Time-dependent fluorescence depolarization and lifetime studies of myosin subfragment-one in the presence of nucleotide and actin

Abstract

Time-dependent fluorescence depolarization and lifetime studies have been made on myosin subfragment 1 to obtain information about mobility changes and dye environment changes when different nucleotides are added. Data are reported for static and actively hydrolyzing systems containing G- and F-actin. Preliminary data indicate that myosin labeled with the fluorophore 1, 5 IAEDANS and treated with DTT preserves its actin-activated Vmax. S1 prepared in this manner gives lifetime changes which are nearly identical for all systems studied. S1 labeling without DTT addition gives a pattern of lifetimes similar, though not identical to ESR work. Either type of labeling produces no observable change in the polarization decay, and we set an upper limit of 15% length change for the elongate S1. An unusually long fluorescence decay lifetime for the S1-Mg++ ATP-G-actin system is found which may indicate a new acto-S1 state stabilized by G-actin. The method for obtaining the bound fraction of S1's in the presence of actin is presented and applied to the S1-F-actin-Mg++ ATP system. Qualitative agreement is obtained with other methods.