Nuclear magnetic resonance, heteronuclear

Probably, one of the most valuable advances in this field has dealt with the first chemoenzymatic synthesis of the stable isotope-enriched heparin from a uniformly double labelled 13C, 15N /V-acetylheparosan from E. coli K5. Heteronuclear, multidimensional nuclear magnetic resonance spectroscopy was employed to analyze the chemical composition and solution conformation of N-acety 1 hcparosan, the precursors, and heparin. Isotopic enrichment was found to provide well-resolved 13C spectra with the high sensitivity required for conformational studies of these biomolecules. Stable isotope-labelled heparin was indistinguishable from heparin derived from animal tissues and might be employed as a novel tool for studying the interaction of heparin with different receptors.30... 

Nuclear magnetic resonance (NMR) has proved to be a very useful tool for structural elucidation of natural products. Recent progress in the development of two-dimensional 1H- and 13C-NMR techniques has contributed to the unambiguously assignment of proton and carbon chemical shifts, in particular in complex molecules. The more used techniques include direct correlations through homonuclear (COSY, TOCSY, ROESY, NOESY) [62-65] and heteronuclear (HMQC, HMBC) [66. 67] couplings. 

Fig. 8. Heteronuclear single-quantum coherenc (HSQC) spectrum of the hypothetical protein of the flowering locus T protein produced in the cell-free system. The FT protein was synthesized in the same way as in Fig. 6 except that Ala, Leu, Gly, and Gin in both translation and substrate mixture were replaced with their -labeled forms (Isotec, Inc ). After incubation for 48 h, the reaction mixture (1 mL) was dialyzed against 10 mMphosphate buffer (pH 6.5) overnight, and then centrifuged at 30,000g for 10 min. The supernatant containing 30 xMof the protein was directly subjected to nuclear magnetic resonance spectroscopy. The spectrum was recorded on a Broker DMX-500 spectrometer at 25°C, and 2048 scans were averaged for the final H- WHSQC spectrum.

This chapter describes protocols for preparing 15N-labeled proteins (ubiquitin is used as an example) using Escherichia coli cells (with purification) and the wheat germ cell-free system (without purification). A comparison of I I-15N heteronuclear single-quantum coherence (HSQC) spectra of yeast ubiquitin prepared using each method indicates that this wheat germ cell-free system may be used for rapid nuclear magnetic resonance analyses of proteins without purification. 

Clore GM, et al. Analysis of the backbone dynamics of interleukin-1 beta using two-dimensional inverse detected heteronuclear 15N-1H NMR spectroscopy. Biochemistry 1990 29 7387-7401. Aue WP, Bartholdi E, Ernst RR. Two-dimensional spectroscopy. Application to nuclear magnetic resonance. J. Chem. Phys. 1976 64 p. 2229-2246. 

Sattler M, Schleucher J, Griesinger C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Progress Nuclear Magnet. Reson. Spectros. 1999 34 93-158. 

M. Bjerring, J. T. Rasmussen, R. S. Krogshave and N. C. Nielsen, Heteronuclear coherence transfer in solid-state nuclear magnetic resonance using a 7-encoded transferred echo experiment. J. Chem. Phys., 2003, 119, 8916-8926. 

Due to the great complexity of this class of molecules, nuclear magnetic resonance (NMR) and mass spectroscopy (MS) are the tools most widely used to identify cucurbitacins. Both one- and two-dimensional NMR techniques have been employed for the structural elucidation of new compounds 2D NMR, 1H-NMR, 13C-NMR, correlated spectroscopy (COSY), heteronuclear chemical shift correlation (HETCOR), attached proton test (APT), distortionless enhancement by polarization transfer (DEPT), and nuclear Overhauser effect spectroscopy (NOESY) are common techniques for determining the proton and carbon chemical shifts, constants, connectivity, stereochemistry, and chirality of these compounds [1,38,45-47]. 

M. Ikura, A. Bax, G. M. Clore and A. M. Gronenborn, Detection of nuclear Overhauser effects between degenerate amide proton resonances by heteronuclear 3-dimensional nuclear-magnetic-resonance spectroscopy, J. Am. Chem. Soc., 1990,112, 9020-9022. 

We present a solid-state nuclear magnetic resonance (NMR) experiment that allows the observation of a high-resolution two-dimensional heteronuclear correlation (2D HETCOR) spectrum between aluminum and phosphorous in aluminophosphate molecular sieve VPI-5. The experiment uses multiple quantum magic angle spinning (MQMAS) spectroscopy to remove the second order quadrupolar broadening in Al nuclei. The magnetization is then transferred to spin-1/2 nuclei of P via cross polarization (CP) to produce for the first time isotropic resolution in both dimensions. 

Battiste JL, Wagner G (2000) Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear Overhauser effect data. Biochemistry 39(18) 5355-5365... 

Clore GM, Gronenbom AM (1994) Multidimensional heteronuclear nuclear-magnetic-resonance of proteins. Nucl Magn Reson Pt C 239 349-363... 

Abstract Nuclear Magnetic Resonance (NMR) relaxation is a powerful technique that provides information about internal dynamics associated with configurational energetics in proteins, as well as site-specific information involved in conformational equilibria. In particular, N relaxation is a useful probe to characterize overall and internal backbone dynamics of proteins because the relaxation mainly reflects reorientational motion of the N-H bond vector. Over the past 20 years, experiments and protocols for analysis of N Ri, R2, and the heteronuclear N- NOE data have been well established. The development of these methods... 

Eden M, Levitt MH (1999) Pulse sequence symmetries in the nuclear magnetic resonance of spinning solids application to heteronuclear decoupling. J Chem Phys 111 1511-1519... 

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