Position of Phenylalanine in the Relay Loop is Important for Myosin Motor Action

Abstract

We have scanned phenylalanine in the relay loop of Dictyostelium discoideum myosin to study the role of the relay loop in the myosin ATPase actin activation. It is known that C-terminus of the relay loop is on the actin binding interface. Myosin crystal structures show that F506 of the relay loop interacts with F487 of the relay helix in the post-recovery stroke structural state. This interaction is absent in the pre-recovery stroke state due to the linear shift of the relay loop along the relay helix. We hypothesized that actin binding facilitates this conformational change in the relay loop-helix region, thus activating myosin ATPase rate. The goal of the phenylalanine scan was to simulate the loop-helix shift and populate myosin pre- or post-recovery stroke structural states, mimicking proposed effect of actin binding. Two myosin mutants were designed (F506A:D505F and F506A:G507F), along with the wild type myosin (F506) placing phenylalanine in the three consecutive positions within the relay loop. We have used transient time-resolved FRET, myosin intrinsic fluorescence, pyrene labeled actin, and chemical quench to characterize myosin kinetics in detail and conclude on the role of F506 in myosin relay loop. Supported by NIH AR59621.