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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR032961-27
Application #
7798599
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (03))
Program Officer
Boyce, Amanda T
Project Start
1983-12-01
Project End
2014-02-28
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
27
Fiscal Year
2010
Total Cost
$507,677
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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
Rohde, John A; Thomas, David D; Muretta, Joseph M (2017) Heart failure drug changes the mechanoenzymology of the cardiac myosin powerstroke. Proc Natl Acad Sci U S A 114:E1796-E1804
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
Avery, Adam W; Crain, Jonathan; Thomas, David D et al. (2016) A human ?-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding. Sci Rep 6:21375
Swanson, Carter J; Sommese, Ruth F; Petersen, Karl J et al. (2016) Calcium Stimulates Self-Assembly of Protein Kinase C ? In Vitro. PLoS One 11:e0162331
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
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
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

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