Xanthmonas campestris pv. campestris 未知功能蛋白之NMR結構解析

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研究生：	林富揚 Fu-Yang Lin
論文名稱：	Xanthmonas campestris pv. campestris 未知功能蛋白之NMR結構解析 NMR Structure Determination of Proteins of Unknown Function In Xanthmonas campestris pv. campestris
指導教授：	周三和 Shan-Ho Chou 黃雪莉 Shir-Ly Huang
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
畢業學年度：	93
語文別：	英文
論文頁數：	115
中文關鍵詞：	Bartar 、PKS 、BolA 、核磁共振、結構基因體、蛋白質結構
外文關鍵詞：	Structural genomics, protein structure, nmr, BolA, PKS, Barstar
相關次數：	點閱：16 下載：0
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快速成長的序列資料庫留下許多未被註解功能的基因。蛋白質的三級結構決定了它的功能。因些，決定一個未知功能蛋白質的結構可以幫助我們預測它的功能。Xanthomonas campestris 結構基因體計畫是以鑑定出具有新穎結構及功能的蛋白為目標。Xanthomonas campestris 為一株感染十字花科造成十字花科黑腐病之植物病原菌，造成農業上的重大損失。X. campestris 的基因體中共被預測出 4182 個 ORF, 其中1474個基因未被註解有任何功能。我們利用高場核磁共振技術(NMR)來解析未知功能蛋白的結構及預測其功能。在本論文中，共研究了3個未知功能蛋白: XC975, XC5375, 和XC1692。XC975是一個由逆境誘發的 morphogen。在Pfam中屬於BolA Family。BolA蛋白在 E. coli的生理功能已有初步研究。當BolA表現時會誘發下游基因的表現，使細菌轉變為圓形的型態。XC975結構和E. coli的BolA和mouse的BolA-like蛋白有很類似的結構，但彼此序列的相似度不高(~30%)，它們的功能推測和DNA的結合有關。XC5357被註解為 tetracenomycin polyketide synthesis protein，但活性未知。比對了XC5357的折疊，我們找出兩個和XC5357相關的結構:一為 oxalate decarboxylase，一為 mannose-6-phosphate isomerase。然而這是兩個很不同的酵素，進行不同的化學反應。必須進行更多生化的試驗後，才可對XC5357的功能做進一步解釋。而XC1692也是一個未知功能蛋白。由初步NMR和生物資訊分析，發現它很可能屬於一個ribonuclease Sa inhibitor—barstar。同樣地，XC1692和barstar有很低的序列相似度，但XC1692具有類似 barstar和ribonuclease結合的保留性胺基酸，且俱相同的二級結構。不同的是XC1692在N端多了具有二級結構的40個胺基酸。 Pull-down assay 和 chemical perturbation experiment 確定它可與X. campestris genome中的ribonuclease Sa (XC1691) 蛋白結合。

The rapidly growing sequence database results in a large number of genes remaining to be annotated. Since the biological function of a protein is determined by its three-dimensional structure, it is thus reasonable to believe that the unknown function of a protein can be predicted from its determined structure.
A structural genomic project of the plant pathogen Xanthomonas campestris has been initiated to identify and characterize the structures and functions of unknown proteins. Xanthomonas campestris is a gram-negative bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. Among the 4182 ORFs predicted in the Xanthomonas campestris pv. campestris str. ATCC 33913 genome, 1474 of the them have no assigned function. Three proteins XC975, XC5357, and XC1692 have been studied using high field NMR in this work. XC975 is a hypothetical protein classified as a stress-induced morphogen in the COG database. The determined structure of XC975 is similar to that of mouse BolA-like protein that exhibits its function through binding with DNA. The determined fold of XC5357 was found to correlate with two very different enzymes, an oxalate decarboxylase and a mannose-6-phosphate isomerase. More functional assays are needed to further determine and confirm its function. XC1692 is another hypothetical protein with a suggested function of ribonuclease inhibitor adopting a barstar-like (secondary) structure. Its potential target was identified and a pull-down assay and a chemical perturbation experiment are used to confirm its interaction with the ribonuclease Sa like protein, XC1691 in the X. campestris genome.

Chapter1. Introduction	1
1-1 Structural Genomics Project of Xanthomonas campestris pv.campestris	1
1-2. Background of the Targeting Genome	2
1-3 Protein NMR in Structural Genomics	2
1-4 High-Throughput NMR Structure Determination	4
Chapter2. Materials and Methods	5
2-1 Target Selection	5
2-2 NMR Sample Preparation	6
2-2-1 XC975	6
2-2-2 XC5357	6
2-2-3 XC1692	7
2-3 NMR Data Collection and Data Processing	9
2-4 Backbone Assignments	10
2-4-1.Resonance Assignments	10
2-4-2. Backbone resonance assignment by triple resonance experiments	11
2-4-3. Automated Backbone Resonance Assignment By AutoAssign Program	12
2-4-5. Protocols of Automated Backbone Assignment Using Sparky and	17
AutoAssign	17
2-4-4. Validation of Manual and AutoAssign assignment Results	17
2-5 Side-chain Chemical Shift Assignment	18
2-5-1 Side-chain Chemical Shift Assignment	18
2-5-2 Stereospecific Assignment	20
2-6 Secondary Structure Determination	21
2-7 Distance Constraints, NOESY Assignment and Structure Calculation	23
2-7-1 Principles	23
2-7-2 Automated NOESY Assignment and Structure Calculation	25
2-8 Additional Restraints	26
2-8-1 Hydrogen Bond Restraints	26
2-8-2 Phi Angle Restraints from HNHA Experiment	27
2-8-3 TALOS Derived Torsion Angle Restraints	27
2-8-4 Other Types of Restraints	28
Chapter3. Results and Discussions	30
3-1 Solution Structure Determination of XC975: A Putative DNA Binding Protein Belonging to BolA Family	30
3-1-1 Introduction	30
3-1-2 Primary sequence analysis	32
3-1-3 Chemical Shift Assignment	33
3-1-4 Secondary Structure Determination, H-bond restraints and Torsion Angle Restraints	34
3-1-5 NOESY Assignment and Structure Calculation	34
3-1-6 Structural Description	35
3-1-7 Discussion	35
3-2. Fold determination of XC5357: A Domain-Swapped Dimer of Tetracenomycin Polyketide Synthesis Protein Adopted the Cupin Fold?	38
3-2-1 Introduction	38
3-2-2 Primary sequence analysis	39
3-2-3 Chemical Shift Assignment	40
3-2-4 Secondary Structure Determination, H-bond restraints and Torsion Angle Restraints	41
3-2-5 NOESY Assignment and Structure Calculation	41
3-2-6 Structure Description	42
3-2-7 Discussions	43
3-3. NMR Analysis and Bioinformatic Survey of XC1692:	45
A Conserved Hypothetical Protein Turns out to Be A	45
Barstar-like Ribonuclease Inhibitor?	45
3-3-1 Introduction	45
3-3-2 Chemical Shift Assignment	46
3-3-3 Secondary Structure Determination, H-bond restraints and Torsion Angle Restraints	46
3-3-4 NOESY Assignment and Structure Calculation	47
3-3-5. Primary Sequence and Secondary Structure Analysis	47
3-3-6 Chemical Shift Perturbation	49
3-3-7 Discussion	49
References	51

                                

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