NMR studies on DNA binding specificity of the lac repressor

Abstract

The thesis describes NMR structures of two protein-DNA complexes. The first structure shows how the protein, the DNA binding domain of lac repressor, recognizes its natural DNA binding site, by adaptation and read out of the nucleotide sequence. The second one shows how the DNA binding specificity of the same protein was altered to that of the gal repressor, by altering both the DNA and the protein binding surfaces. The gal repressor is another transcription regulator highly homologous to the lac repressor. Gal and lac repressors, however, recognize different DNA sequences. Finally we used hydrogen-deuterium exchange to follow the kinetics of amide proton exchange with the solvent. We focused mostly on one alpha-helix of the lac repressor-DNA complex, and we show evidences that the backbone amide protons in the core of this helix exchange in one single event, this demonstrates a correlated unfolding transition that occurs in the time scale of several hours. Overall, these results give us further insight on protein-DNA recognition