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研究生： 亞倫
Arun Gupta
論文名稱： S100之專一性蛋白質間交互作用與結構探討
Understanding Structural Basis for Unique S100 protein-protein Interactions
指導教授： 余靖
口試委員: 余靖
莊偉哲
陳金榜
江昀緯
洪嘉呈
學位類別： 博士
Doctor
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2013
畢業學年度： 102
語文別： 英文
論文頁數： 140
中文關鍵詞： S100B 、FGF2 、ITC 、NMR 、RAGE
相關次數： 點閱：3  下載：0 
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    • 对人类健康的影响的各种疾病状态了解原子和功能的电平信息的S100蛋白 - 蛋白相互作用形成的大分子复合物，以及在下游的信号级联相关的变化，将是非常宝贵。 S100蛋白特异性的基础定义差分行为，与靶蛋白相互作用仍不清楚。大分子复合物的结构洞察S100蛋白蛋白复合物之间的相互作用较弱的情况下，尤其是在中医药的研究，以确定和选择潜在目标的直接后果。要回答这些重要的问题，我们选择， S100B S100A1和S100A6蛋白属于我的研究工作感兴趣的蛋白质S100蛋白家族。

    本论文概述S100A6 - RAGE V域和S100B - FGF2复杂的蛋白质 - 蛋白质相互作用在细胞外空间的结构基础的描述，采用HADDOCK对接方式对接是由两个大分子生物物理信息。结构层次的信息披露在目前的工作都带来了有趣的可能性，这些协会的蛋白质 - 蛋白质复合物的功能后果。

    在第1章中，我们简要地描述人类S100A1和S100A1拮抗剂药物研究发展的新兴权益的生物学作用。实质性的差异而得到的氨基酸序列的S100A1不同物种之间的形式形成的基础着手研究人类S100A1谐振分配。的NMR分配信息在本研究报告将是有益的理解的S100A1和它的潜在的拮抗剂的界面功能。

    在第2章中，我们将重点放在了解S100A6和RAGE V区的残留层次的互动。生物分子核磁共振技术，利用共振分配的突变C3S人类S100A6和表征其与RAGE V区的互动。进一步结合常数突变S100A6与RAGE V域之间由恒温滴定热量的研究。是用来产生HADDOCK异源S100A6 RAGE突变V区复杂的模型。这项研究提供了结构性洞察S100A6配体识别和结合，从而强调目标RAGE受体的治疗方法的潜在作用。

    在第3章中，我们研究非同源的互动S100B与FGF2使用多维核磁共振光谱。我们阐明的界面区域，为S100B FGF2复杂。我们利用计算对接的方法HADDOCK计算S100B FGF2的异源复合基于NMR实验限制。类比推断FGF2停靠S100B四聚体复杂的蛋白质 - 蛋白质接触面和FGF2 -D2域FGFR1互动接口之间，我们推测， S100B蛋白结合位点识别类似的FGF2与FGFR1 D2域。 S100B相关的假说FGFR1抑制FGF2结合位点突变和功能分析研究网站后来被证实。这些结果带来新的视角S100B和FGF2交互的理解，提出一个新颖的角色S100B在FGFR1受体灭活。

    Chapter 1 NMR Resonance Assignment of Human Halo S100A1 1.1 Introduction 1 1.1.1 Background 2 1.1.2 S100A1 Structural features 2 1.1.3 Importance of S100A1 structure determination 6 1.2 Experimental Section 7 1.2.1 Clone construction of S100A1protein 7 1.2.2 Preparation of the 15N and 13C-labeled S100A1 9 1.2.3 Expression and purification of S100A1 10 1.2.4 Sedimentation Velocity Experiments 13 1.2.5 NMR Experiments 13 1.3 Results and Discussion 14 1.3.1 Determination of S100A1 protein Oligomeric form 14 1.3.2 Experiments for backbone and side chain assignment of S100A1 16 1.3.3 NMR Resonance Assignment 26 1.4 Concluding Remarks 28 1.5 References 29 Chapter 2 Interaction of the S100A6 mutant (C3S) with the V domain of the Receptor for Advanced Glycation End Products (RAGE) 2.1 Introduction 32 2.1.1 S100A6 description 32 2.1.2 RAGE receptor 34 2.2 Experimental Section 37 2.2.1 Clone construction of S100A6 protein 37 2.2.2 Expression and characterization of S100A6 38 2.2.3 Expression and characterization of RAGE V domain 41 2.2.4 Preparation of the 15N and 13C-labeled S100A6m and RAGE V domain 44 2.2.5 Resonance Assignments 44 2.2.6 Isothermal Titration Calorimetry 45 2.2.7 1H-15N HSQC titration protocol 46 2.2.8 Size Exclusion Chromatography 47 2.2.9 Docking Studies 48 2.2.10 Cell Culture 50 2.2.11 Reactive oxygen species assay 50 2.3 Results 50 2.3.1 NMR Assignments 50 2.3.2 Binding Thermodynamics 53 2.3.3 Protein-Protein Complex Size determination 2.3.4 Mapping the binding interface 56 2.3.5 Structure of S100A6m-RAGE V domain complex 61 2.3.6 S100A6m-RAGE V domain interface 66 2.3.7 S100A6m induced ROS formation 67 2.4 Discussion 69 2.5 Concluding Remarks 71 2.6 References 72 Chapter 3 E Structural Insights into the Interaction of Human S100B and Basic Fibroblast Growth Factor (FGF2) 3.1 Introduction 77 3.2 Experimental Section 83 3.2.1 Clone construction of FGF2 protein 84 3.2.2 Expression and purification of FGF2 85 3.2.3 Clone construction of S100B protein 87 3.2.4 Expression and purification of S100B 88 3.2.5 NMR Chemical Shift Assignments 91 3.2.6 Isothermal Titration Calorimetry 91 3.2.7 1H-15N HSQC titration protocol 92 3.2.8 Docking Calculations 93 3.2.9 Molecular Dynamics Simulation 94 3.2.10 Cell Culture 95 3.2.11 Cell Proliferation Studies 95 3.2.12 Cell Lysate Extraction 96 3.2.13 Immunoprecipitation and Western Blot 97 3.3 Results 98 3.3.1 NMR ResonanceAssignments 98 3.3.2 Binding Thermodynamics 100 3.3.3 Mapping the S100B and FGF2 Interface 102 3.3.4 Structure of S100B-FGF2 complex 108 3.3.5 Structural stability of S100B-FGF2 complex 114 3.3.6 S100B-FGF2 Interfacial Features 117 3.3.7 Significance of S100B-FGF2 Interface 121 3.3.8 Functional studies with S100B 123 3.3.9 Mutagenesis Studies 127 3.4 Discussion 130 3.5 Concluding Remarks 134 3.6 References 135

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