Structure Determination by NMR—An Example

Carbon resonances in the 13C spectrum were numbered 1-32 in order of chemical shift from the farthest upfield (cl) to the farthest downfield (c32). Using the HSQC spectrum, the protons were named according to the carbon to which they are correlated, using a and 

There are clearly three one-proton olehnic peaks (6.1-6.6 ppm), two coupled to each other with a 6.7 Hz coupling and a third coupled to one of the first two with a long-range coupling 

3 2D HSQC Spectrum (Upfield Region Fig. 11.56 Center and Downfield Regions Fig. 11.57) 

and 12 quaternary carbons, of which five are sp3-hybridized carbons without oxygen and seven are sp2-hybridized carbons. 

Thus the HSQC data confirm that, compared to Pristimerin, LGJC3 has one additional CH3 group (a methoxy) and one oxygenated quaternary sp3-hybridized carbon that corresponds to a sp2-hybridized carbon in Pristimerin. 

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The situation is different for other examples—for example, the peptide hormone glucagon and a small peptide, metallothionein, which binds seven cadmium or zinc atoms. Here large discrepancies were found between the structures determined by x-ray diffraction and NMR methods. The differences in the case of glucagon can be attributed to genuine conformational variability under different experimental conditions, whereas the disagreement in the metallothionein case was later shown to be due to an incorrectly determined x-ray structure. A re-examination of the x-ray data of metallothionein gave a structure very similar to that determined by NMR. 

NMR is particularly suitable for the study of peptidic toxins because these molecules are typically small in size (less than 50 amino acids), are usually highly soluble and very often have well-defined structures stabilised by disulphide bonds, which predispose them to have excellent dispersion in their NMR spectra. Indeed, small disulphide-rich peptides are perhaps the one area of structural biology where NMR dominates over X-ray crystallography as the preferred structural technique. The Protein Data Bank (PDB), for example, shows that of the approximately 50,000 structures deposited, less than 20% have been determined by NMR, but if the analysis is done over peptides smaller than 50 amino acids then the proportion of NMR structures is approximately 90%. An example of the important role of NMR in structure determination of peptide toxins involves those from marine cone snails known as conotoxins. Of the 125 conotoxin structures... 

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