本論文試圖以核磁共振光譜的技術，對核苷酸和蛋白質的結構進行瞭解，全文共包含以下三個部份：
第一章：經過化學修飾的反序列(antisense)核苷酸是具有發展潛力的治療藥物，而在2'羥基(hydroxyl group)上進行修飾的寡核糖核苷酸，更能提高穩定度、和ＲNA的親和力及提高對核苷酶的抗力，故被視為相當有前景，可抑制基因表現的抗癌藥物。本篇實驗中吾人利用二維核磁共振光譜，模擬升溫焠熄(simulated annealing)及設限分子動態模擬(restraint molecular dynamics)之分析方法，決定2'羥基以甲基化修飾的DNA/RNA嵌合雙股螺旋 [d(CGC)r(amamam)d(TTTGCG)]2 之水溶液三度空間立體結構。該結構顯示只有DNA/RNA交界處的第七胸腺嘧啶(thymine, 7T)甲基，其糖基結構為O4'-endo，其餘核苷酸包括DNA/RNA交界處的第三胞嘧啶(cytosine, 3C)，其糖基結構皆如同一般DNA分子為所常見C2'-endo或C1'-exo。利用NOE光譜發現，2'羥基甲基化的第四胞嘧啶(adenine, 4am)和分子內的H1'原子的NOE訊號強度，遠小於2'羥基甲基化的第五、六胞嘧啶(adenine, 5am、6am)和分子內的H1'原子的NOE訊號強度。此結果顯示，第四胞嘧啶(adenube, 4am)的甲基採取trans的構形並朝向次溝槽（minor groove)，而第五、六胞嘧啶(adenine, 5am、6am)的甲基採取gauche(+)的構形。在分子的水合現象(hydration pattern)的研究上，不同於DNA/RNA嵌合雙股螺旋 [d(CGC)r(amamam)d(TTTGCG)]2，特定的水合訊號會分別出現在次溝槽的腺嘌呤(adenine）H2及H1'原子以及主溝槽(major groove)的第七胸腺嘧啶(thymine, 7T)甲基附近，甲基化的DNA/RNA嵌合雙股螺旋 [d(CGC)r(amamam)d(TTTGCG)]2 則沒有明顯的水合訊號，可能的原因在於，2'羥基做甲基化之後，產生了一個疏水性的空間，阻止的水分子的交互作用，或者是2'羥基甲基化之後，使得螺旋結構中間部分的次溝槽之寬度變大，使得水分子無法長時間的停留。此外，在熱穩定性的測試方面，經過2'羥基甲基化後的嵌合雙股螺旋，Tm(melting tempature)可由48.5℃上升至51.9℃，符合之前的研究所示，2'羥基做甲基化確實可以提高熱穩定性。由以上的實驗可以得知，2'羥基甲基化後的嵌合雙股螺旋結構特性和水合現象特徵，此項發現將有助於DNA/RNA嵌合雙股螺旋和2'羥基上進行修飾的寡核糖核苷酸，在治療藥物上的進一步研究開發。
第二章：黃病毒屬(flavivirus)的封套蛋白(envelope protein)在其致病機制上具有相當重要的地位，舉凡與宿主細胞受器的結合，病毒本身的複製，或是酸性環境下與宿主細胞的融合皆與封套蛋白有所關聯。封套蛋白更是引出宿主細胞抗體的主要抗原，在免疫反應上更是扮演了舉足輕重的角色。本篇實驗中利用核磁共振光譜的技術，解出日本腦炎病毒封套蛋白第三區塊(domain III of Japanese encephalitis envelope protein)的水溶液三度空間立體結構。日本腦炎病毒封套蛋白第三區塊，形成了β-barrel形式的結構，包含了六個反向平行的β-strand，其結構類似於免疫球蛋白的不變區(constant region)。為了進一步找出日本腦炎病毒封套蛋白第三區塊的抗原決定位置(epitope)，吾人利用了化學位移擾動(chemical Shift perturbation)的方式，發現殘基302-312，322-339，360-372 及385-392的位置有相當大的化學位移，此結果顯示β2-β3，β3-β4和β5-β6的loop區域和抗體(mAb E3.3)的結合有相當大的關聯。而後此實驗結果，經由本實驗室吳致緯同學利用單點突變(site-directed mutagenesis)配合西方墨點法(Western Blot)而得到了進一步的確認。經由本實驗所得到的日本腦炎病毒封套蛋白第三區塊的水溶液三度空間立體結構及抗原決定位置，將有助於了解免疫反應的機制，並對黃病毒屬(flavivirus)的疫苗研發提供了有價值的資訊。
第三章：幽門螺旋桿菌(Helicobacter pylori)是相當為人所熟悉的致病菌，和胃炎、消化性潰瘍等消化道的疾病，均有相當密切的關係。而在1997年幽門螺旋桿菌的基因序列已被完整解出，而在完整序列被解出之後，下一步的發展重點及在於如何利用現有的資訊，進一步對不同細胞和分子的功能進行了解。幽門螺旋桿菌的HP0495蛋白是一個86個氨基酸，分子量10193道耳吞，等電點8.7，但生物功能未知的假設性蛋白質(Hypothetical protein)，經由psi-blast資料庫的搜尋分析，結果顯示僅有四筆和HP0495序列相似度較高的蛋白質出現，但此四個蛋白質亦均為生物功能未知的假設性蛋白質。本篇實驗中吾人利用旋光儀(CD)和核磁共振(NMR)光譜的技術，決定了HP0495的水溶液三度空間立體結構。HP0495的結構包括了三個β-strand和三個α-helix(排列順序βααββα)，形成(α+β)的折疊方式。此外，由蛋白質交互作用結果顯示，HP0495和HP1205有很強的交互作用，而HP1205和轉譯延長因子(translation elongation factor,. EF-Tu)的序列具有高度相似，故猜測HP0495可能參與了蛋白質的合成步驟，經由本實驗所得到的HP0495的三維結構，希望將有助於對幽門螺旋桿菌有進一步的瞭解。

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.

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