Fluorescence depolarization of actin filaments in reconstructed myofibers: the effect of S1 or pPDM-S1 on movements of distinct areas of actin.
Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.
Borovikov
Yu S
YS
Institute of Cytology, Russian Academy of Science, St. Petersburg 194064, Russia.
Dedova
I V
IV
dos Remedios
C G
CG
Vikhoreva
N N
NN
Vikhorev
P G
PG
Avrova
S V
SV
Hazlett
T L
TL
Van Der Meer
B W
BW
D008841:Actin Cytoskeleton / Q000737:chemistry*; D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D001703:Biophysics / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal; D009132:Muscles / Q000737:chemistry* / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D009218:Myosins / Q000737:chemistry; D010590:Phalloidine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013053:Spectrophotometry; D013997:Time Factors
D000199:Actins; D000596:Amino Acids; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D008301:Maleimides; D015879:Myosin Subfragments; D010590:Phalloidine; D000244:Adenosine Diphosphate; C011328:N,N'-4-phenylenedimaleimide; D009218:Myosins; D008239:Lysine