Linear dichroism of acrylodan-labeled tropomyosin and myosin subfragment 1 bound to actin in myofibrils.
Muscle contraction can be activated by the binding of myosin heads to the thin filament, which appears to result in thin filament structural changes. In vitro studies of reconstituted muscle thin filaments have shown changes in tropomyosin-actin geometry associated with the binding of myosin subfragment 1 to actin. Further information about these structural changes was obtained with fluorescence-detected linear dichroism of tropomyosin, which was labeled at Cys 190 with acrylodan and incorporated into oriented ghost myofibrils. The fluorescence from three sarcomeres of the fibril was collected with the high numerical aperture objective of a microscope and the dichroic ratio, R (0/90 degrees), for excitation parallel/perpendicular to the fibril, was obtained, which gave the average probe dipole polar angle, Theta. For both acrylodan-labeled tropomyosin bound to actin in fibrils and in Mg2+ paracrystals, Theta congruent to 52 degrees +/- 1.0 degrees, allowing for a small degree of orientational disorder. Binding of myosin subfragment 1 to actin in fibrils did not change Theta; i.e., the orientation of the rigidly bound probe on tropomyosin did not change relative to the actin axis. These data indicate that myosin subfragment 1 binding to actin does not appreciably perturb the structure of tropomyosin near the probe and suggest that the geometry changes are such as to maintain the parallel orientation of the tropomyosin and actin axes, a finding consistent with models of muscle regulation. Data are also presented for effects of MgADP on the orientation of labeled myosin subfragment 1 bound to actin in myofibrils.
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Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
Lehrer
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D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000199:Actins / Q000737:chemistry; D000244:Adenosine Diphosphate; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D005456:Fluorescent Dyes; D008956:Models, Chemical; D009210:Myofibrils / Q000737:chemistry; D015879:Myosin Subfragments / Q000737:chemistry*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000737:chemistry*
D000199:Actins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D014335:Tropomyosin; D000244:Adenosine Diphosphate; C038518:acrylodan; D015081:2-Naphthylamine