Excimer fluorescence of pyrenyliodoacetamide-labeled tropomyosin: a probe of the state of tropomyosin in reconstituted muscle thin filaments.
Rabbit skeletal tropomyosin (Tm) specifically labeled at cysteine groups with N-(1-pyrenyl)-iodoacetamide (PIA) exhibits excimer fluorescence. The excimer fluorescence was sensitive to the local conformation of Tm, to actin binding, and, in reconstituted thin filaments, to the Tm state change induced by binding of myosin subfragment 1 (S1). The properties of PIATm were similar to previously studied pyrenylmaleimide-labeled Tm (PMTm) [Ishii, Y., & Lehrer, S.S. (1985) Biochemistry 24, 6631] except that S1 binding to actin-Tm increased the excimer fluorescence in contrast to the time-dependent decrease seen for PMTm. The fluorescence properties of PIATm are sensitive to the Tm chain-chain interaction via equilibria among pyrene configurations and nonfluorescent dimer as well as the monomer and excimer-forming configurations. The effect of bound troponin (Tn) on the excimer fluorescence of PIATm in the reconstituted systems was dependent on ionic strength with a slight Ca2+ dependence. S1 titrations in the absence and presence of Tn and Ca2+ indicated that the excimer fluorescence probes the state change of Tm from the weak S1 binding state to the strong S1 binding state which is facilitated by Ca2+ [Hill et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186]. Binding of MgADP-S1 and MgAMPPNP-S1 produced the same total excimer fluorescence change as for nucleotide-free S1, showing that the strong S1 binding state of Tm-actin is independent of nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)
Ishii
Y
Y
Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
Lehrer
S S
SS
D008841:Actin Cytoskeleton / Q000032:analysis*; D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000032:analysis; D003599:Cytoskeleton / Q000032:analysis*; D007460:Iodoacetamide / Q000031:analogs & derivatives*; D007461:Iodoacetates; D007700:Kinetics; D009132:Muscles / Q000032:analysis*; D011817:Rabbits; D013050:Spectrometry, Fluorescence / Q000379:methods; D014335:Tropomyosin / Q000032:analysis*
D000199:Actins; D007461:Iodoacetates; D014335:Tropomyosin; C029358:N-(1-pyrenyl)iodoacetamide; D002118:Calcium; D007460:Iodoacetamide