Detecting Structural Changes in Myosin using Bifunctional Spin Labels

Abstract

Actomyosin interaction is responsible for driving a vast number of cellular processes, including force generation during muscle contraction. In this context, myosin is the molecular “motor” that produces muscle contraction. The myosin motor domain is comprised from three major subdomains: the force-generating domain (relay helix, SH-1, and converter), the ATP-binding pocket and the actin-binding cleft. During the cycle of force generation, myosin undergoes a series of structural rearrangements including “bending” of the relay helix, which communicates the biochemical state of the ATP binding pocket to the lever arm. The relay helix has previously been shown to reflect changes in distance, dynamics and orientation by electron paramagnetic resonance (EPR) with nucleotide state of the myosin catalytic domain. We now observe relay helix conformational changes using two rigidly attached bifunctional spin labels (BSL) on the relay helix (492.496) and in the lower 50 kDa domain (639.643) of the Dictyostelium myosin II motor domain by double electron electron resonance spectroscopy (DEER). We observe changes in relay helix conformation with various allosteric effectors - including potent myosin II inhibitors and physiological inhibitors such as site-directed oxidation. This approach allows for characterization of perturbations in chemo-mechanical coupling within the actomyosin complex under inhibitory conditions, as well as showing the reliability of relay helix spin probe sensitivity to the changes in myosin structural dynamics.