The structural dynamics of force generation in muscle, probed by electron paramagnetic resonance of bifunctionally labeled myosin.

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The structural dynamics of force generation in muscle, probed by electron paramagnetic resonance of bifunctionally labeled myosin.

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Thompson, Andrew Russell

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2009-05

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Thesis or Dissertation

Abstract

Two proteins in muscle, actin and myosin, are the key structural components that interact in order to produce muscle contraction. Myosin is a molecular motor that utilizes the chemical energy of ATP to undergo conformational changes that translate actin linearly, resulting in mechanical work. While previous studies have provided high-resolution measurement of these structural changes, many are unable to do so in intact muscle or in systems where myosin and actin can interact. This project seeks to make high-resolution structural measurements of myosin in actomyosin complexes during the different biochemical states associated with contraction. These measurements are being made using electron paramagnetic resonance (EPR), a spectroscopic technique sensitive to protein dynamics and orientation. In order to study myosin with EPR, a spin label is chemically attached to cysteine within the protein structure. In certain cases, native cysteines are used for spin labeling whereas in others, mutant protein is created with cysteines engineered in desired locations, a process known as site-directed spin labeling.Traditional spin probes attach via a single, flexible bond. This monofunctional attachment limits the sensitivity of EPR to protein orientation and dynamics because the resultant spectra are a mixture of probe and protein states. This project, on the other hand, uses a novel bifunctional spin label that is rigidly coupled to the protein via attachment to two engineered cysteines. Due to this rigid coupling, high-resolution structural measurements can be made with a degree of sensitivity not available to other techniques.

Keywords

Bifunctional spin label

Dynamics

Electron Paramagnetic Resonance

Muscle

Myosin

Orientation

Physics

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University of Minnesota Ph.D. dissetation. May 2009. Major: Physics. Advisor: David D. Thomas. 1 computer file (PDF); vi, 94 pages.

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Thompson, Andrew Russell. (2009). The structural dynamics of force generation in muscle, probed by electron paramagnetic resonance of bifunctionally labeled myosin.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/52599.

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