Determination of the three-dimensional structure of an intact homeodomain protein, and probing of the internal molecular dynamics of duplexed DNA specifically bound to the homeobox segment of a related protein is proposed. The full-length protein chosen for structure determination is the 193-residue NKx2.8 (MW=21.9 kD), recently discovered as native to chicken heart, which is one of the smallest known members of the NK2 class of homeodomain proteins. Internal dynamics of the canonical NK2 class recognitions DNA sequence will be characterized in part, with the sequence complexed to the 80-residue vnd/NK-2 homeobox protein. Homeodomains proteins bear the 60-residue long homeobox DNA-binding sequence that seems highly conserved, evolutionarily, across all eukaryotic species including humans. They are sequence-specific DNA-binding factors that generally trigger the timing of gene activation in contexts of embryonic development under ordinary circumstances, thus play roles in inducing cell type discrimination, morphogenesis, and growth control. Further, chromosomal translocations that substitute homeoboxes for normally occurring DNA- binding segments of transcription regulators have been linked with oncogenic activation of certain proto-oncogenes (as in some T-cell lymphomas). No structure of an intact homeodomain protein exists as yet. We propose to use methods of high resolution nuclear magnetic resonance spectroscopy (NMR) to determine the solution structure of recombinantly generated NKx2.8 in its free state. In addition to the homeobox itself, NKx2.8 contains two other sub-sequences that have been evolutionarily conserved to a significant degree. These are the 12-residue TN domain, upstream of the homeobox near the N-terminus of the protein, and the 23- residue NK2-SD domain downstream of the homeobox towards the C-terminus. Results recently reported in the literature are only beginning to offer clues as to the functional significance of these domains. No structural data exists for either of these domains as yet. Thus, the development of a detailed description of the structure of NKx2.8 will have great impact on identifying mechanisms by which the sub-sequence factors responsible for the functioning and specificity of homeodomain proteins act. The internal dynamics of DNA are commonly speculated to be of great importance in governing specificity of protein-DNA interactions. We will employ solid-state 2H NMR methods to probe, selectively, dynamics of nucleotide bases, found within the 16 bP recognition sequence of the vnd/NK-2 homeobox, that are known to be critical to the protein-DNA interface in this complex. We will characterize dynamics of both free duplexed forms of the recognition oligonucleotide and forms bound to vnd/NK-2.

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