ATP analogues of various types will be prepared to help characterize the nature of nucleotide-protein interactions in muscle proteins. A new class of analogues will be prepared which exploit the ability to form a stable carbamoyl linkage to the 2'(3')hydroxyls of ATP. New ATP/ADP affinity columns will be utilized to prepare variously modified myosin subfragment l (S1) derivatives for potential crystallization studies. These derivatives include S1 cross-linked via two kinetically reactive cysteines and S1 photolabeled with ATP photoaffinity derivatives. The chemical nature of reductively methylated S1 will be defined by use of [3H]H2CO as a means to help prepare more homogeneous S1 preparations for crystallization studies with bound nucleotide. Photoaffinity labeling studies with ATP analogues and scallop myosin will explore the recently discovered differential labeling which occurs in the presence of regulatory amounts of Ca2+. Mutant light chains will be incorporated into scallop myosin to test the hypothesis that Ca2+ binding to the essential light chains affects the active site in a direct manner. The mechanism of vanadate photocleavage and photomodification of myosins active site will be explored. This study should serve as a paradigm for other studies of vanadate promoted photocleavage of proteins which bind phosphorylated substrates. Other studies will prepare ribose-modified heavy metal derivatives of ATP and GTP (Re complexes) for use in single crystal x-ray studies and ATP with Au11 complexes attached to the 2'(3') ribose hydroxyls to be used to define thick filament structure by electron microscopy and image reconstruction studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK005195-35
Application #
2134447
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Sechi, Salvatore
Project Start
1976-05-01
Project End
1998-06-30
Budget Start
1995-07-01
Budget End
1996-06-30
Support Year
35
Fiscal Year
1995
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
Lawson, J David; Pate, Edward; Rayment, Ivan et al. (2004) Molecular dynamics analysis of structural factors influencing back door pi release in myosin. Biophys J 86:3794-803
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
Wang, D; Luo, Y; Cooke, R et al. (1999) Synthesis of a spin-labeled photoaffinity ATP analogue, and its use to specifically photolabel myosin cross-bridges in skeletal muscle fibers. J Muscle Res Cell Motil 20:743-53
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
Xiao, M; Li, H; Snyder, G E et al. (1998) Conformational changes between the active-site and regulatory light chain of myosin as determined by luminescence resonance energy transfer: the effect of nucleotides and actin. Proc Natl Acad Sci U S A 95:15309-14
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

Showing the most recent 10 out of 32 publications