9419049 Palmer Protein motifs recognizing specific DNA sequences are ubiquitous components of proteins that regulate gene expression; consequently, explication of the molecular basis for specific recognition is necessary for development of a molecular theory of cellular transformation. The long term goal of the research is to elucidate quantitatively the dynamical and entropic aspects of DNA recognition by protein domains. The initial phase of this research will use NMR spectroscopy to characterize the conformational and dynamical properties of the GCN4 bZip homodimer, in the presence and absence of DNA. In the bZip classes of transcriptional activators, the basic domain recognition helices are stable only in the presence of DNA. Dynamical features of DNA-recognition are hypothesized to include: (i) surface exposed, presumably mobile, side chains in the free protein are buried in the major groove of the DNA upon complexation, (ii) low energy deformation of the DNA upon protein binding implies increased conformational flexibility of the free DNA at the site of the deformation, (iii) conformational changes in the backbone of the protein may be necessary to accommodate the -helix into the major groove of the DNA, and (iv) ancillary amino acid residues that interact with the minor groove or phosphate backbone may be disordered in the absence of DNA and ordered in the complex. The (nascent) structure and conformational dynamics of the GCN4 bZip domain in the absence of DNA will be characterized using J-coupling measurements and spin relaxation techniques, including homonuclear NOESY and ROESY experiments, and heteronuclear R1, R2, R1p, and NOE experiments. In the presence of DNA, spin relaxation experiments will be used to obtain information on the dynamical properties of the complex that is complementary to the existing crystallographic structural results. Comparison of the dynamical properties of the free energy of DNA binding from configurational entropy of individual amino acid residues. %%% Proteins that bind to specific DNA sequences are critical for regulation of gene expression. Explication of the molecular basis for sequence-specific recognition is crucial to a fundamental understanding of cellular transformation. In the past, the three- dimensional structures of DNA-binding proteins in the absence and presence of DNA have been studied to determine the interactions between the protein and DNA that confer sequence specificity. The main hypothesis of the present research is that internal motions of the proteins and DNA (i.e. time-dependent changes in the structures of the molecules) also are important in modulating sequence- specific recognition. The hypothesis will be tested for the DNA- binding domain of the protein GCN4, a member of class of proteins called bZip transcriptional activators. GCN4 is an ideal candidate for testing the above hypothesis, because the portions of the protein that recognize the DNA molecule are presumed to be disordered (with large amplitude motions) in the absence of DNA and rigid (with small amplitude motions) in the presence of DNA. Nuclear magnetic resonance spectroscopy (NMR), which analyzes the properties of atomic in very strong magnetic fields, will be used characterize the dynamical properties of GCN4. ***
