The goal of this project is to determine the role of actomyosin structural dynamics in the molecular mechanism of force generation in muscle. Emphasis is placed on the use of high-resolution and time- resolved (TR) spectroscopy to test and revise detailed mechanistic models for the functional interaction of myosin and actin. Several hypotheses inform all aims: (a) Crystal structures and electron micrographs of myosin and actin provide high-resolution models for the structural dynamics of actomyosin, which must be tested and revised by site-directed spectroscopy in functional protein complexes, using computational simulations to connect models with experiment. (b) The weak-to-strong (W-S) transition is fundamental and occurs in both actin and myosin. (c) To understand the physical basis of these transitions, the weak and strong states of actin and myosin must be characterized by dynamics and disorder as well as structure. There are five aims: (1) Development of improved methods for site-directed spectroscopic analysis of myosin and actin structural dynamics. New spectrometers, recently acquired or developed, have raised this project to a new technological level: Pulsed electron paramagnetic resonance (EPR), high-frequency EPR, and high- throughput pulsed fluorescence, performed under both steady-state and transient biochemical conditions, opens new opportunities to measure angles and distances, and to resolve structural states and transitions between them. Spectroscopy will be coordinated with collaborative studies of computational simulations, x- ray crystallography and electron microscopy (EM). (2) Structural dynamics of the myosin catalytic domain will be probed to test and revise mechanistic models. Focusing on three distinct regions (force-generating, actin-binding cleft, nucleotide pocket) and performing experiments under transient conditions, coordination of motor parts will be analyzed in space and time. Functional mutations will be used to distinguish weak and strong states. (3) Structural dynamics of the light chain (LC) domain will be probed to characterize the previously unknown structure of the N-terminal phosphorylation domain (PD) of the myosin regulatory light chain (RLC), and to determine its internal and global dynamics as affected by phosphorylation, ATP, and actin. (4) Actin structural dynamics, both global and internal, will be perturbed by weak and strong interaction with myosin, including myosin isoforms and functional mutants, to correlate structural dynamics with function. (5) The actomyosin interface will be mapped by probes on both proteins, and EM will be used to integrate the data into structural models for the weak and strong states. This work is of fundamental importance for understanding muscle function, and the technology and concepts generated here are already being used elsewhere, by this group and others, to provide insight into muscle malfunction and therapy at the molecular level. More generally, the lessons learned in this project are applicable to a wide range of problems in the biophysics of cellular movement.

Public Health Relevance

The proposed research brings together a powerful combination of techniques, from molecular biology to biochemistry to biophysics, to solve the molecular mechanism of force generation in muscle. This work is of fundamental importance for understanding muscle function, and the technology generated here is being applied elsewhere to provide molecular insight into muscle malfunction. More generally, this well-defined system serves as a model for studying the role of molecular dynamics and interactions in motor proteins, and the approaches we are developing should prove effective in the analysis of a wide range of problems in this field.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-B (03))
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Boyce, Amanda T
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University of Minnesota Twin Cities
Schools of Medicine
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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
Trivedi, Darshan V; Muretta, Joseph M; Swenson, Anja M et al. (2015) Direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin V. Proc Natl Acad Sci U S A 112:14593-8
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
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
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
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
Colson, Brett A; Petersen, Karl J; Collins, Brittany C et al. (2015) The myosin super-relaxed state is disrupted by estradiol deficiency. Biochem Biophys Res Commun 456:151-5
Petersen, Karl J; Peterson, Kurt C; Muretta, Joseph M et al. (2014) Fluorescence lifetime plate reader: resolution and precision meet high-throughput. Rev Sci Instrum 85:113101

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