PROTEIN NMR STRUCTURE AND DYNAMICS VIA ISOTOPIC LABELING

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Various isotopic labeling techniques are being developed for the NMR analysis of structure and dynamics of proteins and protein-DNA complexes. Much of the effort is focused on establishing selective deuteration procedures so that the labeling patterns of the various individual residue types can be, nearly independently, simultaneously controlled within a given experimental growth. This entails the appropriate synthesis of the deuterated amino acids as well as construction of a suitable auxotrophic expression system. Our previous work indicates that in addition to the anticipated enhancement in spectral resolution, there will be a 4-6 fold enhancement in the sensitivity of crosspeaks involving carbon bound protons. This will substantially increase the molecular weight range of macromolecules amenable to detailed structural analysis. Other potentially useful labeling patterns (such as an alternating 12C-13C enrichment) will also be investigated. These labeling patterns will be used to determine the solution conformation of eukaryotic transcription factors. Although several cysteine-zinc binding domains have been analyzed by NMR, structural determination of a larger protein domain is needed so as to include the regions specifying DNA sequence-specific binding. Insight into this process will come from the study of a 148 residue HAP1 fragment binds two dissimilar control sequences using distinct but overlapping binding sites. In a related project, -100 residue domains of Fos and Jun have been shown to bind selectively to the AP-1 DNA binding site as a heterodimer. As the DNA complex will be 40kD, it is anticipated that the selective deuteration approach described will be essential for detailed NMR structural analysis of this model system for oncogene function. Protein dynamics studies will involve amide trapping experiments on E. coli and T4 thioredoxins in order to monitor the detailed kinetics of the folding process. Equilibrium analysis of the conformation and dynamics of a model "molten globule" protein folding intermediate state will make critical use of the selective deuteration techniques developed here in order to overcome the severe spectral resolution problem.