Effect of Actin and Nucleotide on the Movement of A1-Type Myosin Essential Light Chain, Detected by Time-Resolved FRET

Abstract

We have used site-directed time-resolved FRET to detect structural changes of the N-terminal extension on the A1 isoform of ELC, during active interaction with actin. Skeletal muscle myosin subfragment 1 (S1) has two ELC isoforms, A1 and A2, which differ by the presence of 40-45 additional residues at the N-terminus of A1. Removal of ELC from myosin results in a loss of movement of actin filaments, and a reduction in isometric force. It has been proposed that the N-terminal extension of A1 interacts directly with actin to modulate actomyosin kinetics. We have used time-resolved FRET to explore the structural details of this modulation. We recently showed, using probes on actin and ELC, that the amplitude of the myosin power stroke is much greater for A1 than A2. We have now engineered single cysteine (C16, in the N-terminal extension) and double cysteine (C16-C180, in the C-terminal lobe) mutants of A1-ELC and used FRET to determine the distance from C16 to actin 374 or to C180, as affected by the power stroke. Labeled ELCs were exchanged into S1, and the pure isoenzyme (S1A1) was isolated using Talon affinity resin. Labeling and exchange preserved the functional properties of S1A1. Intermolecular FRET between C374 of actin and C16 of A1 showed that the distance increases from 2.9 nm to 3.5 nm upon addition of ATP. Intramolecular FRET between C16 and C180 does not change in the presence of ATP or actin. We conclude that (1) the N-terminus of ELC moves away from actin during the transition between strongly and weakly bound states of acto-S1, and (2) the converter/C-terminal domain of myosin/ELC and the N-terminal extension move as a rigid body during the power stroke.