Work in this laboratory has been focused on the determination of three-dimensional structures of macromolecules in solution by NMR. Methods are being developed to increase the precision with which structures can be determined, the molecular weight range of proteins that can be analysed, and the efficiency of the computational methods used to determine the structures on the basis of the NMR data. In particular, we have demonstrated the applicability of heteronuclear 3D NMR to the study of proteins in the range 15-25 kDa, and recently, we have demonstrated the utility of heteronuclear 4D NMR to assign nuclear Overhauser effects in virtually an automated manner which will permit the extension of the method to even larger proteins. High resolution solution structures of a number proteins have been determined. These include the cytokine interleukin-8, human thioredoxin and the zinc finger domain of a human enhancer binding protein. Extensive use in these studies has been made of systematic conformational searches to obtain stereospecific assignments and torsion angle restraints which have enabled us to obtain structure of much greater precision and accuracy than was heteretofore possible. The typical accuracy attainable is 0.3-0.4 A for the backbone atoms and 0.4-0.6 A for the internal side chains. Work is in progress on determining the solution structures of a number of other proteins. These include the DNA binding protein ner from phage Mu, human interleukin-lo and a trypsin inhibitor from Ascaris. In the case of human interleukin-1-beta, extensive use of 3D and 4D heteronuclear NMR has been made to resolve problems associated with spectral overlap and proton chemical shift degeneracy.
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