臺灣博碩士論文加值系統

除了X-光晶體繞射外，核磁共振技術是另一個被廣為運用來決定生物巨分子構造的一項利器。在本篇論文裡，我們表現並純化出大量所欲探討的重組蛋白質，利用碳-13與氮-15同位素來標定這些重組蛋白，以旋光光譜及異核多維核磁共振技術來探討這些蛋白質的結構與其功能的相關性，內容如下：
[第一部分]
微精漿蛋白被發現存在於許多不同的物種，此蛋白質的特色是不含醣基且具有多對雙硫鍵，但其胺基酸序列保留度並不高。根據我們所得的NOEs發現，豬的微精漿蛋白所形成的雙硫鍵與先前以質譜技術分析出駝鳥蛋白的雙硫鍵位置不一致；此外，旋光光譜顯示出豬跟人類的微精漿蛋白結構皆具有相當高的熱及化學穩定性。透過分析核磁共振光譜所得到的1018個設限(restraints)，我們成功地解出了豬微精漿蛋白的三級結構，其主要藉由全為β-摺板組成的N端與C端區塊(domain)所形成，除了利用此兩區塊間長距 NOEs來確定其立體結構計算外，我們也運用殘存磁偶矩的設限(RDCs)加以證實這兩個區塊所組成的三級立體結構。根據豬微精漿蛋白表面的電荷分布推測，這兩個區塊間的作用力主要應透過其表面電荷相互吸引所造成。我們所解出的豬微精漿蛋白三級結構，是微精漿蛋白質成員裡第一個被決定出的蛋白質結構，同時透過DALI、CATH及 CE等方式搜尋目前已知的蛋白質資料庫，證實這個豬微精漿蛋白質的三級結構是一個全新的立體結構。
[第二部分]
Lon蛋白酶的功用主要是負責清除細胞中結構摺疊錯誤或受損的蛋白質，已知位於Lon蛋白酶的ATPase上alpha區塊可辨識其受質蛋白，也可與去氧核糖核酸結合。在我們所探討的兩個Lon蛋白酶alpha區塊研究結果中，旋光光譜顯示出alpha區塊蛋白的二級結構及熱穩定性都很容易受到pH值的影響而有非常大的變化；在pH值為5.8的條件下，我們解出了枯草芽孢桿菌(Bacillus subtilis) Lon蛋白酶alpha區塊的三級結構，其主要由四段α-螺旋與一對β-摺板所組成。分析其所形成的三級結構，我們發現這個alpha區塊蛋白並不屬於常見的去氧核糖核酸結合結構，如helix-turn-helix或winged-helix，不過經電泳凝膠遲緩分析(gel mobility shift assay)與核磁共振光譜中化學位移的擾動實驗結果顯示，此一alpha區塊蛋白確實可與去氧核糖核酸結合。根據核磁共振光譜發現，位於第三段α-螺旋與其所連接的區段所聚集形成的表面正電荷群，應是這些alpha區塊主要負責結合去氧核糖核酸的位置，同時藉由幾個不同物種的alpha區塊表面電荷分布的比較，我們推測其他的Lon蛋白酶alpha區塊也應具有相似的去氧核糖核酸結合能力。

