The spatial relationships between Lys‐61, Cys‐374 on actin or SH1 on myosin subfragment‐1 (S1) and Cys‐190 on tropomyosin or Cys‐133 on troponin‐I (TnI) in a reconstituted thin filament were studied by fluorescence resonance energy transfer. 5‐(2‐Iodoacetylaminoethyl)aminonaphthalene 1‐sulfonic acid (IAEDANS) attached to Lys‐190 on tropomyosin or to Cys‐133 on TnI was used as a donor. Fluorescein 5‐isothiocyanate (FITC) attached to Lys‐61 or 5‐(iodoacetoamido)fluorescein (IAF) attached to Cys‐374 on actin and 4‐dimethylaminophenyl‐azophenyl 4′‐maleimide (DABMI) attached to SH1 on S1 were used as an acceptor. The transfer efficiency between AEDANS attached to Cys‐190 on tropomyosin and FITC attached to Lys‐61 on actin was 0.42 in the absence of troponin, 0.46 in the presence of troponin and Ca2+ and 0.55 in the presence of troponin and absence of Ca2+. The corresponding distances between the probes were calculated to be 4.7 nm, 4.6 nm and 4.3 nm respectively, assuming a random orientation factor K2= 2/3. A large difference in the transfer efficiency from AEDANS attached to Cys‐133 on TnI to FITC attached to Lys‐61 on actin was observed between in the presence (0.52) and absence (0.70) of Ca2+. The corresponding distances between the probes were calculated to be 4.5 nm in the presence of Ca2+ and 3.9 nm in the absence of Ca2+. The distance between Cys‐190 on tropomyosin and Cys‐374 on actin was measured to be 5.1 nm and the transfer efficiency (0.35) did not change upon addition of troponin whether Ca2+ is present or not, in agreement with the previous report [Tao, T., Lamkin, M. & Lehrer, S. S. (1983) Biochemistry 22, 3059–3064]. The distance between Cys‐133 on TnI and Cys‐374 on actin was measured to be 4.4 nm. No detectable change in transfer efficiency (0.58) was observed between values in the presence and absence of Ca2+. These results suggest that a relative movement of the two domains of actin monomer in a reconstituted thin filament occurs in response to a change in Ca2+ concentration. The transfer efficiencies between DABMI attached to SH1 on S1 and AEDANS attached to Cys‐190 on tropomyosin or Cys‐133 on TnI were too small (less than 2%) for an accurate estimation of the distances, suggesting the distances are longer than 7.3 nm. Copyright © 1990, Wiley Blackwell. All rights reserved
