臺灣博碩士論文加值系統

在本研究中，我們探討了人體纖維母細胞生長因子的熱力學性質及及骨架動態。人體纖維母細胞生長因子是一個約 16 kDa的蛋白質，它參與了器官發育，血管生成，及細胞生長等過程，因此，它也與癌細胞的生成有關。為了探討人體纖維母細胞生長因子在活體外的穩定性，我們將此蛋白質置於會造成蛋白質開散的環境中，如提高外界環境溫度或加入變性劑（如尿素），並觀察蛋白質的螢光（fluorescence）光譜及圓二色（circular dichroism）旋光光譜的變化，我們發現此蛋白不僅在高熱環境下會開散，在接近冰點時也有開散的趨勢。此性質我們亦由二維1H-15N HSQC核磁共振（NMR）光譜證實。我們亦探討人體纖維母細胞生長因子在自由狀態及有配位子(ligand)存在下的骨架動態。我們發現在自由狀態下的人體纖維母細胞生長因子可以進行不同形態（conformation）間的轉換，但一旦與配位子結合，結構就變得比較剛硬（rigid），我們相信這是因為蛋白質為了減少在沒有活性（inactive）及具有活性（active）結構間能階差所作的設計。此外，位於103至111的殘基在有配位子（如SOS）的存在下的自由度變得比較大。氫—氘交換（hydrogen-deuterium exchange）實驗可以幫助我們了解在自然狀態下蛋白質的整體（global）及區域性（local）結構在毫秒(millisecond)至數日間的運動情形。我們發現人體纖維母細胞生長因子的12個b摺板並不是一致活動的，b摺板 IV, V, VIII 和 IX具有區域性的運動。此發現有助於我們解釋蛋白質的折疊的相關問題。

In this study, the thermodynamic and backbone dynamics properties of human acidic fibroblast growth factor (hFGF-1) are described. hFGF-1 is a ~ 16 kDa, all b-barrel protein which plays key roles in several important cellular processes related to morphogenesis, development and angiogenesis. The thermodynamic parameters characterizing the conformational stability of the human acidic fibroblast growth factor (hFGF-1) have been determined by isothermal urea denaturation and thermal denaturation at fixed concentrations of urea using fluorescence and far-UV CD circular dichroism (CD) spectroscopy. Temperature denaturation experiments in the absence and presence of urea show that hFGF-1 has a tendency to undergo cold denaturation. Two-dimensional 1H-15N HSQC spectra of hFGF-1 acquired at sub zero temperatures clearly show that hFGF-1 unfolds under low-temperature conditions. To describe the internal motions of hFGF-1 ranging from pico- to millisecond, 15N NMR relaxation data have been used to characterize the backbone dynamics of hFGF-1 in its free and sucrose octasulfate (SOS) bound states. Significant conformational exchange (Rex) is observed for several residues in the free form of the protein. However, the conformational exchange behavior of hFGF-1 is tremendously reduced upon SOS binding. Also, the receptor-binding segment comprising residues 103-111 shows increased flexibility in the presence of SOS. To investigate the internal motions of hFGF-1 range from millisecond to days, hydrogen-deuterium exchange experiments in the absence and low concentrations of denaturant were performed. In contrast to the equilibrium unfolding events monitored by optical probes, native-like state hydrogen exchange data shows that the beta-trefoil architecture of hFGF-1 does not behave as a single cooperative unit. There are at least two structurally independent units with differing stabilities in hFGF-1. Beta-strands I, II, III, VI, VII, X, XI and XII fit into the global unfolding isotherm. By contrast, residues in beta-strands IV, V, VIII and IX exchange by the sub-folding isotherm and could be responsible for the occurrence of high-energy partially unfolded state(s) in hFGF-1. The work described here elucidates the correlation between the structural dynamics with biological function of hFGF-1. This work also proves that the slow exchanging residues in hFGF-1 do not represent the folding nucleus of the protein.

