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John Rohde, Graduate Student

 

Research Interests

ATPase kinetic cycleThe motor protein myosin is essential for cellular motility and muscle contraction. Myosin undergoes a conformational change called a "lever arm swing" during its ATPase kinetic cycle. Thousands of myosin molecules, moving in concert in the sarcomere, undergo this structural change and lead to muscle contraction. My graduate work focuses on the biochemical and structural kinetics associated with this lever arm swing in skeletal and cardiac muscle myosin, both in health and disease. Our lab has developed a novel technique to study this transient and reversible structural change in real-time through the use of site-directed labeling and time-resolved fluorescence resonance energy transfer (FRET).

TR2FRETWe call this technique TR2FRET. An example of a labeling scheme to detect lever arm swing is shown below: the acceptor fluorophore is attached to a nucleotide bound in the nucleotide-binding pocket on the catalytic head domain of the myosin heavy chain (Cy3ATP/Cy3ADP); the donor fluorophore is attached to an expressed RLC, which is exchanged onto tissue-extracted myosin (AF488RLC). I am very interested in how the structural kinetics of myosin differ in situ in intact muscle fibers, and especially in muscle fibers under load.

Summary of Research