The functional architecture of Lambda cro repressor mutants will be analyzed in solution at the level of atomic resolution using the techniques of two-dimensional NMR spectroscopy and distance geometry. An overproducer plasmid strain that has been genetically engineered so that any position in the repressor can be replaced with any amino acid will be used to produce mutant repressors for studying the effects of strategic mutations on the structure, stability, and specificity of the repressor. The solution structure of the mutant repressors will be determined at ca. 1 Angstrom resolution using the distance constraints of time-dependent NOESY experiments, combined with distance geometry calculations, to generate the three-dimensional structure. Repressor:operator complexes will also be studied by 2DNMR in an attempt to define the precise atomic contacts at the surface of the DNA:protein interface. Based on current models of specific DNA recognition, complementary mutations in the operator DNA sequence will be synthesized in an attempt to restore the affinity and specificity of mutant repressors. This will lead to the design and engineering of repressor proteins capable of binding to any pre-determined DNA sequences. The long term goal of this project is a detailed understanding, at the atomic level, of sequence-specific DNA recognition by repressor proteins, as well as a fundamental understanding of the effects of amino acid sequence on protein structure and stability.
