The goals of this research is to utilize the Cro protein-operator system of bacteriophage Lambda to develop an 15N nuclear magnetic resonance method for studying DNA-protein interactions. 15N labeled A, T, C, and G nucleotide derivatives which can be used in the normal phosphoramidite chemistry for synthesizing oligomers of DNA will be prepared. Complementary double strand 17-mers containing one AT or one GC base pair enriched with 15N (>90%) will be synthesized. 15N NMR resonances are very sensitive to hydrogen bonding. Thus, when the protein binds to the DNA with formation of hydrogen bonds between the N-atoms of the bases and the amino acid side chains, the resonances from individual N atoms will undergo shifts. In this way, the specific atoms of the bases in the DNA which are interacting with the protein will be mapped. The method will be generalized to predict the nature of DNA/protein interactions involving proteins of unknown structure. %%% DNA-protein interactions are central to control of all aspects of the life of the cell. Nevertheless, short of X-ray crystallography, contemporary biologists have few tools with which to study the details of these important interactions. The work proposed involves use of stable 15N isotope-substituted DNA molecules and nuclear magnetic resonance to provide detailed information about the interactions between proteins and DNA. Such studies may lead a rapid means for obtaining unprecedentedly detailed information about such interactions.
