Myosin is a molecular motor binding ATP and actin to produce work by causing relative translation of the two proteins. Myosin contains a lever arm probably executing a power stroke by rotating through an angle of ~70o to translate actin against resistive force. ATP hydrolysis at myosin's active site energizes contraction by influencing lever arm movement and is influenced by allostery with actin in actin-activation of myosin ATPase. The influences are conducted through the protein matrix by coupling pathways investigated by mutation (naturally occurring and computation inspired), molecular dynamics simulation (MD), and structure/function assays. Two coupling pathways identified for study mediate actin-activation of myosin ATPase and conformation change triggering tryptophan nucleotide sensitivity that might link small active site displacements to the larger lever arm movement. The goal of the project is to elucidate the native relationships among actin binding, active site conformation, lever arm rotation and protein displacement and then to observe how these relationships are affected by modifications introduced to coupling pathways. Human skeletal myosin variants play a fundamental role in exercise physiology, human disease, and population diversity. The variants involve widely dispersed amino acid substitutions covering several regions essential to function and are naturally embedded clues to discovering functional domain interconnectedness through the coupling pathways. They implicate sites for mutagenesis in model proteins and are essential for correlation of myosin functional alteration to phenotype. Myosin MD simulation provides complementary insights into how coupling pathways perform. MD introduces the causality test identifying source, path, and termination of coupling networks in sequential time that is an integral part of the competent motor. Causality testing applied to tryptophan nucleotide sensitivity has converged with experimental findings from a tonic smooth muscle myosin to suggest tryptophan nucleotide sensitivity could disconnect from lever arm movement in native myosin. A new experimental causal rotation/displacement metric, quantifying completion of a productive myosin cycle, will correlate myosin lever arm rotation with displacement of a bound actin filament (F-actin) in an in vitro assay. The two-molecule technique utilizes a green fluorescent protein (GFP) on myosin and nanometer resolution localization of a fluorescent probe bound to F-actin. Myosin variants that are, adapted to specialize function, implicated in human disease, or sourced in population diversity, are mined for insight into functional divergence. MD simulation introduces causality to characterize myosin coupling networks and produces experimentally testable hypotheses. A causal two-molecule assay tests completion of a productive myosin cycle and characterizes myosin's ability to displace actin. These analytical tools are next-generation methods addressing transduction and motility in muscle myosin.

Public Health Relevance

Skeletal myosin is the motor in muscle powering contraction. Its ability to convert chemical energy to useful movement is fundamental to our ability to lead happy and productive lives. The proposed research promotes understanding of its design for energy conversion shaping approaches for how to repair an ailing motor and how to adapt it to applications where muscle productivity is limiting human potential.

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
Mayo Clinic, Rochester
United States
Zip Code
Wang, Y; Burghardt, T P (2017) In vitro actin motility velocity varies linearly with the number of myosin impellers. Arch Biochem Biophys 618:1-8
Burghardt, Thomas P; Ajtai, Katalin; Sun, Xiaojing et al. (2016) In vivo myosin step-size from zebrafish skeletal muscle. Open Biol 6:
Wang, Yihua; Ajtai, Katalin; Kazmierczak, Katarzyna et al. (2016) N-Terminus of Cardiac Myosin Essential Light Chain Modulates Myosin Step-Size. Biochemistry 55:186-98
Burghardt, Thomas P; Sun, Xiaojing; Wang, Yihua et al. (2015) In vitro and in vivo single myosin step-sizes in striated muscle. J Muscle Res Cell Motil 36:463-77
Wang, Yihua; Ajtai, Katalin; Burghardt, Thomas P (2014) Analytical comparison of natural and pharmaceutical ventricular myosin activators. Biochemistry 53:5298-306
Wang, Yihua; Ajtai, Katalin; Burghardt, Thomas P (2014) Ventricular myosin modifies in vitro step-size when phosphorylated. J Mol Cell Cardiol 72:231-7
Sun, Xiaojing; Ekker, Stephen C; Shelden, Eric A et al. (2014) In vivo orientation of single myosin lever arms in zebrafish skeletal muscle. Biophys J 107:1403-14
Wang, Yihua; Ajtai, Katalin; Burghardt, Thomas P (2013) The Qdot-labeled actin super-resolution motility assay measures low-duty cycle muscle myosin step size. Biochemistry 52:1611-21
Ajtai, Katalin; Mayanglambam, Azad; Wang, Yihua et al. (2013) Human Tonic and Phasic Smooth Muscle Myosin Isoforms Are Unresponsive to the Loop 1 Insert. ISRN Struct Biol 2013:634341
Burghardt, Thomas P; Sikkink, Laura A (2013) Regulatory light chain mutants linked to heart disease modify the cardiac myosin lever arm. Biochemistry 52:1249-59

Showing the most recent 10 out of 33 publications