The long-term goal of this research is to understand the role of protein rotational motions in the generation of force during muscle contraction. Site-selective molecular probes (spin labels or luminescent dyes) will be attached to myosin or actin, and spectroscopy experiments (electron paramagnetic resonance (EPR) and time-resolved optical anisotropy) will provide direct information about the orientation and rotational motions of he labeled proteins. These experiments will be performed on purified proteins, in which biochemical conditions can be precisely controlled and monitored, and also on skinned muscle fibers, in which mechanical performance can be controlled and monitored. Experimental conditions will be chosen to provide direct tests for the proposed motions of myosin heads (cross-bridges) and actin subunits during the generation of force. The high orientational resolution of EPR and time resolution of optical anisotropy will be used to resolve the multiple conformations present under complex physiological conditions. Although the primary emphasis will be on these applications, an integral apart of this project is the development of the spectroscopic methods. The five closely related principal aims of the project are (1) development of EPR techniques (both conventional and saturation-transfer) (2) application of these EPR techniques to detect the orientations and rotations of myosin and actin, (3) development of time-resolved optical anisotropy techniques (transient absorption, phosphorescence, and fluorescence), (4) application of these optical techniques to resolve the rotational motions of myosin and actin, and (5) correlation of the molecular dynamics with structural, mechanical, and biochemical measurements on the same preparations. These studies should provide direct insight into the role of molecular dynamics in this fundamental physiological process, and the technology we are developing should be applicable to a wide range of other biophysical system in which conformational dynamics are coupled to enzyme action.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Lymn, Richard W
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University of Minnesota Twin Cities
Schools of Medicine
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Muretta, Joseph M; Reddy, Babu J N; Scarabelli, Guido et al. (2018) A posttranslational modification of the mitotic kinesin Eg5 that enhances its mechanochemical coupling and alters its mitotic function. Proc Natl Acad Sci U S A 115:E1779-E1788
Elam, W Austin; Cao, Wenxiang; Kang, Hyeran et al. (2017) Phosphomimetic S3D cofilin binds but only weakly severs actin filaments. J Biol Chem 292:19565-19579
Guhathakurta, Piyali; Prochniewicz, Ewa; Roopnarine, Osha et al. (2017) A Cardiomyopathy Mutation in the Myosin Essential Light Chain Alters Actomyosin Structure. Biophys J 113:91-100
Colson, Brett A; Thompson, Andrew R; Espinoza-Fonseca, L Michel et al. (2016) Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics. Proc Natl Acad Sci U S A 113:3233-8
Espinoza-Fonseca, L Michel; Alamo, Lorenzo; Pinto, Antonio et al. (2015) Sequential myosin phosphorylation activates tarantula thick filament via a disorder-order transition. Mol Biosyst 11:2167-79
Guhathakurta, Piyali; Prochniewicz, Ewa; Thomas, David D (2015) Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain. Proc Natl Acad Sci U S A 112:4660-5
Alamo, Lorenzo; Li, Xiaochuan Edward; Espinoza-Fonseca, L Michel et al. (2015) Tarantula myosin free head regulatory light chain phosphorylation stiffens N-terminal extension, releasing it and blocking its docking back. Mol Biosyst 11:2180-9
Muretta, Joseph M; Rohde, John A; Johnsrud, Daniel O et al. (2015) Direct real-time detection of the structural and biochemical events in the myosin power stroke. Proc Natl Acad Sci U S A 112:14272-7
Muretta, Joseph M; Jun, Yonggun; Gross, Steven P et al. (2015) The structural kinetics of switch-1 and the neck linker explain the functions of kinesin-1 and Eg5. Proc Natl Acad Sci U S A 112:E6606-13
Binder, Benjamin P; Cornea, Sinziana; Thompson, Andrew R et al. (2015) High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label. Proc Natl Acad Sci U S A 112:7972-7

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