The proposed investigation is designed to extend the use in NMR studies of proteins of a general system, developed in this laboratory, for the production of samples with specific patterns of labeling with stable isotopes. The labeled samples will be used to probe aspects of protein dynamics. The two, structurally related, thioredoxins form E. coli and from bacteriophage T4 have been chosen for study. There are high resolution x-ray structures of both proteins, extensive data on the solution properties of the E. coli protein, mutants of the E. coli protein known to affect folding kinetics, and differential stability of the oxidized and reduced forms. The sequential assignment of the T4 protein will be completed following the methodology already successfully used on the E. coli protein: Spectral editing using residue-specific enrichment with 2H, 13C and 15N labeled amino acids and 2D NOESY and COSY spectra on the 75% randomly deuterated proteins. The latter samples are central to the sequential assignment procedures and to the future dynamic studies on both proteins. They yield spectra of comparable sensitivity to the natural abundance protein with substantial enhancement of resolution due to reduced natural linewidths and reduced passive spin coupling. Reduced spin diffusion renders these random fractionally deuterated samples clearly superior to those at natural abundance for quantitative NOE distance analysis. Procedures for chiral beta deuteration will be developed. When combined with 15N enrichment, such samples should serve to extend both assignment and NOE structure determinations. Unidirectional rate constants will be determined for both the redox and denaturation transitions via chemical exchange and magnetization transfer experiments with samples possessing selected alpha and sidechain protonated positions in a perdeutero background. Such experiments will potentially both detect and provide the structural information on folding intermediates.

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National Institute of General Medical Sciences (NIGMS)
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Biophysics and Biophysical Chemistry B Study Section (BBCB)
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Northwestern University at Chicago
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McNaughton, Lynn; Hernandez, Griselda; LeMaster, David M (2003) Equilibrium O2 distribution in the Zn2+-protoporphyrin IX deoxymyoglobin mimic: application to oxygen migration pathway analysis. J Am Chem Soc 125:3813-20
LeMaster, D M (1997) Assessment of protein solution versus crystal structure determination using spin-diffusion-suppressed NOE and heteronuclear relaxation data. J Biomol NMR 9:79-93
LeMaster, D M (1996) Structural determinants of the catalytic reactivity of the buried cysteine of Escherichia coli thioredoxin. Biochemistry 35:14876-81
LeMaster, D M; LaIuppa, J C; Kushlan, D M (1994) Differential deuterium isotope shifts and one-bond 1H-13C scalar couplings in the conformational analysis of protein glycine residues. J Biomol NMR 4:863-70
Homer, R J; Kim, M S; LeMaster, D M (1993) The use of cystathionine gamma-synthase in the production of alpha and chiral beta deuterated amino acids. Anal Biochem 215:211-5
Chanatry, J A; Schafer, P H; Kim, M S et al. (1993) Synthesis of alpha, beta-deuterated 15N amino acids using a coupled glutamate dehydrogenase-branched-chain amino acid aminotransferase system. Anal Biochem 213:147-51
Kushlan, D M; LeMaster, D M (1993) Resolution and sensitivity enhancement of heteronuclear correlation for methylene resonances via 2H enrichment and decoupling. J Biomol NMR 3:701-8
Katti, S K; LeMaster, D M; Eklund, H (1990) Crystal structure of thioredoxin from Escherichia coli at 1.68 A resolution. J Mol Biol 212:167-84
Wang, J F; LeMaster, D M; Markley, J L (1990) Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence-specific assignments of hydrogen-1 signals and solution structure of the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex. Biochemistry 29:88-101
LeMaster, D M (1990) Uniform and selective deuteration in two-dimensional NMR of proteins. Annu Rev Biophys Biophys Chem 19:243-66

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