陳長謙臺灣大學：化學研究所黃人則Huang, Jen-TseJen-TseHuang2007-11-262018-07-102007-11-262018-07-102004http://ntur.lib.ntu.edu.tw//handle/246246/51797蛋白質只有位於自然狀態下才會表現出正常的弁遄C有關初期動力學的研究，對於了解蛋白質開始摺疊的情形，扮演著關鍵性的角色。在本計劃中，我們的目的是要建立一種新的方法來模擬蛋白質摺疊的初期現象(可快至數個奈秒)。 這個新的方法是利用一個對光不穩定的化合物來環化胜肽，使之成為非自然的狀態。我們發展出一種對光不穩定物（Br-AcCMB），利用其高量子產率（ΦCh = 0.72）的優點，我們環化了數種不同種類的胜肽至非自然狀態，來研究在早期蛋白質摺疊時，各種不同結構單元的形成。利用紫外光脈衝雷射，我們快速地(~10-12 s)打斷此一對光不穩定連結物，再使用光聲波熱卡計【photoacoustic calorimetry】或是光熱雷射折射儀【photothermal beam deflection spectroscopy】，追蹤整個摺疊動力學。而無論是利用光聲波熱卡計（從奈秒至數微秒），或是光熱雷射折射儀（從微秒至數毫秒），整個時間的解析度都大幅提昇了。 我們成左漲X成出數種環化的胜肽，其中包括了含有三種不同轉彎序列的β摺板型胜肽(c-E12C, c-P6DE12C, and c-19merE11C)，以及一種β髮夾型胜肽(c-D6C)，和兩種Proteins do not perform their proper functions unless they can reach their native states. Studies of the early kinetic events play a key role because they provide a means to explore the beginning of the free energy landscape of protein folding. The aim of the project is to develop a new method to monitor the early kinetic events in protein folding (as fast as a few nanoseconds). This methodology involves the deployment of a photolabile linker to “cage” a polypeptide chain to form a non-native state. In this thesis, we have developed a photolabile compound, Br-AcCMB, to serve as the cage. Taking advantage of the high quantum yield of this photolabile linker (ΦCh = 0.72), we have caged several different peptide models in their non-native states in order to address certain fundamental issues related to the formation of structural motifs during the early stages of protein folding. By the use of a pulse UV laser (~10-12 s), we can break the photolabile linker and monitor the refolding process by using photoacoustic calorimetry (PAC) or photothermal beam deflection (PBD). The time resolution is greatly improved by the use of PAC (from nanoseconds to a few microseconds) and PBD (from a few microseconds to several milliseconds). We have synthesized various caged peptides including three b-sheets with different turn sequences (c-E12C, c-P6DE12C, and c-19merE11C), one b-hairpin (c-D6C), and two a-helices (c-VHPM12C and c-aE9C). By the use of nuclear magnetic resonance spectroscopy (NMR) and circular dichroism spectroscopy (CD), we have studied the structural alteration of these caged peptides before and after photolysis. Except for the b-hairpin model c-D6C that apparently has a slow folding rate, the folding kinetics of the other caged peptides all fall within the time resolution of PAC. After de-convolution, the time constant of the individual refolding process could be obtained. Temperature dependent PAC measurements were carried out in order to obtain certain thermodynamic parameters, including the volume change (ΔV), enthalpy change (ΔH), and kinetic parameters, including the activation energy (Ea). Kinetic measurements using PBD on the peptide c-D6C are still on going. Because cyclization constraint limits the initial state of the caged peptide to a subset of conformational space, our approach does not allow us to monitor the global refolding. However, it has the distinct advantage of allowing one to study a system beginning from a well-defined state. Specifically, this method allows direct observation and comparison of the structural change of the peptides folding with different driving forces or nucleation centers (such as the nucleation at the turn or by hydrophobic interaction) from the same initial state in real time.Table of Contents i Acknowledgements iv Abstract vi Chinese Abstract vii Abbreviation x Chapter 1: The Protein Folding and its Significance 1 1.1 Introduction 2 1.2 The Nature of