Studies on the spatial arrangement of muscle thin filament proteins using fluorescence energy transfer.

The distance between a pair of fluorophores attached to Cys-36 of beta-tropomyosin and Cys-373 of actin in reconstituted muscle thin filaments was measured by fluorescence energy transfer. Two pairs of donor/acceptor fluorophores, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid/5-iodoacetamidofluorescein and N-(1-pyrene)maleimide/dimethylamino-4-maleimidostilbene, were covalently attached to tropomyosin and actin. The energy transfer efficiencies in various reconstituted systems were determined from decrease in donor lifetime using nanosecond pulse fluorimetry. Based on the 8 to 13% energy transfer efficiency observed for the first donor/acceptor pair labeled on tropomyosin and actin, respectively, and a calculated critical distance of 45 A (assumed kappa 2 = 2/3) the distance between Cys-373 of actin and Cys-36 of beta-tropomyosin was estimated to be 65 A. Fluorescence energy transfer experiments using other donor/acceptor pairs gave similar results. Since Cys-373 of actin is thought to be in the myosin head-binding sites, the minimal distance between tropomyosin and the myosin-binding site on actin is estimated to be about 34.5 A. These results place some constraints on possible spatial arrangements of thin filament proteins.