Chapter 1 Introduction ...……………………………….…………….. 1
1.1 A physical picture of protein motions …………...…………………….... 1
1.2 Protein stability ...……………………………………………………….… 5
1.2.1 Solvent Denaturation of Proteins …………………………………… 7
1.2.2 1.2.2 Thermodynamics of Protein Folding ……………………...… 10
1.2.3 Heat Capacity and Cold Denaturation Phenomenon of Proteins ….. 13
1.3 Dynamic parameters of proteins ……………………………………...…. 20
1.3.1 Time Windows for Nuclear Magnetic Resonance (NMR) ………… 21
1.3.2 Relaxation Parameters …………………………………………… 24
1.3.3 Spectral Density Function and the Model-free Formalism ………... 31
1.3.4 Backbone Dynamics and Biological Function of Proteins ………... 45
1.4 Hydrogen-deuterium exchange ………………………………………….. 47
1.4.1 Hydrogen-Deuterium Exchange at Equilibrium …………………... 49
1.4.2 EX1 versus EX2 limit ……………………………………………... 51
1.4.3 Hydrogen-deuterium Exchange Mechanisms …………………….. 54
1.5 Fibroblast growth factors ………………………………………………... 60
Chapter 2 Expression and Purification of Human Acidic Fibroblast Growth Factor …………………………………………… 65
2.1 Clone construction of hFGF-1 …………………………………………... 65
2.2 Expression of hFGF-1 …………………………………………………… 67
2.2.1 Expression of hFGF-1 ……………………………………….. 67
2.2.2 Expression of 15N-labeled hFGF-1 ………………………………... 68
2.3 Purification of hFGF-1 …………………………………………………... 69
Chapter 3 Thermodynamic Properties of hFGF-1 ………………… 74
3.1 General Properties of hFGF-1 …………………………………………... 74
3.2 Equilibrium Unfolding Experiments ……………………………………. 75
3.3 Stability of hFGF-1 with pH …………………………………………….. 76
3.4 Urea-induced Unfolding of hFGF-1 …………………………………….. 80
3.4.1 Two-state Unfolding of hFGF-1 ……………...…………………… 80
3.4.2 Noncooperative Unfolding of hFGF-1 …………………………….. 84
3.5 Thermal Denaturation of hFGF-1 ……………………………………….. 87
3.5.1 Thermal Denaturation of hFGF-1 at Various pH ………………….. 87
3.5.2 Stability Curve and Evidence of Cold Denaturation of hFGF-1 ….. 90
3.6 NMR Evidence for Cold Denaturation ………………………………………… 98
Chapter 4 Backbone Dynamics of hFGF-1 ……………………….. 101
4.1 Interactions of hFGF-1 with Sucrose Octasulfate (SOS) ……………… 101
4.2 Measurement of the Relaxation Parameters for Free and SOS-bound hFGF-1 …………………………………………………………….…… 105
4.2.1 Longitudinal (R1) and Transverse (R2) Relaxation Rates ………… 112
4.2.2 {1H}-15N NOE Data ……………………………………………… 115
4.3 Estimation of the Overall Correlation time tm …………..…………….. 116
4.4 Analyzing the Relaxation Parameters Using the Modelfree Formalism 121
4.4.1 Motions on the Microsecond-Millisecond Time Scale …………... 125
4.4.2 Estimation of the Generalized Order Parameter …………………. 129
4.4.3 Effective Correlation Time te ……………………………………. 133
4.5 Dynamics and FGF-Receptor Interaction Sites ……………………….. 135
4.6 Comparison of the Backbone Dynamics of Interleukin 1b and hFGF-1 136
Chapter 5 Stability of hFGF-1 Measured by Hydrogen-Deuterium Exchange …………………………………………...….. 137
5.1 Measurement of Hydrogen-deuterium Exchange Rates of hFGF-1 ….... 137
5.1.1 NMR Spectroscopy of 15N-labeled hFGF-1 at Various Concentrations of Urea ………………...………………………………………... 137
5.1.2 H/D Exchange Experiments ………………..………………...……138
5.1.3 Analysis of Amide Proton Exchange Rates ………………....…….138
5.2 Equilibrium Unfolding of hFGF-1 ………………………………………144
5.3 H/D Exchange of hFGF-1 at Native State ……………………………... 146
5.4 Correlation between Temperature Coefficient and Protection Factor …. 156
5.5 H/D Exchange and FGF-Receptor Interaction Sites …………………… 160
5.6 H/D Exchange of hFGF-1 in the Presence of Urea ……………………. 163
5.6.1 Global, Local and Subglobal Unfolding in hFGF-1 ……………. 167
5.6.2 Discrepancies between DGex and DGu ………………………….. 175
5.7 Relationship with Folding Events ……………………………………… 176
Future Perspectives ………………………………………………… 181
References …………………………………………………………… 182
Publication List ……………………………………………………... 199

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