Determination of Large Protein Structures from NMR Data: Definition of the Solution Structure of the TRP Repressor
We have determined the solution structure of the E. Coli trp repressor (a 25 kD dimer) from NMR data. This is the largest protein structure thus far determined by NMR. The determination of the solution structure of larger proteins (MW ±15 kD) by NMR requires a different methodology, both experimental and computational, than that developed for peptides and small proteins. This structure determination illustrates a general paradigm for this purpose. We have found it necessary to first, use isotopic spectral editing and second, to develop new strategies for sequential assignment of the resonances. Third, since the protein contains more than one peptide chain it was necessary to distinguish between intra-chain and inter-chain contacts by spectroscopy of isotopically labeled hybrids. Fourth, in order to efficiently search a very large conformational space with a relatively small data set, we have used a hierarchical method of data analysis: the heuristic refinement method coded in PROTEAN has proven both accurate and computationally efficient in solving the structure of the trp repressor and placing the bound L-tryptophan ligand. Our NMR structure has the same general topology, and same binding site for the L-tryptophan molecule as the previously reported crystal structures. There are, however, some differences in the backbone trace. In addition, the solution structure shows significant structural uncertainty in the DNA binding region of the molecule.
KeywordsDistance Constraint Nuclear Overhauser Effect Dime Core Report Crystal Structure Tryptophan Molecule
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