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研究生:羅元超
研究生(外文):Yuan-Chao Lou
論文名稱:布魯頓式酪胺酸激脢SH3區塊及D型肝炎抗原N端白胺酸重複片段之結構,折疊及動力學研究
論文名稱(外文):Structure, Folding and Dynamics of BTK SH3 Domain and the N-terminal Leucine-Repeat Region of Hepatitis Delta Antigen
指導教授:程家維
指導教授(外文):Jya-Wei Cheng
學位類別:博士
校院名稱:國立清華大學
系所名稱:生命科學系
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:2002
畢業學年度:90
語文別:中文
論文頁數:88
中文關鍵詞:核磁共振光譜布魯頓氏酪胺酸激脢SH3區塊折疊
外文關鍵詞:Nuclear Magnetic Resonance SpectroscopyBruton''''s Tyrosince KinaseSH3 domainNOESYFolding
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  • 收藏至我的研究室書目清單書目收藏:1
這篇論文包含了三個部份,第一章是:D型肝炎病毒抗原N端白胺酸重複蛋白片段之結構研究。D型肝炎病毒所製造的蛋白質稱為:D型肝炎抗原,此抗原N端區域內含有許多白胺酸,不僅可與D型肝炎病毒RNA結合,且會抑制D型肝炎病毒的核脢活性。我們之前的研究發現D型肝炎抗原序列24到50這段胜月太,可與D型肝炎病毒RNA結合,且其在含有TFE溶液中的結構為a-螺旋,不過,當溶液中沒有TFE時,其a-螺旋性質卻降到5 %以下;為了增加這段具有生物活性的胜月太在水溶液中的穩定性,我們合成了D型肝炎抗原序列24到38的胜月太,並在N端加入了capping box,經由旋光儀及核磁共振光譜的分析,這段胜月太的a-螺旋性質可提升到64 %,而其結構也顯示了capping box所特有的L形彎曲,這些分子層次上的發現,對於發展D形肝炎的治療而言,提供了有意義的幫助。
第二章是:人體布魯頓氏酪胺酸激脢 (BTK) 的SH3區塊和一段富含脯胺酸的胜月太p120cbl的複合物的結構研究。人體內布魯頓氏酪胺酸激脢中的突變,會造成一種遺傳性的疾病:XLA,其起因是因為突變使得布魯頓氏酪胺酸激脢失去功能,進而使B細胞的分化失敗,造成病人體內的抗體不足而容易受到細菌的反覆感染;在這些突變當中,有一個點突變會造成布魯頓氏酪胺酸激脢SH3區塊C端14個殘基的消失,為了了解布魯頓氏酪胺酸激脢在B細胞發育過程中所扮演的角色,我們進行了布魯頓氏酪胺酸激脢SH3區塊和一段富含脯胺酸的胜月太p120cbl的複合物的結構研究,BTK SH3區塊有五段b-strands互相包傅成兩塊b-sheets而形成了一個b-barrel的結構,對BTK SH3區塊上217-217殘基和p120cbl胜月太6-12的殘基的骨架上的原子來說,結構上誤差的均方根值為 0.87 A (± 0.16 A). 根據化學位移的改變和分子間NOE的觀察,我們發現和這段胜月太進行結合的區域為RT loop,n-src loop及b4和b5之間的helical loop,另外我們也發現這段胜月太是以class I的方位來和SH3區塊進行結合,在我們之前的研究中,我們發現刪除了C端14個殘基的SH3區塊和p120cbl胜月太的結合會減弱,而現在的研究結果提供了很好的解釋,也了解了C端區域對於SH3區塊產生結合時的重要性。
第三章是:布魯頓氏酪胺酸激脢SH3區塊的動力學及折疊研究。布魯頓氏酪胺酸激脢SH3區塊整體的穩定性可利用旋光儀(CD)及螢光(fluorescence)來決定,HD交換實驗及四個溫度下蛋白質骨架的動態研究則可得到各個殘基的穩定度及熱容(Cp);在蛋白質的折疊研究上,則利用stopped-flow CD, fluorescence及 quenched-flow pulse labeling來進行,我們的結果顯示布魯頓氏酪胺酸激脢SH3區塊的折疊是屬於兩階段的過程,而且二級結構和三級結構的形成是同步的,經由熱力學及動力學的研究,其折疊的自由能約等於3.87 ± 0.07 kcal/mol,HD交換實驗的結果指出,b2,b3及b4是最穩定的區域,且其交換的自由能大於整個蛋白質折疊的自由能,這代表在去活性的狀態下,SH3區塊擁有比完全伸展還要來的緊密的結構,這也可以由熱容差的值得到證實;骨架原子間的動態活動所造成的熱容值,在b1,b5及loop區較高,這可以造成布魯頓氏酪胺酸激脢SH3區塊擁有較高的去活性溫度,quenched-flow pulse labeling的實驗則指出b2,b3及b4也是折疊的中心區域,diverging turn及b5接著形成,最後則是b1及RT loop區域。
Chapter 3 : The stability of BTK SH3 domain has been characterized by equilibrium circular dichroism (CD), and fluorescence. Hydrogen-deuterium exchange experiments and backbone dynamics at four temperatures are also performed to monitor the stability and heat capacity at the level of individual residues. The folding kinetics of BTK SH3 domain is investigated with stopped-flow CD, fluorescence and quenched-flow pulse labeling. Our results indicate that BTK SH3 folds via a two-state process and the formation of secondary and tertiary structure are concurrent. The free energy of unfolding, DGUH2O, estimated from guanidine denaturation is 3.87 ± 0.07 kcal/mol, which is in good agreement with those estimated from thermal denaturation, and kinetic experiments. Hydrogen-deuterium (HD) exchange