Myosin is believed to generate force and movement by the rotation of a long a-helical region that extends from the C-terminus of the motor domain, and is stabilized by the essential light chain (ELC) and the regulatory light chain (RLC). The role of the light chain-binding domain or """"""""lever arm"""""""" is to amplify small conformational changes originating at the nucleotide binding site into larger movements of the lever arm. Despite recent advances in kinetic and structural approaches, many aspects of the communication pathway between ATP hydrolysis, actin-binding, and the lever arm remain unresolved. This is due, in part, from an absence of structural information regarding the flexible N-terminal regions of the light chains, and the actin- myosin interface for which no atomic structure exists. Here we propose advanced techniques in electron cryomicroscopy (cryoEM) and image analysis, fluorescence microscopy and transient kinetics to provide further insights into the mechanism of mechanochemical coupling.
Specific Aim 1 will examine the binding of the N-terminal extension of ELC to the SH3 (src-homology 3) domain. To date, the function of the SH3-like (3-barrel domain in myosin is unknown. We will test the hypothesis that SH3 mediates the communication pathway between the ELC-1 isoform, actin, and the catalytic site, by using gold-labeled- (for cryoEM) and fluorescent-labeled (for spectroscopy) mutants of expressed ELC. Any conformational changes in ELC will be correlated with steps in the ATPase cycle by stopped-flow kinetics.
Aim 2 will examine how phosphorylation of the RLC leads to activation of smooth muscle myosin from its inhibited, dephosphorylated state. The hypothesis that the N-terminus undergoes a major conformational change will be tested by introducing labeled cysteine residues into RLC and ELC to facilitate determination of length changes and sites of interaction by fluorescence spectroscopy.
Aim 3 will characterize the actomyosin interface by high resolution cryoEM, and new computational methodologies, using actin filaments decorated with wild type and mutant cardiac myosin isoforms. Pathophysiological conditions involving abnormal expression of the light chains, as well as cardiomyopathies resulting from point mutations in the light and heavy chains of myosin, will benefit from a deeper understanding of how the different light chains and domains in myosin interact to perform work with maximum contractile efficiency.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
Project #
Application #
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Boyce, Amanda T
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Vermont & St Agric College
Schools of Medicine
United States
Zip Code
Banerjee, Chaity; Hu, Zhongjun; Huang, Zhong et al. (2017) The structure of the actin-smooth muscle myosin motor domain complex in the rigor state. J Struct Biol 200:325-333
Taylor, Kenneth A; Feig, Michael; Brooks 3rd, Charles L et al. (2014) Role of the essential light chain in the activation of smooth muscle myosin by regulatory light chain phosphorylation. J Struct Biol 185:375-82
Lowey, Susan; Bretton, Vera; Gulick, James et al. (2013) Transgenic mouse ?- and ?-cardiac myosins containing the R403Q mutation show isoform-dependent transient kinetic differences. J Biol Chem 288:14780-7
Lowey, Susan; Trybus, Kathleen M (2010) Common structural motifs for the regulation of divergent class II myosins. J Biol Chem 285:16403-7
Lowey, Susan; Lesko, Leanne M; Rovner, Arthur S et al. (2008) Functional effects of the hypertrophic cardiomyopathy R403Q mutation are different in an alpha- or beta-myosin heavy chain backbone. J Biol Chem 283:20579-89
Nair, Usha B; Joel, Peteranne B; Wan, Qun et al. (2008) Crystal structures of monomeric actin bound to cytochalasin D. J Mol Biol 384:848-64