Tropomyosin-troponin and tropomyosin-actin interactions: a fluorescence quenching study

Rabbit skeletal alpha alpha-tropomyosin was specifically labeled at Cys-190 with the fluorescent probe N-(iodoacetyl)-N'-(1-naphthyl-5-sulfo)ethylenediamine (1,5-IAE-DANS). The fluorescence decay of the resultant AE-DANS-labeled alpha alpha-tropomyosin (Tm) was monoexponential with a lifetime of 13.55 ns. When acrylamide was used as the quencher, the apparent Stern-Volmer quenching constant Ksv' for Tm was measured to be 5.78 M-1 and the quenching rate constant kq to be 3.20 X 10(8) M-1 s-1. The presence of troponin reduced the magnitude of Ksv' to 4.14 M-1 and induced the appearance of a second decay component. This second component had an amplitude of approximately 20% of the total intensity, a lifetime of approximately 20 ns, and a kq of 4.5 X 10(-7) M-1 s-1. Similarly, the presence of F-actin induced the appearance of a minor longer lived decay component with a decreased kq. On the basis of the increase in the lifetime and the decrease in kq, the appearance of the long-lived decay component was interpreted to be due to troponin or actin interacting with Tm near the Cys-190 site in both cases. Our results further suggest that the label was capable of equilibrating between an exposed hydrophilic environment on the surface of Tm and a buried hydrophobic environment at the troponin-Tm or actin-Tm interaction interfaces.