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研究生:周俊吉
研究生(外文):Chun-Chi Chou
論文名稱:鋅指蛋白與核酸結合之結構特徵與嵌合模擬研究
論文名稱(外文):Structure characterization and docking study of three-Cys2His2 zinc-finger protein in complex with DNA
指導教授:陳金榜
指導教授(外文):Chinpan Chen
學位類別:博士
校院名稱:國防醫學院
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:116
中文關鍵詞:核磁共振鋅指蛋白核酸辨識複合體模型
外文關鍵詞:NMRzinc finger proteinDNA recognitioncomplex model
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  • 被引用被引用:0
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  • 下載下載:12
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摘要
X射線晶體學和核磁共振光譜是眾所周知用以決定生物大分子三維結構的高解析度方法,提供原子等級的資訊用以瞭解蛋白質與核酸的相互作用。在本論文中,我們主要是進行核磁共振實驗,用以深入瞭解睪丸鋅指蛋白與核酸的結合特性。此外,我們也提供電腦模擬計算的方法以獲得鋅指蛋白複合體模型。

[第一章]
小鼠睾丸鋅指蛋白C端鋅指結構區域(TZD)與aurora-C家族的Aie1基因啟動子上5'-TGTACAGTGT-3'的結合具有很高的特異性。有趣的是,鋅指結構區域的序列很獨特,具有TGEKP和GAAP兩個不同的連接序列,並且在每個鋅指蛋白預期的核酸結合位點具有不同的殘基,尤其是鋅指蛋白3。自由態鋅指結構區域的核磁共振結構顯示,每個鋅指蛋白形成一個典型的ββα摺疊。而關於與核酸結合方面,鋅指蛋白3的化學位移擾動及R2的橫向弛豫率明顯小於鋅指蛋白1和2,這表示鋅指蛋白3與核酸結合的親和力較弱。此外,鋅指結構區域與同源序列的核酸或是突變核酸結合的化學位移擾動顯示,鋅指蛋白3與核酸的結合與序列無關,值得注意的是,當鋅指結構區域的連接序列由GAAP突變成TGEKP時,鋅指蛋白3與核酸結合的化學位移擾動卻沒有明顯的變化,這進一步表示,鋅指蛋白3在特定序列的核酸辨識上並沒有扮演關鍵角色。鋅指結構區域與核酸序列專一以及非序列專一結合同時發生的模式提供相當價值的訊息,用以更加地了解蛋白質與核酸的識別。

[第二章]
運用核磁共振光譜和X射線晶體學的方式決定蛋白質與核酸的複合體結構在許多情況下依然充滿挑戰。高度模糊限制條件(restrains)所驅動的嵌合模擬(HADDOCK)是一種以訊息驅動的嵌合模擬,並且已經成功地被運用在許多不同結合模式的蛋白質核酸複合體,然而纏繞於核酸的鋅指蛋白具有複雜的結合幾何構形,因此決定模糊交互作用的限制條件是很關鍵的。在這項研究中,我們合理地在個別區域選擇具有良好幾何分佈的兩個限制條件用以產生複合體模型。根據對已知晶體結構複合體所做的模擬研究結果,我們獲得鋅指蛋白-核酸複合體模型的建構方法。將相同的建構方法運用在未知複合體的Sp1蛋白,用以產生複合體模型,由此種方法計算出的複合體模型,其蛋白質與核酸的交互作用與之前報導的實驗結果吻合。因此,根據我們的嵌合模擬結果顯示,對於具有三個鋅指結構區域蛋白的未知複合體結構,其蛋白以纏繞核酸的方式結合,經由在個別區域選擇具有良好幾何分佈的兩個限制條件足以用來產生精確的複合體模型。
Abstract
It is well known that X-ray crystallography and NMR spectroscopy are high resolution methods used to determine the 3D structure of biomacromolecule and provide atomic-level information to understand the protein-DNA interaction. In this dissertation, we mainly carried out NMR experiments to gain insight into the binding characteristic on testis zinc finger protein. Besides, we also provide a computational approach to attain complex models for zinc finger proteins.
