Fluorescence studies of the conformation of pyrene-labeled tropomyosin: effects of F-actin and myosin subfragment

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Abstract
The fluorescence of pyrene-TM [rabbit skeletal tropomyosin (TM) labeled at Cys with N-(1-pyrenyl)maleimide] consists of monomer and excimer bands [Betcher-Lange, S., and Lehrer, S. S. (1978) J. Biol. Chem. 253, 3757-3760]; an increase in excimer fluorescence with temperature is due to a shift in equilibrium from a chain-closed state (N) to a chain-open state (X) associated with a helix pretransition [Graceffa, P., and Lehrer, S. S. (1980) J. Biol. Chem. 255, 11296-11300]. In this study, we show that the presence of appreciable excimer fluorescence at temperatures below the N .fwdarw. X pretransition (initial excimer) is due to perturbation of the TM chain-chain interaction by the pyrenes as Cys-190. Fluorescence and ATPase titrations indicated that the label caused a decrease in TM binding to F-actin primarily due to reduced end to end TM interactions on the actin filament. Under conditions where pyrene-TM was bound to F-actin, however, the excimer fluorescence did not increase with temperature, indicating that F-actin stabilizes tropomyosin by inhibiting the N .fwdarw. X transition. The binding of myosin subfragment 1 (S1) to pyrene-TM-F-actin at low ratios to actin caused time-dependent changes in fluorescence. After equilibrium was reached, the initial excimer fluorescence was markedly reduced and remained constant over the pretransition temperature range. Further stabilization of tropomyosin conformation on F-actin is therefore associated with S1 binding. Effects of the binding of S1 to the F-actin-tropomyosin thin filament on the state of tropomyosin were studied by monitoring the monomer fluorescence of pyrene-TM. Samples with low and high labeling ratios (0.17 and 1.7, respectively) both gave an 18% increase in pyrene monomer fluorescence with the same S1 titration profile, indicating a lack of preferential interaction of S1 with the probe. The fluorescence change, which saturated much before the 1:1 stoichiometric binding to actin was complete, followed a curve described by the random binding of between one (or more) and two (or more) S1'' to seven actin subunits, showing that the state (or position) of tropomyosin is changed by the strong binding of a small number of myosin heads to a tropomyosin-actin unit. The low [S1]/[actin] ratios that were sufficient for the changes in monomer and excimer fluorescence indicate that myosin can produce long-range effects on tropomyosin in the thin filament.