Protein Folding 2 1.3 The Folding Models 4 1.4 Methods fot the Rapid Triggering of Protein Folding 7 1.4.1 Temperature Jump Methods 9 1.4.2 CO Flash PHotolysis 11 1.4.3 Photoreduction of the Cytochrome c Heme 12 1.4.4 pH Jump Methods 12 1.5 Significance of Protein Folding 13 1.5.1 The Role of Protein Folding 13 1.5.2 Some Disease Related to Incorrect Protein Folding 14 Chapter 2: Synthesis of Photolabile Linkers based on Benzoin 19 2.1 Introduction 20 2.2 Caging Strategies and the Development of a Photolabile Linker 23 2.2.1 The Side-Chain Cage Strategy 26 2.2.2 The Head-to-Side Chain Cage Strategy 29 2.3 Material and Methods 32 2.3.1 General 32 2.3.2 Synthesis of Br-AcCMB 33 2.3.3 Synthesis of Linker Derivative 36 2.3.4 Quantum Yield Determination of the Photolysis Reaction of Br-AcCMB 37 2.4 Results and Discussion 38 Chapter 3: Monitoring the Folding Kinetics of "Caged Peptides" 43 3.1 General Strategy to Synthesize the "Caged Peptides" 44 3.1.1 Standard Procedures of the Synthesis 44 3.1.2 Improvements on the Synthesis 46 3.2 Materials and Methods 48 3.2.1 General 48 3.2.2 Solid Phase Peptide Synthesis 50 3.2.3 Purfication of Caged Peptides 51 3.2.4 Identification of the Caged Peptides by Mass Spectroscopy 52 3.2.5 Circular Dichoism Spectroscopy 52 3.2.6 NMR Spectroscopy 52 3.2.7 Laser-Flash Photolysis and Photoacoustic Calorimetry 53 3.3 Monitoring the Peptide Refolding Process 54 3.3.1 Photoacoustic Calorimetry (PAC) 54 3.3.2 Photothermal Beam Deflection (PBD) 58 Chapter 4: Refolding of b-Sheets Directed by the Turn Formation 61 4.1 Introduction 62 4.1.1 Two Designed Peptides including WT-20mer and P6D Mutant 63 4.1.2 Experimental Design 65 4.2 Material and Methods 67 4.2.1 Synthesis of Wild Type and Caged Peptides 67 4.2.2 Purification and Identification 68 4.2.3 Circular Dichroism Spectroscopy 68 4.2.4 Photoacoustic Calorimetry 68 4.3 Results 69 4.3.1 Purification and Identification of Caged Peptides 69 4.3.2 Examination the Structural Althertion of the Peptides after Cyclization by CD Spectroscopy 69 4.3.3 Examination the Structure Alteration of the Peptides after Cyclization by NMR Spectroscopy 74 4.3.4 Spontaneous Hydrolysis 75 4.3.5 Refolding Kinetics of the Peptide by Photoacous Calorimetry 78 4.4 Discussion 84 Chapter 5: Peptide Refolding Nucleated by Hydrophobic Collapse 87 5.1 Introduction 88 5.1.1 The Small b-hairpin from the Protein G B1 Domain 88 5.1.2 The Small b-sheet 19mer 89 5.2 Material and Methods 92 5.2.1 Synthesis of Wild Type and Caged Peptides 92 5.2.2 Circular Dichroism Spectroscopy 93 5.2.3 Photoacoustic Calorimetry 93 5.3 Results 69 5.3.1 Purification and Identification of c-D6C and c-19merE11C 94 5.3.2 Examination the Structural of the Caged Peptides 98 5.3.3 Refolding Kinetics of c-D6C and c-19merE11C 101 5.4 Discussion 110 Chapter 6: Refolding Dynamics of Peptides Containing a-Helical Structure 111 6.1 Introduction 112 6.1.1 The Villin Headpiece Subdomain 112 6.1.2 The Single a-helix from the ata Peptide 113 6.2 Material and Methods 114 6.2.1 Peptide Synthesis and Caged Peptide Sythesis 114 6.2.2 Purification and Identification 115 6.2.3 Circular Dichoism Spectroscopy 115 6.2.4 Photoacoustic Calorimetry 115 6.2.5 Photothermal Beam Deflection 116 6.3 Results 117 6.3.1 Purification and Identification 117 6.3.2 Examing the Structural Alteration of Caged Peptides after Photolysis 117 6.3.3 Refolding Kinetics of c-VHPM12C and c-aE9C 120 6.4 Discussion 131 Chapter 7: Overall Summary 134 Reference 140en-US光熱射折射儀光聲波熱卡計蛋白質蛋白質摺疊胜肽photoacoustic calorimetrypriteinphotothermal beam deflectionprotein foldingpeptidethesis