Isotopic labeling resonance assignment

Benzoates. The selective debenzoylation of sucrose octabenzoate [2425-84-5] using isopropylamine in the absence of solvents caused deacylation in the furanose ring to give 2,3,4,6,1/3/6 -hepta- and 2,3,4,6,1/6 -hexa-O-benzoyl-sucroses in 24.1 and 25.4% after 21 and 80 hours, respectively (54). The unambiguous assignment of partially benzoylated sucrose derivatives was accompHshed by specific isotopic labeling techniques (54). Identification of any benzoylated sucrose derivative can thus be achieved by comparison of its C-nmr carbonyl carbon resonances with those of the assigned octabenzoate derivative after benzoylation with 10 atom % benzoyl—carbonyl chloride in pyridine. 

In addition to the in vitro studies of well-defined systems that have been discussed here, NMR spectroscopy can also be applied to living systems or complex substance mixtures (146). This broad applicability is an advantage of NMR spectroscopy over X-ray crystallography. Early in vivo NMR studies that mostly identify small metabolite molecules by P or H ID experiments have led to different applications. In-cell NMR uses isotopic labeling combined with two or higher dimensional NMR experiments for structural studies of the phosphorylation state of a given protein in the cellular environment or of intrinsically unstructured proteins. In-cell NMR applications in prokaryotic systems cover structural and functional studies (i.e., protein-protein interactions, protein dynamics, automated structure determination, and de novo resonance assignments (147-149)). 

The essentially complete assignment of H, 13C and 15N resonances of recombinant human ubiquitin were obtained by analysis of HNCA, HN(CO)CA, HNCO and HCCH-TOCSY spectra and reference to previously reported H (Di Stefano and Wand, 1987) and 15n (Schneider et al., 1992) resonance assignments (Wand et al., 1996). Stereospecific assignments of leucine 5-CH3 and valine y-CHj groups were obtained using the isotopic labeling strategy developed by Neri et al. (1990). This approach provided unequivocal prochiral... 

There are two general approaches through which stage (1), resonance assignment, can be accomplished, and the choice between them is determined essentially by the molecular mass of the protein. The first approach, which was pioneered in the laboratory of Kurt Wiithrich34 and which led to his award of the 2002 Nobel Prize in Chemistry, involves the use of 2D 1H—1 H (homonuclear) NMR experiments such as COSY, NOESY, and TOCSY (see below). This approach is still widely used today, but only for proteins smaller than lOkDa that cannot be isotopically labeled. Nevertheless, the homonuclear NMR strategy is suitable for studying peptides purified from natural sources, and this will be the focus of Section 9.09.3. 

K.M., and Spiro, T.G. (1993) Complete assignment of cytochrome c resonance Raman spectra via enzymic reconstitution with isotopically labeled hemes. Journal of the American Chemical Society, 115, 12446-12458. 

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