Functional and Structural Impact of Site-Directed Methionine Oxidation in Myosin

Abstract

We have examined the functional and structural perturbations of methionine (Met) oxidation in Dictyostelium (Dicty) myosin II. Protein oxidation by reactive oxygen species (ROS) is a critical element of cell function. However, elevated production of ROS can lead to the accumulation of oxidatively modified proteins, contributing to a decline of function in biologically aged or diseased muscle. For Dicty myosin containing all native methionines, peroxide-treatment (used as an oxidative agent) decreases actin-activated myosin ATPase activity, consistent with the decline in actomyosin function previously observed in biologically aged or peroxide-treated muscle. We have recently identified a single Met that is responsible for this functional decline, Met 394, located near the myosin cardiomyopathy loop in the actin-binding interface. Oxidation of this Met also induces a redistribution of existing myosin structural states of the actin-binding cleft. We have now expanded the understanding of how oxidation at M394 affects myosin function and structure by using site-directed mutagenesis (from M to Q) to mimic oxidation. We then characterized actomyosin interaction and changes in structural states of both the force-generating region and actin-binding cleft of myosin. Our goal is to bridge our understanding of site-specific Met oxidation and muscle dysfunction with molecular-level insights into actomyosin interaction.