3D Orientation of Alpha Helix in Muscle Myosin Regulatory Light Chain Using Bifunctional Electron Paramagnetic Resonance

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3D Orientation of Alpha Helix in Muscle Myosin Regulatory Light Chain Using Bifunctional Electron Paramagnetic Resonance

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Savich, Yahor

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2021-06

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Abstract

Muscle contraction is a coordinated work of nanometer-sized force generators, myosin molecules. These molecules are out of equilibrium: they use the energy stored in the form of ATP to move collectively along the track protein actin. The myosin molecules transfer their work via lever arms that connect force generators to their cargo. Orientation of these lever arms has been studied thoroughly since 1) their structural dynamics is fundamental for understanding the muscle contraction and 2) their particular orientations are associated with disease states of cardiac and skeletal muscle. Electron microscopy, fluorescence polarization, and X-ray diffraction have provided insight into the structure of muscle, but there is still no high-resolution data of the vertebrate lever arm orientation available at ambient (not vitrified or crystallized) conditions. The present work establishes a method of measuring the orientation of the alpha helices in three dimensions using electron paramagnetic resonance (EPR). Chapter 3 introduces the use of EPR with bifunctional spin labels attached to different helices of the myosin regulatory light chain (RLC) protein with and without ATP. Demembranated skeletal muscle fibers were aligned with the slowly-varying magnetic field; RLC was chemically substituted by labeled RLC; axial orientational dynamics of the probe with respect to the muscle axis was determined. Chapter 4 utilizes 1) directional statistics that replaces the previous use of a Gaussian distribution and provides new insights into the degree of disorder and 2) a new bifunctional probe that adds an azimuthal dimension to the orientational data. Together, these techniques allow determination of the tilt and roll angles of the alpha helix without relying on the myosin structure.

Keywords

bifunctional spin label

directional statistics

EPR

muscle

myosin

spectroscopy

Description

University of Minnesota Ph.D. dissertation. June 2021. Major: Physics. Advisor: David Thomas. 1 computer file (PDF); x, 96 pages.

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Savich, Yahor. (2021). 3D Orientation of Alpha Helix in Muscle Myosin Regulatory Light Chain Using Bifunctional Electron Paramagnetic Resonance. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/258657.

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