studies of BTK SH3 indicate that the opening process leading to exchange is highly cooperative and b2, b3, and b4 are the most stable regions for exchange. The free energies of exchange (DGHD) of slowly exchanging protons are higher than the free energy of unfolding (DGUH2O). This may indicates that the denatured state might be more compact than the extended conformation, and it is also confirmed from the fact that the DCp value from thermal denaturation is higher than that from empirical calculation. The contributions to the heat capacity (Cp) from fast backbone dynamics have been determined from the temperature dependence of order parameters. The higher mean Cp values of N-terminal b1 and C-terminal b5 at folded state may contribute to the high denaturation temperature of BTK SH3 domain. Quenched-flow pulse labeling experiments indicate that the slow exchange core is also the folding core. b2, b3 together with b4 forms the folding core, and the following protected regions are the diverging turn and the C-terminal b5. N-terminal b1 and first half of RT loop are protected most slowly. From these studies, we know that the deletion on SH3 domain, which loses b4 and b5, have large effects kinetically and thermodynamically.
Abbreviations
Chapter 1 Solution Structure of an N-Capping Peptide from the N-terminal Leucine-Repeat Region of Hepatitis Delta Antigen
Introduction
Materials and Methods
Peptide synthesis, purification, and analysis.
Circular dichroism (CD) spectroscopy.
NMR spectroscopy.
Structural constraints and structure calculations.
Results
dAgCap24-38am forms a stable a-helix in solution.
NMR data of dAgCap24-38am.
NMR evidence of capping.
Solution structure of dAgCap24-38am.
Discussion
Reference
Chapter 2 Solution Structure of the Human BTK SH3 Domain Complexed with a Proline-Rich Peptide from p120cbl
Introduction
Materials and Methods
Protein preparation and purification.
Peptide synthesis.
NMR spectroscopy.
Experimental restraints.
Structure Calculation.
Results
NMR data.
Solution structure of BTK SH3 domain.
Solution Structure of BTK SH3 domain / p120cbl peptide complex.
Discussion
Reference
Chapter 3 Events of Folding and Dynamics of the BTK SH3 Domain
Introduction
Materials and Methods
Protein expression and purification.
Equilibrium CD and fluorescence experiments
Kinetic stopped-flow experiments.
NMR spectroscopy
HD exchange experiments
Backbone dynamics and heat capacities
Quenched-flow pulse labeling
Results
Equilibrium denaturation
Stopped-flow fluorescence and circular dichroism
HD exchange experiments
Temperature dependence of order parameters
Contribution to heat capacity from backbone dynamics
Quenched-flow hydrogen exchange experiments
Discussion
Two-state folding process
The denatured state of BTK SH3 domain
Heat capacity and protein stability
The relationships between kinetics and thermodynamics
Reference
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