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. ? ? ?
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| 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 |
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