The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Inferences from biochemical kinetic data suggest the actomyosin interaction consists of a series of states for which each intermediate in the degradation of ATP corresponds to a unique actomyosin conformation. Crystal structures of the myosin globular head or subfragment 1 (S1) representing the ATP hydrolysis intermediates show an articulated molecule made from nearly intact N-terminal catalytic and C-terminal lever-arm domains that change their relative position due to localized deformations in the intervening peptide. The system works by amplifying small structure changes in the catalytic domain active site where ATP is hydrolyzed into the large lever-arm domain movement. Elucidating the mechanism for propagation and amplification of the active site structure changes into the lever-arm movement is the principal aim of the proposal to be accomplished by construction of an explicit model. The model involves individual residue participants in energy transduction such that the effect of a specific residue modification or mutation may be realistically analyzed.
In Aim 1, spectroscopy from strategically located extrinsic probes and an intrinsic ATP-sensitive tryptophan detect myosin conformation during transduction.
In Aim 2, biochemical and molecular biological approaches probe the role of a structured surface loop in the actomyosin interaction.
Aim 3 is to build the atomic structures representing skeletal actomyosin conformation during the contraction cycle from crystallographic structures mined from the protein database combined with structural implications from Aims 1 and 2 data using the energy transduction model. The model satisfies simultaneously, multiple independent requirements from crystallography, spectroscopy, and biochemistry/molecular-biology to arrive at a best solution for the myosin transduction intermediate structure. Additionally, the model is subject to cumulative improvement as new data becomes available.
Aim 4 is to implement verifiably reliable routines for translating peptide structure to a spectroscopic signal, a process whose accuracy is critical to the proposed model building. The structure to spectrum translation routines have broader implications for structure sensing optical spectroscopy in biophysics. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR049277-02
Application #
6869632
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Nuckolls, Glen H
Project Start
2004-04-01
Project End
2009-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
2
Fiscal Year
2005
Total Cost
$324,500
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
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