It is well known that NMR, in addition to X-ray crystallography, is another powerful technique used to determine the 3D structure of biomacromolecule. In this dissertation, several recombinant unlabeled as well as 15N- or 15N/13C-labeled proteins were expressed and purified in large quantities. We then carried out circular dichroism (CD) and heteronuclear multidimensional NMR experiments to gain insight into the structure-function relationships on these proteins, as described below.
Part I. β-microseminoproteins (MSPs), identified from diverse species, are all non-glycosylated and disulfide bond-rich, but show a relatively low level of conservation. Although all Cys residues are conserved, the disulfide bond pairings of porcine MSP determined based on NOEs are different from those of ostrich MSP derived based on mass spectrometric analysis. CD titration spectra revealed that both porcine and human MSPs are thermally and chemically stable. The solution structure of porcine MSP determined on the basis of 1018 restraints showed that it exhibits a β-sheet-rich structure with two distinct domains, an N-terminal domain consisting of one double-stranded and one four-stranded antiparallel β-sheets, and a C-terminal domain consisting of two double-stranded antiparallel β-sheets. The orientation of the two domains was derived mainly on the basis of long-range NOEs and verified using residual dipolar coupling data. A number of charged residues were found in close proximity between the two domains, indicating that electrostatic interaction may be the key contact between the two domains. Also, structure of porcine MSP is the first 3D structure reported among all MSPs, and contains a novel fold according to the structural comparison using DALI, CATH, and CE methods.
Part II. Lon protease is mainly responsible for eliminating misfolded or damaged proteins in cells. A small α-domain, localized in the sub-domain of the ATPase domain for Lon, is thought to carry the substrate-recognition and DNA-binding sites. CD spectra revealed that secondary structure and structural stability of Lon α-domains are dramatically altered at extreme pH values. Solution structure of the Bacillus subtilis Lon α-domain at pH 5.8 is consisted of four α-helices and a short two-stranded parallel β-sheet. Although this α-domain contains neither the canonical helix-turn-helix motif nor the winged-helix motif, as usually observed in the DNA-binding protein, it was confirmed that this α-domain indeed binds with DNA based on gel mobility shift assay and NMR shift perturbation experiment. It is suggested based on NMR data that the positively charged residues in α3 helix and in the both loops connecting with α3 are the major sites responsible for DNA binding. Structural comparisons of various Lon α-domains further revealed that other Lon α-domains are very likely to possess similar DNA-binding characteristics.

Table of Contents
List of Abbreviations………………………………………………………….……i
List of Tables……………………………………………………………………..…ii
List of Figures……………………………………………………………………...iii

Part 1:
Solution Structural Studies of β-Microseminoproteins
1-1. Introduction………………………………………………………….……….2
1-2. Materials and Methods…………………………………………………….8
1-2-1. Preparation of porcine and human MSPs…………………………………8
1-2-2. Circular dichroism (CD) experiment…………………………………….10
1-2-3. NMR experiments and resonance assignments………………………….11
1-2-4. Residual dipolar coupling analysis………………………………………13
1-2.5. Structure calculation and analysis………………………………………..14
1-2-6. Relaxation data analysis…………………………………………………15
1-3. Results………………………………………………………………...………20
1-3-1. Conformational stability based on CD analysis of pMSP and hMSP……20
1-3-2. Comparison of NMR data and resonance assignments…………………20
1-3-3. Disulfide pairings and proline cis-trans conformation………………….21
1-3-4. Secondary structure and amide proton exchange rate……………….....22
1-3-5. Structure determination for pMSP………………………………………23
1-3-6. Structure description for pMSP…………………………………………24
1-3-7. Backbone dynamics analysis……………………………………………26
1-3-8. Structural similarity search………………………………………………27
1-4. Discussion……………………………………………………………………57
Part 2:
Structure and DNA-binding of the Bacillus subtilis Lon protease α-domain
2-1. Introduction…………………………………………..……………………..71
2-2. Materials and Methods…………………………………………………...76
2-2-1. Protein expression and purification……..………………………………76
2-2-2. Circular dichroism (CD) experiment………..…………………………77
2-2-3. NMR experiments and resonance assignments…………………………78
2-2-4. Structure calculation and analysis……………………………………….79
2-2-5. Relaxation data analysis…………………………………………………79
2-2-6. Protein-DNA interactions monitored by NMR…………………………80
2-2-7. Homology model…………………...……………………………………80
2-3. Results………………………………………………………………...………83
2-3-1. Conformational stability based on CD analysis of Lon α-domains…..…83
2-3-2. Resonance assignment and secondary structure…………………………83
2-3-3. Structure determination……………………………….…………………85
2-3-4. Backbone dynamics analysis………….…………………………………86
2-3-5. Mapping of interactions between Bs-Lon α-domain and DNA…………87
2-4. Discussion………………………………………..…………………………106

References…………..……………………………………………………………112
Appendix……………...……………………………………………..................…119

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