Site Directed Spin Labeling to Probe Acto-myosin Binding

University Of Minnesota Twin Cities, Minneapolis MN

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Abstract

The long-term goal of my research is to understand the dynamic interactions between myosin and the regulated thin filament (actin- troponin-tropomyosin) that are critical for muscle contraction. The present proposal focuses on the interaction between actin and myosin. I will use site-directed spin labeling to test directly two detailed structural models for the changing interactions between actin and myosin during muscle contraction. This project has three aims: (1) Mutate specific actin residues, which are predicted to form the actin-myosin interface, into cysteine and attach spin labels selectively to these residues. (2) Use EPR to detect interactions between these sites and myosin, and determine how these interactions change when nucleotides induce the transition between weak and strong actin-myosin interactions. (3) Mutate selected complementary surface residues in myosin for site directed spin labeling, and use EPR to test models for the weak-to-strong structural transition. This work will set the stage for future work that probes the molecular mechanisms of diseases, such as familial hypertrophic cardiomyopathy and dilated cardiomyopathy, that are proposed to be caused, in some cases, by mutations in the actin-myosin interface.