Isotope effects on chemical shift are well known, and have been shown to reflect the chemical environment of the observed nucleus. This effect has been exploited to study hydrogen bonding within small molecules in organic solvents. In this project we wish to investigate the utility of the isotope shifts for probing hydrogen bonds within protein in aqueous solution. There has been a great deal of recent interest in the possible role of """"""""strong"""""""" hydrogen bonds in catalysis. Protons believed to be involved in these strong hydrogen bonds have NMR resonances that are strongly downfield shifted. Initial experiments are to examine the isotope effect on chemical shift of some of these strongly shifted protons, and establish the degree of correlations between isotope shift, chemical shift and functional behavior. For small molecules it has often been possible to measure the primary isotope effect on shift by comparing hydrogen and deuterium. The linewidths for deuterium are substantially larger due to its quadrupole coupling, but for short correlation times the lines are sharp enough to accurately determine the peak centers. However for proteins, with their much longer correlation times, deuterium lines are so broad that they cannot be detected, and the comparison must be done between proton and tritium. Since the hydrogens involved are labile (exchanging relatively rapidly with solvent) the measurements must be done in tritiated water (ca. 2% T in H or D). Detection then requires suppression of the bulk tritium signal from water, but this is done in the same way that solvent suppression is normally done for detection of proton signals in protonated water. Initial studies have been done on RNase A and chymotrypsin. For RNase at 10 mM concentration the fairly broad, downfield shifted resonances could easily be detected in the tritium spectrum. Lines at 13.736, 13.162 and 12.470 ppm had isotope shifts of -0.177, -0.116 and -0.099 ppm respectively. Thus there does seem to be a general correlation of chemical shift and isotope shift. In chymotrypsin the sample was only 2 mM, and the proton resonance at about 18.5 ppm in the inhibited enzyme was not visible in the tritium spectrum. Thi s experiment will be repeated with effort to optimize the sensitivity of detection, and with longer acquisition time if needed. An additional experiment is scheduled to do T-15N correlations to detect isotope effects on both T and 15N in a sample of Staphylococcus nuclease.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
3P41RR001237-16S1
Application #
6220453
Study Section
Project Start
1998-08-01
Project End
2000-07-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
16
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Lawrence Berkeley National Laboratory
Department
Type
DUNS #
078576738
City
Berkeley
State
CA
Country
United States
Zip Code
94720
Singh, Navneet; Moody, Alan R; Zhang, Bowen et al. (2017) Age-Specific Sex Differences in Magnetic Resonance Imaging-Depicted Carotid Intraplaque Hemorrhage. Stroke 48:2129-2135
Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Ikemoto, Noriaki et al. (2005) Probing a putative dantrolene-binding site on the cardiac ryanodine receptor. Biochem J 387:905-9
Wang, Huimin; Shimizu, Eiji; Tang, Ya-Ping et al. (2003) Inducible protein knockout reveals temporal requirement of CaMKII reactivation for memory consolidation in the brain. Proc Natl Acad Sci U S A 100:4287-92
Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Bhat, Manjunatha B et al. (2002) Identification of a dantrolene-binding sequence on the skeletal muscle ryanodine receptor. J Biol Chem 277:34918-23
Westler, William M; Frey, Perry A; Lin, Jing et al. (2002) Evidence for a strong hydrogen bond in the catalytic dyad of transition-state analogue inhibitor complexes of chymotrypsin from proton-triton NMR isotope shifts. J Am Chem Soc 124:4196-7
Tomizawa, M; Wen, Z; Chin, H L et al. (2001) Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [(3)H]azidoneonicotinoid. J Neurochem 78:1359-66
Than, C; Morimoto, H; Williams, P G et al. (2001) Preparation, NMR characterization, and labeling reactions of tritiated triacetoxy sodium borohydride. J Org Chem 66:3602-5
Saljoughian, M; Williams, P G (2000) Recent developments in tritium incorporation for radiotracer studies. Curr Pharm Des 6:1029-56
Cianci, C; Yu, K L; Dischino, D D et al. (1999) pH-dependent changes in photoaffinity labeling patterns of the H1 influenza virus hemagglutinin by using an inhibitor of viral fusion. J Virol 73:1785-94
Palnitkar, S S; Bin, B; Jimenez, L S et al. (1999) [3H]Azidodantrolene: synthesis and use in identification of a putative skeletal muscle dantrolene binding site in sarcoplasmic reticulum. J Med Chem 42:1872-80

Showing the most recent 10 out of 28 publications