吳世雄臺灣大學：生化科學研究所吳煜森Wu, Yu-SenYu-SenWu2007-11-262018-07-062007-11-262018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/52808膜融合是釵h生物反應的重要步驟之一。對具有外套膜的病毒而言，寄主的細胞膜與病毒的外套膜之間的融合，是這類病毒感染寄主的第一步。凝血素（Hemagglutinin）是流行性感冒病毒外套表面的醣蛋白，它可使病毒的外套膜和細胞內含體（endosome）的膜融合，內含體中的微酸性環境使得凝血素的結構轉為可產生膜融合的狀態，使病毒的內含遺傳物質得以進入細胞質內。 凝血素的結構可分為兩個弁鈰炩禲GHA1和HA2。HA2的前20個胺基酸序列稱為融合胜肽，其結構主要由第2到第11個序列所形成的Membrane fusion plays an important role in many fundamental biological processes. The infection of enveloped virus such as Influenza virus, membrane fusion between host cell membrane and viral envelope is a key point of these infection processes. Hemagglutinin (HA) is the enveloped glycoprotein of influenza virus which mediates the fusion activity. HA causes the fusion of the viral envelope with the endosomal membrane to enable the passage of the viral genome into the host cytoplasm. The mildly acidic pH in the mature endosome induces the transition of the HA structure into fusogenic state.The first 20 residues of the HA2 domain in HA have been identified as the membrane-fusion peptide, which is the only portion to insert into the target membrane at an oblique angle and thereby triggers membrane fusion. All four membrane fusion peptide analogs carried out in this study were based on the sequence of the E5 fusion peptide analog. The substitution of one proline for either Gly12, Gly13 and for both Gly12 and Gly13 are named G12P, G13P, and monoP, respectively. According to the experimental results, the strict kink which is composed of one proline shows restriction on the rotation of the COOH-terminal residues, thus the little difference in fusion activity are observed in monoP analogs at various pH. The kink composed of one or two Gly is more flexible than the kink composed of only Pro. A flexible kink may allow a greater rotation of COOH-terminal residues, thus the negative charged residues in this region could rotate to against the phophorus group of membranes; the strict kink in monoP restricts the rotation of COOH-terminal residues. The COOH-terminal modification of HGGH shows little influence at its fusion activity. This may indicate that even the COOH-terminal helix exists at neutral pH, the rotation of residues in COOH-terminus are not suitable for fusion activity.摘 要 2 ABSTRACT 3 I. INTRODUCTION 4 1.1 INFLUENZA VIRUS 4 1.2 HEMAGGLUTININ 4 1.3 FUSION PEPTIDE OF HEMAGGLUTININ 7 II. MATERIALS AND METHODS 10 2.1 SAMPLE PREPARATION 10 2.2 SAMPLE PURIFICATION 12 2.3 CIRCULAR DICHROISM (CD) EXPERIMENTS 13 2.4 FLUORESCENCE MEASUREMENTS 14 2.5 NUCLEAR MAGNETIC RESONANCE SPECTRUM (NMR) EXPERIMENTS 15 2.6 STRUCTURE CALCULATION 17 III. RESULTS 19 3.1 SAMPLE PREPARATION AND PRODUCT PURIFICATION 19 3.2 CONFORMATIONAL CHANGES AT DIFFERENT PH VALUES 19 3.3 THE INSERTION DEPTH OF TRYPTOPHAN AT DIFFERENT PH VALUES 20 3.4 RESONANCE ASSIGNMENTS AND SECONDARY STRUCTURE DETERMINATION 21 IV. DISCUSSION 24 V. APPENDIX 28 VI. REFERENCE 541233864 bytesapplication/pdfen-US膜融合胜肽Membrane FusionStructureotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52808/1/ntu-93-R91242032-1.pdf