Fluorescence studies of the conformation of pyrene-labeled tropomyosin: effects of F-actin and myosin subfragment 1.
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., & 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., & 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----X pretransition (initial excimer) is due to perturbation of the TM chain-chain interaction by the pyrenes at 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----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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000199:Actins / Q000378:metabolism*; D000818:Animals; D005456:Fluorescent Dyes / Q000494:pharmacology*; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology*; D009132:Muscles / Q000378:metabolism*; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014335:Tropomyosin / Q000378:metabolism*
D000199:Actins; D005456:Fluorescent Dyes; D008301:Maleimides; D015879:Myosin Subfragments; D010446:Peptide Fragments; D014335:Tropomyosin; C003944:N-(3-pyrene)maleimide; D009218:Myosins