ATP analogs of various types will be prepared to help characterize the nature of nucleotide-protein interactions in contractile proteins with the long-term goal of explaining the molecular basis of muscle contraction. A new class of second generation ATP photoaffinity analogs will be synthesized which carry with them attached reporter groups. These groups will include spin labels, fluorescent and luminescent probes. ATP analogs will be photo-incorporated after first trapping them at the active site by use of the phosphate analogs, vanadate, AlF4, or BeFx. The attached analogs can be displaced from the active site by actin treatment to yield fully active myosin either in solution or in glycerinated muscle fibers. The probes, attached to either side of the active site, will be used to measure distances to other defined parts of myosin (or to actin) by use of the new technique of luminescence resonance energy transfer in collaboration with P. Selvin and R. Cooke. Specifically, changes in the relative orientation of the motor and light chain domains of subfragment 1 as influenced by nucleotides or actin binding will be assessed. Related experiments will utilize a new class of non-nucleoside triphosphate analogs to introduce specific spin probes at myosin's active site in muscle fibers to probe for global orientation changes in heads of myosin during the contraction cycle utilizing EPR spectroscopy. The properties of new thiol mutants of Dictyostelium myosin will be determined with the goal of preparing crystals of thiol-crosslinked subfragment for x-ray structure determination.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK005195-38A1
Application #
2856697
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Sechi, Salvatore
Project Start
1976-05-01
Project End
2003-03-31
Budget Start
1999-04-15
Budget End
2000-03-31
Support Year
38
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Washington State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
041485301
City
Pullman
State
WA
Country
United States
Zip Code
99164
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Chen, Xiaoru; Grammer, Jean; Lawson, J David et al. (2002) A novel restricted photoaffinity spin-labeled non-nucleoside ATP analogue as a covalently attached reporter group of the active site of Myosin subfragment 1. Biochemistry 41:2609-20
Chen, X; Grammer, J; Cooke, R et al. (2000) Synthesis and characterization of novel spin-labeled photoaffinity nonnucleoside analogues of ATP as structural and EPR probes for myosin. Bioconjug Chem 11:725-33
Gulick, A M; Bauer, C B; Thoden, J B et al. (2000) X-ray structures of the Dictyostelium discoideum myosin motor domain with six non-nucleotide analogs. J Biol Chem 275:398-408
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Chen, X; Siems, W F; Asbury, G R et al. (1999) Fingerprint patterns from laser-induced azido photochemistry of spin-labeled photoaffinity ATP analogs in matrix-assisted laser desorption/ionization mass spectrometry. J Am Soc Mass Spectrom 10:1337-40
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Deng, H; Wang, J; Callender, R H et al. (1998) Raman difference spectroscopic studies of the myosin S1.MgADP.vanadate complex. Biochemistry 37:10972-9
Rayment, I; Smith, C; Yount, R G (1996) The active site of myosin. Annu Rev Physiol 58:671-702
Grammer, J C; Loo, J A; Edmonds, C G et al. (1996) Chemistry and mechanism of vanadate-promoted photooxidative cleavage of myosin. Biochemistry 35:15582-92

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