Abstract
The thesis focuses on structural studies of nucleic acids and proteins using NMR spectroscopy. This thesis consists of three chapters: Chapter 1 : The Solution Structure of (d(CGC)r(amamam)d(TTTGCG))2 The solution structure and hydration of a DNA•RNA hybrid chimeric duplex (d(CGC)r(amamam)d(TTTGCG))2 in which the RNA adenines were substituted by 2'-O-methylated riboadenines was determined using two-dimensional NMR, simulated annealing, and restrained molecular dynamics. Only DNA residue 7T in the 2’-OMe-RNA•DNA junction adopted an O4'-endo sugar conformation, while the other DNA residues including 3C in the DNA•2’-OMe-RNA junction, adopted C1'-exo or C2'-endo conformations. The observed NOE intensity of 2’-O-methyl group to H1’ proton of 4am at the DNA•2’-OMe-RNA junction is much weaker than those of 5am and 6am. The 2’-O-methyl group of 4am was found to orient towards the minor groove in the trans domain while the 2’-O- methyl groups of 5am and 6am were found to be in the gauche (+) domain. In contrast to the long-lived water molecules found close to the RNA adenine H2 and H1’ protons and the methyl group of 7T in the RNA-DNA junction of (d(CGC)r(aaa)d(TTTGCG))2, there were no long-lived water molecules found for (d(CGC)r(amamam)d(TTTGCG))2. This is probably due to the hydrophobic enviroment created by the 2’-O-methylated riboadenines in the minor groove. Comparing with (d(CGC)r(aaa)d(TTTGCG))2, the melting temperature of (d(CGC)r(amamam)d(TTTGCG))2 increases from 48.5□C to 51.9□C. Since no long-lived water molecules were found close to the 2’-O-methylated RNA adenine H2 and H1’ protons in the hybrid segment and in the methyl group of 7T in the RNA-DNA junction, the increase of melting temperature may be solely due to the hydrophobic interactions between substituents in the minor groove. The characteristic structural features and hydration patterns of this chimeric duplex provide a molecular basis for further therapeutic applications of DNA•RNA hybrid and chimeric duplexes with 2’-modified RNA residues. Chapter 2 : Solution Structure of the Domain III of the Japanese Encephalitis Virus Envelope Protein The flavivirus envelope protein is the dominant antigen in eliciting neutralizing antibodies and plays an important role in inducing immunologic responses in the infected host. We have determined the solution structure of the major antigenic domain (domain III) of the Japanese Encephalitis Virus (JEV) envelope protein. The JEV domain III forms a β-barrel type structure composed of six antiparallel β-strands resembling the immunoglobulin constant domain. We have also identified epitopes of the JEV domain III to its neutralizing antibody by chemical shift perturbation measurements. The NMR result was also confirmed by site-directed mutagenesis experiments and western blot analysis (performed by Chih-Wei Wu). Our study provides a structural basis for understanding the mechanism of immunologic protection and for rational design of vaccines effective against flaviviruses. Chapter 3 : Solution Structure of the Hypothetical Protein HP0495 from Helicobacter Pylori Helicobacter pylori known as gastric pathogenic bacteria are able to cause digestive illnesses including gastritis and peptic ulcer disease. Their genomes have been completely sequenced. The next step of genomic studies after the yielding of the complete genome sequence of the species is to identify both cellular and molecular function of each gene in the genome. The HP0495 gene of H. pylori encodes a hypothetical protein of 86 amino acid residues with a molecular weight of 10,193 Da and a calculated isoelectric point of 8.7. HP0495 has four similar sequences referred to as “hypothetical protein” by PSI-BLAST analysis and no identifiable sequence homology to well-characterized proteins. In our present studies, structural studies of HP0495 were carried out using CD and NMR. Solution structure analysis revealed that the overall fold of this protein consists of three β strands and three □ helices. The HP0495 is classified as typical “□ and β (□ + β)” fold, arranged in a□β□□□□□β□β□□□topology□□□□Moreover, the protein-protein interaction map of the H. pylori showed that HP0495 interacts strongly with HP1205. Since HP1205 is strongly similar to translation elongation factor EF-Tu, HP0495 may be involved in the protein synthesis. Knowledge of the structure of H. pylori protein will help the proteomic studies of the roles of H. pylori in human diseases.