Studying Solution Structure and Dynamics of a Multi-domain Molecular Chaperone by a Hybrid Approach

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研究生：	黃芝婷 Huang, Chih-Ting
論文名稱：	Studying Solution Structure and Dynamics of a Multi-domain Molecular Chaperone by a Hybrid Approach
指導教授：	呂平江 Lyu, Ping-Chiang
口試委員:	徐駿森 Hsu, Chun-Hua 徐尚德 Hsu, Shang-Te Danny
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	76
中文關鍵詞：	Trigger Factor 、Trigger Factor 、核磁共振、小角度散射
外文關鍵詞：	Trigger Factor, Trigger Factor, NMR, small angel scattering
相關次數：	點閱：2 下載：0
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Trigger factor (TF) is a 49 kDa molecular chaperone that is highly conserved in bacteria and is the first to interact with newly synthesized polypeptide chains. TF binds to the ribosomal tunnel exit through the N-terminal ribosome binding domain (RBD), and together the peptidyl-prolyl cis/trans isomerase (PPIase) domain, and together with the C-terminal domain (CTD), to forms a “crouching dragon”-like cradle to facilitate folding of nascent chains as they emerge from the ribosome.
Recent studies have established that TF exists in a three-state equilibrium with the ribosome: as the free TF is in a monomer-dimer equilibrium and the TF-bound form with the ribosome in a 1:1 stoichiometry. Comparison of several TF crystal structures reveals large domain motions, which are particularly observed in pronounced for the PPIase domain. Unlike most molecular chaperones, e.g., Hsp70, Hsp90 and GroEL/GroES, the chaperone activity of TF is independent of ATP hydrolysis, which is the common energy source to trigger conformational rearrangements during substrate binding. The flexibility based on the different domain architectures in TF is used for the substrates binding, and therefore saying their biologically importance.
Here we display the development of a hybrid approach based on the combination of NMR spectroscopy and small angle X-ray scattering (SAXS) techniques to characterize the domain architecture of TF in solution and the dynamics thereof. Our long-term goal is to apply such approach to study TF about its co-translational folding. Importantly, the approach should be generally applicable to structural studies on multi-domain, high molecular weight proteins.

我們所研究的蛋白稱為Trigger Factor (TF)，在多數細菌中具高度保留性。此蛋白大小為49 kDa，並可分成三個區段，N端的ribosome-binding domain (RBD)、C端的chaperone domain （CTD），及peptidyl-prolyl cis/trans isomerase domain （PPIase）。其在生物中主要扮演的角色為輔助新合成胺基酸序列摺疊成三級結構，或是控管細胞內蛋白質的品質。藉RBD domain結合核醣體於新合成胺基酸序列的通道出口，TF蛋白成為新生蛋白質第一個接觸的輔助摺疊蛋白，其形成似搖籃的口袋，在通道口等待幫助新合成胺基酸的摺疊。除了TF蛋白，仍有其它下游輔助摺疊的蛋白，但它們與TF蛋白最大不同，在於TF蛋白利用改變本身結構即可幫助約65-80%的蛋白完成摺疊。
最近研究中發現，TF蛋白在水溶液中具有三態平衡，可以1:1的比例與核糖體結合、自行形成二聚體及維持單體。另外，對於TF蛋白結構研究上，發現在不同狀態下的TF蛋白，如有結合或沒有結合核醣體的TF蛋白，疊合它們的結構，發現各domain之間具有約10度角的旋轉。所以這篇論文主要研究TF蛋白水溶液中的動態運動，觀察其在水溶液中如何形成二聚體，再進一步探討TF蛋白與新合成蛋白序列之間如何作用以幫助摺疊。我們主要是利用核磁共振（NMR）技術研究。然而，我們所研究的分子對NMR而言是屬於大分子量蛋白質，在一般的方法下，會得到解析度較低的光譜而難以分析，因此，我們希望發展出一套由NMR技術結合其他技術，如小角度散射（SAXS）、residual dipolar coupling (RDC)、paramagnetic relaxation enhancement（PRE）…等方法，發展一套觀察大分子蛋白質在水溶液中動態表現的新研究方法，進而應用在其他蛋白質上，突破以往NMR技術只能研究小分子蛋白（≤ 30kDa）的限制。

