We have used phosphorescence anisotropy from erythrosin-5- iodoacetamide covalently attached to rabbit skeletal muscle tropomyosin (Tm) to characterize the molecular dynamics of Tm on the surface of F-actin. Although the probe was loosely bound to Tm, it was able to monitor the molecular dynamics of the F- actin/Tm complex on the microsecond time scale. The steady-state phosphorescence anisotropy of Tm in the complex was 0.025+/- 0.005 at 20 degree(s)C. Since the anisotropy of actin in a similar complex was 0.11+/- 0.01, Tm may be moving independently on the filament surface. Studies of the effect of temperature and solution viscosity provided additional evidence for independent tropomyosin motions. A modified Perrin plot of 1/r versus (tau) T/(eta) (where r is the steady-state phosphorescence anisotropy, (tau) is the phosphorescence lifetime, T is the absolute temperature, and (eta) is solution viscosity) provided evidence for two distinct modes of motion for Tm on F-actin; these modes corresponded to the rotation of segments with significantly different volumes. While the volume of the fast component was comparable to that of a single tropomyosin molecule, providing evidence that Tm moves on the surface of F- actin, the volume of the slow component corresponded to only a small portion of the entire complex.

Date Published: 17 August 1994
PDF: 9 pages
Proc. SPIE 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV, (17 August 1994); doi: 10.1117/12.182760

Published in SPIE Proceedings Vol. 2137:
Time-Resolved Laser Spectroscopy in Biochemistry IV
Joseph R. Lakowicz, Editor(s)