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

Abstract

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-IAEDANS). The fluorescence decay of the resultant AEDANS-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 .times. 108 M-1 s-1. The presence of troponin reduced the magnitude of Ksv'' to 4.14 M-1 and induced the appearance of a 2nd decay component. This 2nd component had an amplitude of .apprx. 20% of the total intensity, a lifetime of .apprx. 20 ns and a kq of 4.5 .times. 10-7 M-1 s-1. 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. Evidently, 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.