Dynamics of the N-Terminal Domain of Myosin V Monitored by FRET

Abstract

The N-terminus is a highly variable region among the myosin superfamily and has been associated with alterations in rotation of the lever arm and step size. We examined the structural dynamics of the N-terminal region of myosin V using steady-state and stopped-flow FRET. We introduced a tetra-cysteine site at the extreme N-terminus of a Myosin V 1IQ construct and labeled it with a bisarsenical fluorescin derivative (FlAsH) (MV NT-FLAsH). Energy transfer between FlAsH and mant or deac labeled nucleotides (dmantATP, dmantADP and deacATP) was monitored. Steady-state FRET experiments with the mant-FlAsH pair demonstrated a high FRET state in the presence of dmantATP and a low FRET state in the presence of dmantADP. Sequential-mix, in which MV NT-FLAsH is first mixed with dmantATP, aged to allow formation of the M.ADP.Pi state, and then mixed with saturating actin allowed us to explore the actin-acitvated product release steps. Biphasic transients with rates corresponding to the fast and slow rate of dmantADP release were observed. The sequential mix experiments with deacATP yielded three phases; a fast, actin dependent, phosphate release phase followed by the two phases of ADP release. Our results suggest the FRET signal monitors a structural change in the N-terminus associated with the sequential release of products. A high FRET state upon binding of ATP is associated with formation of the pre-powerstroke state of the lever arm and a low or no FRET state in the presence of ADP suggests formation of the post-powerstroke state. Our results allow us to hypothesize that movement of the N-terminal domain follows the movement of the converter/lever arm. Further experiments will explore the dynamics of the N-terminal domain and examine how its motion correlates with the conformation of the nucleotide binding pocket and lever arm.