Fluorescence properties of acrylodan-labeled tropomyosin and tropomyosin-actin: evidence for myosin subfragment 1 induced changes in geometry between tropomyosin and actin.
The Cys groups of rabbit skeletal tropomyosin (Tm) and rabbit skeletal alpha alpha Tm were specifically labeled with acrylodan (AC). The probe on Tm is quite immobile yet exposed to solvent as indicated by its limiting polarization (P0 = 0.38) and fluorescence emission spectrum (lambda max = 520 nm) and its accessibility to solute quenching. Changes in the shape of the excitation spectrum with temperature correlated with the helix thermal pretransition and main transition without much spectral change of the emission spectrum. The probe environment of ACTm did not significantly change on binding to F-actin, but fluorescence energy transfer between tryptophan in actin and AC on Tm was indicated by a 15-20% increase in AC fluorescence and a few percent decrease in tryptophan fluorescence. This energy transfer increased when myosin subfragment 1 (S1) was bound to the ACTm-actin filament, in quantitative agreement with the postulated shift in state of Tm associated with the cooperative binding of S1 to actin (Hill et al., 1980). The increase in energy transfer shows that there is a change in the spatial relationship between Tm and actin associated with the S1-induced change in state of Tm.
Lehrer
S S
SS
Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
Ishii
Y
Y
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000199:Actins / Q000378:metabolism*; D000818:Animals; D066298:In Vitro Techniques; 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; D015879:Myosin Subfragments; D010446:Peptide Fragments; D014335:Tropomyosin; C038518:acrylodan; D015081:2-Naphthylamine; D009218:Myosins