臺灣博碩士論文加值系統

在所有多細胞真核生物中，細胞核和細胞質內蛋白質的O連結 beta-N-乙醯基葡萄糖胺在絲胺酸或蘇胺酸殘基的動態醣化作用是很常見的。足夠的證據顯示，此種醣化作用(又名O-乙醯基葡萄糖胺醣化)調控著許多細胞生理過程，包括細胞核內的輸入現象，蛋白質的轉譯，基因的轉錄以及細胞骨架的形成。此外，越來越多的研究顯示，O-乙醯基葡萄糖胺醣化結合磷酸化以共價性修飾來調節許多蛋白質的生理功能。在此篇論文中，我們合成一段alpha螺旋髮夾胜肽(alpha螺旋/轉彎/alpha螺旋)，並利用此胜肽當作一模擬系統去探討醣化作用如何影響胜肽的性質。一分子的 beta-N-乙醯基葡萄糖胺加至於轉彎區域中蘇胺酸殘基的羥基上。我們將此醣化胜肽在水溶液中及在脂雙層中的性質和未醣化比較。二維核磁共振光譜顯示O-乙醯基葡萄糖胺醣化會顯著地影響此胜肽在水溶液中的局部構形。我們也利用了減弱式全反射傅立葉轉換紅外線光譜和氮十五標定固態核磁共振光譜來檢測是否醣化作用會藉由改變螺旋位向來將訊息傳遞過磷脂膜。

The dynamic glycosylation of serine or threonine residues on nuclear and cytosolic proteins by an O-linked beta-N-acetylglucosamine (O-GlcNAc) is abundant in all multicellular eukaryotes. Accumulating evidence implicates this kind of glycosylation (also named O-GlcNAcylation) in the regulation of numerous cellular processes, including nuclear import, protein translation, gene transcription and cytoskeletal formation. Here we synthesized an alpha-helical hairpin peptide (alpha-helix/turn/alpha-helix) and used this peptide as a model system to explore how the glycosylation affects the properties of the peptide. One beta-N-acetylglucosamine was added to the hydroxyl group of the threonine residue in the turn region. The properties of the glycosylated peptide in the aqueous solution and in the lipid bilayer were compared with those of the wild-type peptide. The analysis of 2-D NMR spectra indicates that O-GlcNAcylation substantially affects the local conformation of the peptide in aqueous solution. We also utilized attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and 15N labeled solid state NMR technique to examine if the glycosylation can transfer signals across the membrane by altering the helix orientation.

Contents
Abbreviations i
謝誌 iv
Abstract v
中文摘要 vi
Chapter 1 Introduction
1.1 Glycosylation of proteins 1
1.2 Possible roles of glycosylation 6
1.3 The aim of this project 10
Chapter 2 Materials and Methods
2.1 Materials
2.1.1 Water 15
2.1.2 Chemicals 15
2.1.3 Buffer solutions 18
2.2 Methods
2.2.1 Peptide synthesis, purification, and identification 19
2.2.2 Two-dimensional NMR spectroscopy in aqueous solution 19
2.2.3 Fluorescence quenching 20
2.2.4 Attenuated total reflection Fourier transform
infrared spectroscopy 22
2.2.5 Solid-state NMR spectroscopy 23
Chapter 3 Results and Discussion (I): Purification and Identification of Peptides
3.1 Results of peptide purification by HPLC 26
3.2 Identification of alpha-helical hairpin peptide
by mass spectrometry 29
Chapter 4 Results and Discussion (II): Conformation and Orientation Studies of the alpha-turn-alpha Peptide and the Glycosylated alpha-turn-alpha Peptide
4.1 The effect of glycosylation on the structure in solution
4.1.1 Two-dimensional NMR spectroscopy in aqueous solution 32
4.2 Examining the insertion of the peptide into the membrane
4.2.1 Tryptophan fluorescence quenching by aqueous quenchers 38
4.2.2 Introduction to Solid-state NMR spectroscopy 40
4.2.3 Using solid state NMR to study the conformation of the alpha-helical hairpin peptide in phospholipid bilayers 41
4.3 Measuring the helix tilt angle
4.3.1 Introduction to Attenuated Total Reflection Fourier Transform InfraRed spectroscopy (ATR-FTIR) 47
4.3.2 How to measure the angle of helix axis to membrane normal 51
4.3.3 The overall helix tilt angle determined by ATR-FTIR 57
Conclusions and Future Outlooks 65
References 67

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