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 IAEDANS¹‐and treated with DTT preserves its actin‐activated V_max. 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.