ABSTRACT .............................................................................................................................. 4
INTRODUCTION ................................................................................................................ 8
1. THE BIOLOGICAL FUNCTION OF TF ................................................................................. 9
2 THE STRUCTURE AND DYNAMICS OF TF ......................................................................... 10
2.1. TF provides a cradle for its folding substrates ...................................................... 11
2.2. The conformational flexibility of TF ...................................................................... 11
2.3. Three-state equilibrium in solution ........................................................................ 12
MATERIAL AND METHODS ......................................................................................... 16
1. PROTEIN CONSTRUCT DESIGNS ...................................................................................... 16
1.1. Domain constructs, expression vectors and E. coli expression system ................. 16
1.2. Site-directed mutagenesis of TF ............................................................................ 17
1.2.1. Design strategy for I-to-V and L-to-V mutations ......................................................................... 19
1.2.2. The primer sequence design ......................................................................................................... 19
1.2.3. The condition and procedure of PCR experiment ........................................................................ 21
2. PROTEIN EXPRESSION .................................................................................................... 22
2.1. Unlabelled protein expression ............................................................................... 22
2.1.1. Small-scale expression tests ......................................................................................................... 22
2.1.2. Large-scale expression and purification for sample production .................................................. 22
2.2. Uniform and selective isotope labeling strategy .................................................... 23
2.2.1. Minimum medium of uniform 13C/15N labeling ........................................................................... 23
2.2.2. Uniform protein deuteration ......................................................................................................... 24
2.2.3. Selective protein side-chain methyl group protonation ................................................................ 25
3. PROTEIN PURIFICATION ................................................................................................. 27
3.1. His-tag affinity purification ................................................................................... 27
3.2. Size-exclusion chromatography (SEC) .................................................................. 27
4. BIOPHYSICAL AND BIOCHEMICAL CHARACTERIZATION OF TF ..................................... 28
4.1. The oligomeric state of TF ..................................................................................... 28
4.1.1. SEC ............................................................................................................................................. 28
4.1.2. Analytical ultracentrifugation (AUC) .......................................................................................... 28
4.1.3. Glutaraldehyde crosslinking assay ............................................................................................... 29
4.2. Circular dichroism spectroscopy .......................................................................... 30
4.2.1. In-house CD ................................................................................................................................ 30
4.2.2. Synchrotron radiation CD (SR-CD) ............................................................................................. 30
4.3. Solution state nuclear magnetic resonance (NMR) spectroscopy ......................... 31
4.3.1. Principle of multidimensional NMR spectroscopy ...................................................................... 31
4.3.2. Analysis of NMR data .................................................................................................................. 32
4.4. Small-angel X-ray scattering (SAXS) .................................................................... 33
4.4.1. Principle of SAXS ........................................................................................................................ 33
4.4.2. Analysis and modeling of SAXS data .......................................................................................... 34
RESULT .............................................................................................................................. 36
1. OVERVIEW OF I-TO-V AND L-TO-V MUTANTS OF TF .................................................... 36
2. SMALL SCALE EXPRESSION TESTS OF DIFFERENT TF CONSTRUCTS .............................. 36
3. PURIFICATION OF DIFFERENT TF CONSTRUCTS ............................................................. 37
3.1. Culture in Luria-Bertanl medium .......................................................................... 37
3.2 Culture in D2O M9 minimal medium ...................................................................... 40
4. BIOPHYSICAL AND BIOCHEMICAL CHARACTERIZATION OF TF ..................................... 43
4.1. Analysis oligomerisation state of TF by SEC ........................................................ 43
4.1.1.Protein molecular weight calibration (analytical column and preparative column) ..................... 43
4.1.2. Analysis of the oligomeric state of TF by concentration-SEC ..................................................... 45
4.1.3. Inter-domain Interactions between different TF constructs ......................................................... 48
4.2. Analysis oligomerisation state of TF by AUC ....................................................... 49
4.3. Analysis oligomerisation state of TF by glutaraldehyde crosslinking (A) ............. 50
4.4. Comparison of TF variants by in-house CD and SR-CD spectroscopy ................ 51
4.5. Solution state NMR spectroscopy .......................................................................... 53
4.5.1. Spectral comparison of different domain constructs .................................................................... 53
4.5.2. Backbone assignments of TF-113-432 ......................................................................................... 54

4.5.3. Comparison of I-to-V and L-to-V mutants .................................................................................. 56
4.6. SAXS ....................................................................................................................... 57
4.6.1. Analysis and modeling of TF variants ......................................................................................... 57
DISCUSSION ...................................................................................................................... 60
1 ANALYSIS OLIGOMERISATION STATE OF TF ................................................................... 60
2 THE SOLUTION STATE NMR SPECTROSCOPY ................................................................. 61
3 THE ENVELOPE SHAPE OF FULL-LENGTH DIMER FORM .................................................. 62
SUPPLEMENTARY INFORMATION ........................................................................... 65
1 VECTOR SYSTEM MAPS ................................................................................................... 65
2 THE ELUTION VOLUME OF SEC ...................................................................................... 67
3 THE DATA FOR THE AUC EXPERIMENTS ........................................................................ 69
REFERENCES: ................................................................................................................... 73

                                

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