Studies of structure, hydration and dynamics of biological macromolecules by use of NMR spectroscopy
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Nuclear magnetic resonance (NMR) has emerged as a powerful tool for studying biological macromolecules in aqueous solution. This dissertation describes the efforts to study the structure, hydration and dynamics of biological macromolecules by solution state NMR spectroscopy. There are three projects covered by the dissertation: (i) polypeptide hydration studied by NMR in supercooled water; (ii) structure determination of protein AQ-1857 by NMR spectroscopy and (iii) base flipping mechanism of M. Hha I DNA methyltransferase. Chapter 1 gives brief introduction of NMR applications in biological sciences. Chapter 2 describes the first investigation of polypeptide hydration in supercooled water at temperatures down to -18oC, which allowed identification of the zero crossing points of intermolecular NOEs between hydration water protons and peptide protons. The experimental results were then fitted to spatial motional models to calculate the translational diffusion coefficients of different hydration water molecules. Chapter 3 presents data collection, spectral analysis and structure calculation of the NESGC target protein AQ-1857, pursued in the context of high-throughput structure determination efforts for structural genomics. Chapter 4, the centerpiece of this dissertation, describes the tremendous efforts in investigating the base flipping mechanism of M. Hha I DNA methyltransferase, including enzymatic synthesis of a partially 13 C, 15 N labeled cognate DNA duplex and the NMR studies on the MTase-DNA complexes. The ample NMR information from chemical shifts, line widths and peak intensities, and imino proton exchange rates provided new insights for understanding the base flipping process.