Light Chain Domain Orientation Determined by Time-resolved FRET

Abstract

We have used site-directed time-resolved FRET to determine light-chain domain transitions in expressed Dictyostelium full-length myosin S1. Myosin S1 is proposed to undergo large rotations of the lever-arm in response to the nucleotide state. Previous fluorescence experiments (Shih et al., 2000) have shown that S1 adopts two populations of pre-stroke states and the equilibrium between these states is driven by nucleotide. To observe these population changes in detail, and to observe the effect of actin, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to measure the distribution of distances between the labeled catalytic domain (A250C) and a labeled RLC (M129C). In the present work, we show that the lever arm adopts multiple orientations in solution. In the absence of nucleotide, the light-chain domain probe is at a mean distance of 8.4 nm from the catalytic domain probe. This distance extends to >9.0 nm when flS1 is bound to actin. The addition of ATP gives three roughly equal populations of distances at 3.6, 4.3, and >9.0 nm. ADP.Vi stabilizes the shorter of these populations, indicating that ADP.Pi induces a conformational change of the light-chain domain. This work was supported by grants from NIH (AR32961, AR07612). We thank Igor Negrashov for excellent technical assistance.