臺灣博碩士論文加值系統

離子對作用力是穩定蛋白質重要的作用力之一，穩定離子對作用力主要由帶相反電荷的氨基酸所形成，例如:Arg/Lys and Asp/Glu。為了深入了解離子對作用力的形成以及它們在蛋白質結構中扮演的腳色，我們設計了一個β-hairpin的胜肽系統，測量離子對作用力對β-hairpin結構穩定性的影響。在此，我們研究了側鏈長度對cross-strand diagonal離子對作用力的影響。胜肽透過固相合成，並用反相HPLC純化。根據二維核磁共振光譜(TOCSY，ROESY和DQF-COSY)，確認胜肽所形成的β-hairpin結構並計算出β-hairpin摺疊比例以及自由能。結果顯示β-hairpin的穩定性: HPDAadDap> HPDGluDab～HPDAadDab>HPDGluDap> HPDAspDab>HPDAspDap。即側鏈較長的胺基酸離子對，形成最穩定的β-hairpin 結構。
雙螺旋結構是在自然界中常發現的結構單元，對於其序列和穩定性之間的關係有相當了解，因此常被用來做為研究蛋白質二級和三級作用力的理想系統。許多研究顯示將含氟氨基酸(疏水胺基酸的類似物)併入蛋白質疏水核心的結構中， 可穩定此蛋白質結構。這種穩定通常被稱為fluoro-stabilization effect。為了進一步了解fluoro-stabilization effect對蛋白質穩定性的影響，我們將含氟氨基酸併入一雙螺旋結構胜肽的疏水核心結構中,此一胜肽是根據轉錄因子GCN4設計的序列。

Ion pairing interactions play important roles in protein structure stability. Stabilizing ion pairing interactions are formed between two oppositely charged residues such as Arg/Lys and Asp/Glu. To gain insight into ion pairing interactions and their potential roles in protein structure, we have designed a β-hairpin peptide system, allowing the measurement of the stability effects of individual charged residues and ion pairing interactions. Herein, we study the effect of side chain length on cross-strand diagonal ion pairing interactions.
Peptides were synthesized by solid phase methods and purified by reverse phase HPLC. The sequence specific assignments were obtained based on TOCSY, ROESY, and DQF-COSY spectra. The β-hairpin structures were confirmed by chemical shift deviation, 3JHNα coupling constants, and NOE signals. The fraction folded and ΔGfold of the peptides were derived by comparing the chemical shifts with the folded and unfolded reference peptides. The stability of the peptides followed the trend : HPDAadDab ~ HPDAadDap > HPDGluDab ~ HPDGluDap > HPDAspDab ~ HPDAspDap. The results indicate that pairing the longer side chains provides the most stable β-hairpin.
Coiled coil is a common motif found in nature. These motifs are ideal models for the study of protein secondary and teriary inteactions because the relationship between sequence and stability are well-understood. Incorporation of fluorinated amino acid residues into coiled coil peptides can stabilize coiled coils. This stabilization has been referred to as the fluoro-stabilization effect. To further understand the fluoro-stabilization effect in proteins, peptides based on the leucine zipper region of the yeast transcription factor GCN4 was designed and fluorinated amino acids were incorporated at the hydrophobic positions.

Table of Contents
誌謝 i
中文摘要 iii
Abstract iv
Table of Contents vi
List of Figures xi
List of Tables xii
List of Schemes xiii
Abbreviation xiv
Chapter 1. Introduction 1
1-1. Proteins 2
1-2. Hierarchy of Protein Structure 4
Primary Structure 4
Secondary Structure 5
Tertiary Structure 6
Quaternary Structure 6
1-3. Driving Force of Protein Folding 7
1-4. Thesis Overview 8
1-5. References 9
Chapter 2. Effect of Side Chain Length of Charged Residues on a Diagonal Ion Pairing Interaction in a β-Hairpin 13
2-1. Introduction 14
β-Sheets Structrues 14
β-Sheet Propensity 15
β-Turns 16
Cross-Strand Interactions 17
β-Hairpins 18
Double Mutant Cycle 19
2-2. Results and Disscussion 21 Peptide Design 21
Peptide Structure Analysis by 2D-NMR Spectrometry 25
Characterization of the β-Hairpin Structues: Sequence Specific Assignment
and Chemical Shift Dispersion 25
Characterization of the β-Hairpin Structues: Chemical Shift Deviation 26
Characterization of the β-Hairpin Structues: 3JHNα Coupling Constant 28
Characterization of the β-Hairpin Structues: Nuclear Overhauser Effect
(NOE) 33
Characterization of the β-Hairpin Structues: β-Hairpin Stability 49
Diagonal Cross-Strand Interaction Energy 51
2-3. Disscusion 52
2-4. Conclusion 54
2-5. Future Aspects 54
2-6. Acknowledgements 54
2-7. Experimental Section 55
General Materials and Methods 55
Peptide Syntheis 56
Nuclear Magnetic Resonance Spectroscopy 66
3JNHα Measurement 77
Distance Determination by NOE Intergrations 77
Chemical Shift Deviation 77
Free folding energy 78
Double mutant cycle 78
2-8. Refernces 80
Chapter 3. Ion-Pairing Interaction of β–Hairpin Structure 85
3-1. Introdution 86
Coiled Coils in Biological Systems 86
GCN4 Coiled Coil Motif 87
Coiled Coil Sequence and Structure 88
Knobs-Into-Holes Interactons of a Dimeric Coiled Coil 90
Coiled Coil Stability 91
Coiled Coil with a Fluorous Core 94
Chapter Overview 96
3-2. Results 96
Synthesis of Fmoc-hexafluoroleucine 96
3-3. Discussion 100
3-4. Experimental Section 100
3-5. References 102
NMR Spectrum 106
Appendix A. Effect of Positively Charged Amino Acid Side Chain Length on β-Hairpin Stability 108
A-1. Chemical Shift Deviation 109
A-2. NOE Assignments 110
A-3. Wuthrich Diagrams 113
A-4. 3JHNα Coupling Constant 115
A-5. Fraction Folded and ΔGfold 116
A-6. 1H Chemical Shift Assignment 118
License for Reprint of Figure 121

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