Pulse-fluorometry study on actin and heavy meromyosin using F-actin labelled with N-(1-pyrene)maleimide

Abstract

The single-photoelectron counting technique was used for measurement of the fluorescence decay kinetics of N-(1-pyrene)maleimide conjugated to the fast reactive cysteine of actin. The fluorescence decay curve of the labelled G-actin could not be described by a single-exponential function but by a double-exponential function. Polymerization of actin was accompanied by significant changes in the decay parameters of the two decay components. We found that the ionic strength, which plays an important role in the G-F equilibrium, scarcely affected these parameters provided that the labelled actin exists in the monomeric state. Thus it is suggested that the conformational change of actin protomer occurs at the time of association. When heavy meromyosin was added to the labelled F-actin, the decay parameter changed monotonically on increasing saturation of binding of heavy meromyosin and it levelled-off around a ratio of heavy meromyosin:actin of 0.5 mol/mol. Decay parameters under the influence of heavy meromyosin had values intermediate between those observed for the labelled G-actin and for the labelled F-actin. Therefore, it is suggested that binding of heavy meromyosin to F-actin alters the conformation of actin protomer towards that similar to G-actin.