Protein Structure Determinations by NMR and Joint NMR-X-ray Refinement

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9406065 Hoffman The goal of the proposed research is to analyze the structures of two unusual and biologically significant protein molecules. The specific structures to be investigated are: (1) the "0.3" protein of bacteriophage T7; (2) the RNA-binding protein L9 from the prokaryotic ribosome. In each case, the structural information obtained will provide insight into the mechanism used by the protein in performing its particular function. The 12.5 kDalton 03 gene product of bacteriophage T7 is unique in its ability to bind to E. coli restriction enzymes and RNA polymerase. The protein performs the task of inhibiting the E. coli restriction system, thus permitting the T7 phage to safely inject its genome into the host cells. There is evidence suggesting that the 0.3 protein may function by mimicking the DNA structures that are normally recognized by the E. coli restriction system. The structure of the 0.3 protein will be determined by NMR methods, providing insight into the mechanism by which it binds and inhibits DNA-processing enzymes. L9 is a highly conserved primary RNA-binding protein from the prokaryotic ribosome. In the proposed research plan, NMR and x-ray data will be used to supply structural constraints, and the protein structure will be determined by a simultaneous refinement against both the NMR and x-ray data, or "joint-refinement". This joint-refinement approach should yield a much higher resolution structure, in which both RNA-binding sites are well defined. This analysis will provide a model as to how NMR and x-ray data can be practically and logically combined to determine the structure of a single protein molecule. %%% The principal goal of the proposed research is to analyze the structures of two unusual and biologically significant protein molecules. The specific structures to be investigated are: 1) a protein that is unique in its ability to bind to and inhibit the function of DNA-processing enzymes; and 2) a protein with the ability to recognize and bind specific sequences of ribosomal RNA. In each case, the structural information obtained will provide insight into the mechanism used by the protein in performing its particular function. An additional goal of the proposed project is to develop new methods for determining protein structures, using the combined data from nuclear magnetic resonance (NMR) experiments and x-ray diffraction experiments. It is anticipated that these new methods will be useful in determining protein structures with greater accuracy. ***