Anisotropy Decay of Labelled Actin

Abstract

G actin, labeled presumably on cysteine-373 with the fluorescent chromophore N-iodoacetyl-N''-(5 sulfo-1-naphthyl)-ethylenediamine and purified by Sephacryl S-200 gel chromatography, migrated in 1 band on polyacrylamide gel electrophoresis and had the same polymerizability as unlabeled purified G actin. Anisotropy decays of labeled actin solutions were studied at different ionic strengths and protein concentrations. These anisotropy decays could be fitted by a sum of 2 exponential functions. Under low ionic strength or below the critical concentrations the longer correlation time (45 ns at 3.5.degree. C) was independent of protein concentration and ionic strength. Above the critical concentration, the longer correlation time increased with ionic strength and protein concentration. To take into account that, under these conditions, the solutions contained a mixture of F and G actin at the critical concentration, the anisotropy decays were analyzed as a sum of 3 exponential functions in which the longest correlation time characterized F actin. Since F actin correlation time also depended on actin concentration, an analysis with a sum of 4 exponential functions was performed, in which 2 fixed correlation times (100 ns and 900 ns at 3.5.degree. C) were introduced to characterize the F actin motions. The longer of these correlation times was attributed to regions where 2 actin filaments interact side by side, while the shorter one was attributed to filament regions free from intermolecular interactions. The small value of the free F actin correlation time indicates that the protomer peptide chain is very flexible around its C terminus, probably involving the motion of a molecular lobe. This flexibility might be an important factor in the interaction of actin with myosin during the muscular contraction.