Dynamics of the Lever-Arm Swing in Myosin V

Abstract

Myosins utilize a conserved structural mechanism to convert the energy from ATP hydrolysis to a large swing in the force generating lever-arm. However, there remains an ongoing controversy about the kinetics of lever-arm swing in relation to the steps in the ATPase cycle. To address this question we have developed a novel FRET system in myosin V (MV) that utilizes several donor-acceptor pairs to examine the dynamics of lever arm motion. MV containing a single IQ motif and an N-terminal (NT) tetracysteine site was labeled with the bisarsenical dye FlAsH (MV.NT.FlAsH). The first IQ motif of MV.NT.FlAsH was exchanged either with IAANS labeled CaM, a donor, or QSY-9 labeled CaM, a non-fluorescent acceptor. Steady-state and transient kinetic experiments reveal a decrease in FRET upon ATP binding (recovery stroke) in both donor-acceptor pairs. We utilized transient kinetic experiments to demonstrate that upon mixing the MV.ADP.Pi complex with actin there was a FRET increase that occurred in two phases, and the fast and slow phases correlated well with the release rates of Pi and ADP, respectively. We also labeled the upper-50kDa tetracysteine site with FlAsH (MV.U50.FlAsH) and exchanged the QSY labeled CaM on to the first IQ motif. We observed structural changes during ATP binding that were very similar to the MV.NT.FlAsH results. During actin-activated product release we observed two-phases, a rapid increase in FRET followed by a slower decrease in FRET, which correlated well with ADP release. We find that the force generating motion of the lever arm occurs in two steps which are closely coupled to the product release steps. Our results also indicate that the conformational changes in the lever arm associated with the power stroke may follow a unique pathway that is not simply the reversal of the recovery stroke.