Ca2+-induced movement of tropomyosin in skeletal muscle thin filaments observed by multi-site FRET

To obtain information on Ca2+-induced tropomyosin (Tm) movement in Ca2+-regulated muscle thin filaments, frequency-domain fluorescence energy transfer data were collected between 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid at Cys-190 of Tm and phalloidin-tetramethylrhodamine B isothiocyanate bound to F-actin. Two models were used to fit the experimental data: an atomic coordinate (AC) model coupled with a search algorithm that varies the position and orientation of Tm on F-actin, and a double Gaussian distance distribution (DID) model. The AC model showed that little or no change in transfer efficiency is to be expected between different sites on F-actin and Tm if Ca2+ causes azimuthal movement of Tm of the magnitude suggested by structural data (C. Xu, R. Craig, L. Tobacman, R. Horowitz, and W. Lehman. 1999. Biophys. J. 77:985-992). However, Ca2+ produced a small but significant change in our phase/modulation versus frequency data, showing that changes in lifetime decay can be detected even when a change of the steady-state transfer efficiency is very small. A change in Tm azimuthal position of 17degrees on the actin filament obtained with the AC model indicates that solution data are in reasonable agreement with EM image reconstruction data. In addition, the data indicate that Tm also appears to rotate about its axis, resulting in a rolling motion over the F-actin surface. The DID model showed that the distance from one of the two chains of Tm to F-actin was mainly affected, further verifying that Ca2+ causes Tm to roll over the F-actin surface. The width of the distance distributions indicated that the position of Tm in absence and in presence of Ca2+ is well defined with appreciable local flexibility.