[Chapter 1] The C-terminal three-Cys2His2 zinc-finger domain (TZD) of mouse testis zinc-finger protein binds to the 5’-TGTACAGTGT-3’ at the Aie1 (aurora-C) promoter with high specificity. Interestingly, the sequence of TZD is unique, possessing two distinct linkers, TGEKP and GAAP, and distinct residues at presumed DNA binding sites at each finger, especially finger 3. NMR structure of the free TZD showed that each zinc finger forms a typical ββα fold. On binding to DNA, chemical shift perturbations and the R2 transverse relaxation rate in finger 3 are significantly smaller than those in fingers 1 and 2, which indicate that the DNA binding affinity in finger 3 is weaker. Furthermore, the shift perturbations between TZD in complex with the cognate DNA and its serial mutants revealed that the DNA binding in finger 3 is sequence independent. Remarkably, the shift perturbations in finger 3 on the linker mutation of TZD (GAAP mutated to TGEKP) were barely detected, which further indicates that finger 3 does not play a critical role in DNA sequence-specific recognition. The DNA sequence and nonsequence-specific bindings occurring simultaneously in TZD provide valuable information for better understanding protein–DNA recognition.
[Chapter 2] Determination of protein–DNA complex structures with NMR and X-ray remains challenging in many cases. High Ambiguity-Driven DOCKing (HADDOCK) is an information-driven docking program that has been used to successfully model many protein–DNA complexes. Defining the ambiguous interaction restraints (AIRs) for three-Cys2His2 zinc-finger proteins that wrap around DNA is critical because of the complicated binding geometry. In this study, the complex models we generated on the basis of two AIRs with a good geometric distribution in each domain are rational. We derived the modeling approach for generating three-Cys2His2 zinc-finger-DNA complex models according to the results of docking studies using crystal complex structures. A newly Sp1–DNA complex model was calculated with this approach and the interactions are in good agreement with those previously reported. Our docking data demonstrate that two AIRs with a reasonable geometric distribution in each of the three-Cys2His2 zinc-finger domains are sufficient to generate accurate complex models for unknown complex structures in which the protein wraps around DNA.
Abstract I
摘要 II
Table of contents III
Abbreviations VI
List of tables VII
List of figures VIII
Chapter 1 1
Structure and DNA binding characteristics of the three-Cys2His2 domain of mouse testis zinc finger protein
1-1 Introduction 2
1-2 Materials and Methods 10
1-2-1 Protein expression and purification 10
1-2-2 Circular dichroism experiment 11
1-2-3 NMR experiments and resonance assignments 11
1-2-4 Structure calculation and analysis 12
1-2-5 Relaxation data analysis 12
1-2-6 Residual dipolar coupling (RDC) analysis 13
1-2-7 HADDOCK docking 13
1-3 Results 15
1-3-1 Sample preparation of recombinant protein 15
1-3-2 Conformational investigation by CD analysis 15
1-3-3 Resonance assignment and solution structure of the free TZD 15
1-3-4 TZD is an authentic DNA binding zinc finger protein 17
1-3-5 Mapping of TZD-DNA interaction by CSP and NOE pattern 18
1-3-6 Dynamics between TZD and TZD-DNA complex 20
1-3-7 Effect of linker mutation on DNA binding 22
1-3-8 Molecular basis of sequence specificity mapped with mutant DNAs 22
1-3-9 The complex model of TZD-DNA generated using HADDOCK 24
1-4 Discussion 56
Chapter 2 60
An effective approach for generating a three-Cys2His2 zinc-finger–DNA complex model by docking
2-1 Introduction 61
2-2 Materials and Methods 68
2-2-1 Starting structure of Zif268, YY1, WT1, Aart, Sp1 protein and DNA 68
2-2-2 Calculation of hydrogen bonds and van der Waals contacts 68
2-2-3 AIRs for docking Zif268, YY1, WT1, Aart and Sp1 69
2-2-4 Geometric distribution analysis of different sets of AIRs 70
2-2-5 Docking procedure 71
2-2-6 Analysis of the complex models 71
2-3 Results and discussion 73
2-3-1 Overview of the docking approach 73
2-3-2 Wrap-around conformation of the complex models for AIR sets 74
2-3-3 Localization of AIRs in the complex models and geometric distribution of AIR sets in the reference structure 75
2-3-4 Analysis of complex models based on AIR energy 76
2-3-5 Comparison of the 10 best complex models 77
2-3-6 Complex modeling of other test cases 78
2-3-7 Complex modeling based on the homology modeled structure 79
2-3-8 An efficient docking procedure to generate a zinc-finger protein–DNA complex model 80
2-3-9 Analysis of Sp1–DNA complex model 82
2-4 Conclusions 99
Reference 101
Appendix 107
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