We propose new solid-state rotational-echo double-resonance (REDOR) NMR experiments to characterize the complexes of four proteins: (i) lumazine synthase, (ii) polymerized tubulin, (iii) L-selectin, and (iv) uracil DNA gIycosylase. The systems to be examined are not suited to anaIysis by solution-state NMR and are too large for the total-structure solid-state methods currently under development. Stable-isotope labeling will be used for both the protein and its ligands whenever possible. For lumazine synthase and polymerized tubulin, some relatively low-resolution electron or x-ray diffraction data are avaiIable. For these systems, we will start with the best available structure from diffraction analysis and then use molecular modeling, restrained by a few accurate REDOR distances, to refine or enhance the structure of the liganded binding site. Our goal in these two systems is to define the conformation of the bound ligands with sufficient accuracy that new drug molecules can be developed that have improved bioactivity. L-selectin has low-affinity binding but is still highly selective. The selectivity appears to arise from multivalency; that is, the L-selectin binds ligands using multiple interactions at the same time. These complexes do not crystallize. We propose to use REDOR to determine quantitatively the degree of multi-valency of L-selectin in binding to a natural glycoprotein substrate. This basic information could be crucial in the development of anti-inflammatory drugs. The DNA damage and repair enzyme uracil DNA glycosylase is thought to achieve its specificity by the recognition of the shapes of certain types of molecular defects. This idea will be tested by the REDOR characterization of stable-isotope labeled (non-crystallizable) mimics of transient protein-DNA complexes thought to lie along the recognition pathway. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
9R01EB001964-16
Application #
6678920
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Mclaughlin, Alan Charles
Project Start
1988-07-01
Project End
2007-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
16
Fiscal Year
2003
Total Cost
$292,750
Indirect Cost
Name
Washington University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Yu, Tsyr-Yan; O'Connor, Robert D; Sivertsen, Astrid C et al. (2008) (15)N{(31)P} REDOR NMR studies of the binding of phosphonate reaction intermediate analogues to Saccharomyces cerevisiae lumazine synthase. Biochemistry 47:13942-51
Schaefer, Jacob; Jiang, Hong; Ransome, Aaron E et al. (2007) Multiple active site histidine protonation states in Acetobacter aceti N5-carboxyaminoimidazole ribonucleotide mutase detected by REDOR NMR. Biochemistry 46:9507-12
Paik, Younkee; Yang, Chao; Metaferia, Belhu et al. (2007) Rotational-echo double-resonance NMR distance measurements for the tubulin-bound Paclitaxel conformation. J Am Chem Soc 129:361-70
McDowell, Lynda M; Studelska, Daniel R; Poliks, Barbara et al. (2004) Characterization of the complex of a trifluoromethyl-substituted shikimate-based bisubstrate inhibitor and 5-enolpyruvylshikimate-3-phosphate synthase by REDOR NMR. Biochemistry 43:6606-11
Jiang, Yu Lin; McDowell, Lynda M; Poliks, Barbara et al. (2004) Recognition of an unnatural difluorophenyl nucleotide by uracil DNA glycosylase. Biochemistry 43:15429-38
Mehta, Anil K; Shayo, Yuda; Vankayalapati, Hariprasad et al. (2004) Structure of a quinobenzoxazine--G-quadruplex complex by REDOR NMR. Biochemistry 43:11953-8
McDowell, Lynda M; Poliks, Barbara; Studelska, Daniel R et al. (2004) Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase. J Biomol NMR 28:11-29