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研究生:李銘哲
研究生(外文):Ming-Che Lee
論文名稱:介白素-8相似物之結構研究
論文名稱(外文):Structural study of an interleukin-8 analogue
指導教授:程家維
指導教授(外文):Jya-Wei Cheng
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:英文
論文頁數:57
中文關鍵詞:介白素-8核磁共振
外文關鍵詞:interleukin-8NMR
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摘要
介白質-8(Interleukin-8, IL8)為一72個胺基酸組成,分子量約為8.5千道耳吞的蛋白質。由於其具有引起白血球化學傾向的能力,能誘導發炎反應,因此IL8和許多發炎性疾病有關連。John Gordon教授在近年發現,對IL8進行K11R/G31P兩個點突變後,此突變的IL8相似物對IL8具有功能上的頡抗作用(antagonism)。因此我們決定利用核磁共振儀,對此相似物進行結構上的分析。利用HNCA、HNCO、HN(CO)CA、CBCANH 和 CBCA(CO)NH 光譜,我們可以得到此相似物之骨架序列光譜的前後標定順序關係,而胺基酸上側鏈的氫訊號則是經由TOCSY-HSQC、 HCCH-TCOSY以及HCC(CO)NH等光譜實驗來觀測。除此之外,我們亦完成了氫氘交換的實驗。 利用NOE光譜的共振訊號,氨基上氫氘交換速率快慢,以及化學位移索引(CSI)之統合分析,此相似蛋白質之二級結構可清楚地判斷出來,並得以和前人已研究出的IL8二級結構進行比較,然而結果顯示兩者二級結構並無明顯差異。G31P突變產生局部的結構改變,而我們算出之三維結構和正常IL8結構比較,發現兩者除了在胺基酸30至35間的結構有所改變,其他部分大致相同。因此,此結構上之改變可能就是造成此相似物具頡抗作用功能的關鍵位置。
Abstract
Interleukin-8 (IL8), a kind of CXC chemokines with an ELR motif, was identified in 1987 as a novel type of neutrophil-activating cytokine. IL8 plays an important role in immune system because IL8 can induce inflammation response. IL8 is a molecular weight of 8.5 kDa protein with 72 amino acids. Professor John Gordon had proved that IL8(3-74) K11R/G31P mutation is a potential antagonist. Thus, we used human K11R/G31P as the subject to perform the structural studies. The backbone sequential assignments were accomplished from five triple resonance experiments (HNCA, HNCO, HN(CO)CA, CBCANH, and CBCA(CO)NH). Side chain assignments were obtained from TOCSY-HSQC, HCC(CO)NH and HCCH-TOCSY. Moreover, hydrogen-deuterium exchange experiments were achieved. Secondary structures were identified via NOE correlations, amide proton exchange and CSI analyses. Our results show that the secondary structure is similar to wild type IL8. The G31P mutation induced a local conformational change and the three-dimensional structure roughly displayed the divergent S30-A35 turn of K11R/G31P compared with wild type IL8. Thus, we suggested that this local conformational change is a possible influence making K11R/G31P antagonistic to wild type IL8.
Content
Abstract......................................................................................................I摘要........................................................................................................II List of Figures and Tables......................................................................IV Chapter 1 Introduction.........................................................................1
Cytokines /1
Chemokines /1
Interleukins /3
Interleukin-8 /4
Receptors of ELR+ CXC Chemokines /5
Aims /6
Chapter 2 Materials and Methods.......................................................8
Protein Sample Preparation and Purification
Preparation of Competent Cell /8
Construction of K11RG31P Expression Plasmid /9
Transformation of K11RG31P Expression Plasmid /10
Expression of the Recombinant K11R/G31P /10
Cell Lysis and Purification Procedures /11
NMR Spectroscopy
Protein Sample Condition and General Information /13
NMR Experiments /14
Data Processing and Analysis /15
Structure calculation /16
Chapter 3 Results.................................................................................17
Characterization of K11R/G31P
Expression, Purification and Protein Sample Properties /17
Amino Acid Sequence Alignment of K11R/G31P /18
Resonance Assignment and Structure Calculation
Backbone Assignments /18
Side Chain Assignments /20
Secondary Structure Determination /20
Structure Calculation /22
Chapter 4 Discussions.........................................................................24
Figures.....................................................................................................27
Tables.......................................................................................................47
Reference.................................................................................................54
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