David M. LeMaster

Deuterium labelling in NMR structural analysis of larger proteins

In the last few years since the early NMR structural studies of small proteins such as glucagon (Braun et al. 1983) and lac represser headpiece (Zuiderweg et al. 1984) the quality of the structure determinations have improved considerably. Of major importance has been the introduction of phase sensitive detection in the Tl dimension (States et al. 1982; Marion & Wuthrich, 1983) which has allowed for absorption presentation of 2D data with the resulting enhancement in resolution, accuracy of coupling constant measurements and accuracy of peak volume integrations. Introduction of new pulse sequences, advances in instrumentation and further developments in the structure calculation algorithms have also helped improve the quality of NMR structural analyses of proteins.

Resolution and sensitivity enhancement of heteronuclear correlation for methylene resonances via 2H enrichment and decoupling.

SummaryMonodeuterated methylene positions exhibit substantially superior spectral characteristics in 1H−13C correlation experiments as compared to diprotio signals. A combination of 2H decoupling and multiplet editing of HMQC and HSQC experiments provides resolution enhancement for both stereoselective and random fractionally deuterated samples. For HMQC experiments with [2-2Hr, 2-13C]glycine-labeled E. coli thioredoxin (11.7 kDa), 3-fold increases in both 1H and 13C resolution result in a complementary 9-fold enhancement in sensitivity. Owing to a smaller improvement in 13C resolution, the corresponding enhancements for the HSQC experiment are 2-fold less.

Differential deuterium isotope shifts and one-bond 1H−13C scalar couplings in the conformational analysis of protein glycine residues

SummaryThe one-bond deuterium isotope shift effect for glycine Cα resonances exhibits a conformational dependence comparable to that of the corresponding 1JHC scalar coupling in both magnitude (∼11 Hz at 14.1 T) and dihedral angle dependence. The similarity in the conformational dependence of the 1JHC and deuterium isotope shift values suggests a common physical basis. Given the known distribution of (ϕ,ψ) main-chain dihedral angles for glycine residues, the deuterium isotope shifts and the 1JHC scalar couplings can determine conformations in the left-and right-handed helical-to-bridge regions of the (ϕ,ψ) plane to an accuracy of approximately 13°. In the absence of stereochemical assignments, the differential deuterium isotope shifts and the 1JHC scalar couplings can be combined with limited independent structural information (e.g., the sign of ϕ) to determine the chirality of the deuterium substitution.

Enantiomeric conversion of racemic amino acid mixtures via an oxidase-aminotransferase coupled system

Abstract D-amino acid oxidase, branched-chain amino acid aminotransferase and excess L-glutamate are used to convert racemic mixtures to the L-form enantioner in high chemical and optical yields for a number of both common and non-biological amino acids.

Combining 2H and 13C Selective Enrichment to Probe Protein Dynamics

The dipolar interactions between 13C nuclei and the directly attached 1H nuclei offer a potentially rich set of spectroscopic monitors of the dynamical behavior of biological macromolecules. However, in contrast to the more widely exploited 1H-15N dipoles of the mainchain amide resonances of proteins, additional technical complexities have impeded the more extensive use of 13C relaxation analysis. As for the case of 15N studies, the low sensitivity of natural abundance samples have limited 13C relaxation studies to quite small highly soluble systems. On the other hand uniform enrichment introduces scalar and dipolar interactions between directly bonded 13C nuclei which confound the interpretation of the conventional pulse sequences used in relaxation analysis. Progress has been reported in the development of pulse sequences to circumvent some of the complications arising from geminal 13C nuclei,1,2 but a general solution is not yet available. An alternate approach which allows for extensive high levels of site specific enrichment with minimal occurrence of geminal 13C-13C couplings can be achieved via protein expression in bacterial strains carrying suitable metabolic lesions so as to direct the flow of isotopic label from selectively enriched carbon sources.