Application and development of multi-dimensional NMR spectroscopy in structural studies of oligonucleotides and proteins.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful method of determining three-dimensional (3D) structures of molecules in solution at atomic resolution. This dissertation describes application of NMR to study the structure of a DNA hairpin and the peptide binding domain of protein DnaK along with the development of three new NMR pulse sequences. The first chapter describes the 3D structure determination of a disulfide cross-linked analog of the Dickerson/Drew dodecamer hairpin using two-dimensional (2D) NMR spectroscopy and a combined distance geometry-simulated annealing-iterative relaxation matrix approach (IRMA) method. In the final conformational ensemble that best matches the experiment data, the hairpin stem adapts a B form duplex. The cross-link does not distort the duplex geometry away from the B-like DNA. Evidence of dynamic behaviors in the loop and the termini regions are also discussed. The second chapter describes the backbone resonance assignment of the peptide binding domain of DnaK, a molecular chaperone from E. coli, using two- and three-dimensional heteronuclear NMR spectroscopy. The secondary structure identified from nuclear Overhauser effect (NOE) connectivity patterns and alpha carbon chemical shifts consists of mostly $\beta$-sheets and a long helix near the C terminal region. The topology of its $\beta$ sheets is different from a predicted model structure and appears to represent a new structure fold. The last three chapters describe developments in NMR methodology. These method include: a new three-dimensional method to suppress interfering signals in the 2D NOE experiment, arguably the single most important experiment used in solution structure determination by NMR; significant sensitivity improvement of a critical NMR resonance assignment experiment for large proteins, the HCCH experiment, by using heteronuclear cross polarization; and dramatic improvement of solvent suppression in the HCCH experiment by implementation of pulsed field gradients. The pulsed field gradients also achieved magnetization pathway selection in a single scan.PhDBiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104881/1/9610261.pdfDescription of 9610261.pdf : Restricted to UM users only