NMR & CIDNP STUDIES OF MB MUTANTS & OLIGONUCLEOTIDE LOOP

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This research combines biophysical techniques, notably nmr and photo-CIDNP spectroscopy, and recombinant DNA techniques to determine the structure and dynamics of several key biological molecules. Two different areas have emerged from earlier work: (i) studies of heme-protein interactions in myoglobin, combining site-specific mutagenesis and nmr spectroscopy; and (ii) nmr and photo-CIDNP studies of single-stranded loop regions of oligonucleotides. Myoglobin and hemoglobin are among the most studied proteins. The gene which codes for human Mb has been expressed in large quantities in E. coli, and it is straightforward to change individual amino acids by site-specific mutagenesis. Although the human protein is obviously most relevant for human health, it has not previously been studied in very great detail because it is difficult to obtain in large quantities. This protein will be characterized by X-ray crystallography and nmr spectroscopy for the first time. Many rare natural hemoglobin mutants have been studied. They give rise to a range of pathological conditions due to deficiencies in oxygen binding and cooperativity. The corresponding myoglobin mutants will be prepared by recombinant DNA methods, so that the molecular basis for these changes can be studied in detail. Specific mutants will be prepared to test models which predict trajectories taken by ligands to approach the heme binding site. It will also be possible to prepare novel mutants of amino acids in the vicinity of the heme to explore the limits of environmental control over this ubiquitous prosthetic group. Loop structures are an essential feature of RNA and DNA structure. The average loop conformation will be probed in a series of synthetic oligonucleotides by a range of contemporary nmr methods. Photo-CIDNP is sensitive to single-stranded regions in oligonucleotides. The range of applicability of this nmr method will be tested using carefully chosen oligonucleotide model systems, including dangling bases, mismatches, loops, and Z-DNA.