of added salt there was only one unfolding transition near 33 degrees C for both the native and the labeled tropomyosin. Thus, conformational perturbation by the probe was minor. Despite the specific labeling, the fluorescence decay of didansylcystine-labeled tropomyosin was composed of two components; a major component with a short lifetime (6 ns) and a minor blue shifted component with a relatively long lifetime (17 ns), due to the sampling by the probe of both a polar and a hydrophobic environment, respectively, near Cys190. The fluorescence contribution of the long-lived component increased in the pretransition before decreasing in the main unfolding transition. The increase of the long-lived component appears to be a consequence of a shift in conformational equilibrium of tropomyosin from a "chain-closed" toward a localized "chain-open" state, which allows greater access of the probe to the exposed hydrophobic region.
D000818:Animals; D002942:Circular Dichroism; D003553:Cystine / Q000031:analogs & derivatives; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D006358:Hot Temperature; D009206:Myocardium / Q000032:analysis; D011487:Protein Conformation; D011817:Rabbits; D012965:Sodium Chloride; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D014335:Tropomyosin; C038494:di(dansyl)cystine; D012965:Sodium Chloride; D003553:Cystine