至今，約有一半的藥物開發是以膜蛋白作為設計標的物，而膜蛋白也佔據了約四分之一的已知基因編碼。因此，了解膜蛋白在天然脂質環境下的結構與功能之關聯是當前藥物開發的重要課題。然而，由於膜蛋白不易研究，成功解析的膜蛋白結構僅佔了已知蛋白質結構的1%。本論文旨在描述利用脂質奈米盤及電子自旋共振(ESR)來研究膜蛋白BsYetJ在天然脂質環境的結構與功能之關聯。近來，ESR及奈米盤的結合已成功用以研究不同的膜蛋白在天然脂質環境下的構形。本論文於第一章中介紹一般性的背景知識。於第二章的研究中，我們利用ESR研究膜蛋白BsYetJ在奈米盤提供的脂質環境中研究其結構與功能之關聯。BsYetJ為Bax inhibitor-1 (BI-1) 在細菌上的同源蛋白，在先前的研究中發現BsYetJ為一可受pH值所調控的鈣離子通道膜蛋白。BI-1為transmembrane Bax inhibitor motif-containing (TMIBM) 膜蛋白家族一員，在不同物種的內質網上作為細胞凋亡的抑制者。我們將BsYetJ重組於奈米盤上並以ESR研究其結構，發現BsYetJ的結構在奈米盤中和在界面活性劑所形成的結晶有所不同。另外，當鈣離子梯度消失時BsYetJ對於pH的敏感度大幅下降。我們的結果顯示BsYetJ為一可受pH調控之電位門控型離子通道蛋白。於第三章中介紹利用自旋標記奈米盤改善ESR解析膜蛋白結構的精細度。自旋標記奈米盤在ESR中訊號極佳，呈現均勻且狹窄的距離分佈。我們將自旋標記奈米盤混入膜蛋白的ESR量測中，藉由其訊號極佳之特性以提升膜蛋白ESR的訊號，也因此改善以往ESR研究膜蛋白結構精確度不足的問題。

Membrane proteins (MPs), though encoded by 25–30% of published genomes, account for more than half of pharmaceutical drug targets. However, less than 1% of unique entries in protein structure databases are MPs. A major challenge in MP studies is to obtain detailed structure in the functional cycle within native-like lipid environment. In this dissertation, the problem of MP structure/function characterization is tackled with spin-label ESR techniques enhanced with lipid nanodiscs (ND). In Chapter 1, general background information of this study is presented. In Chapter 2, we report our study on the structure-function relationship of membrane protein BsYetJ in lipid nanodisc. BsYetJ is a bacterial homolog of Bax inhibitor-1 (BI-1), belonging to the transmembrane Bax inhibitor motif-containing (TMBIM) family. We use ESR to uncover the basis of calcium-leak regulation of BsYetJ at various pH and calcium conditions and illuminate how BsYetJ conducts its function in a nanodisc lipid environment. We show that when transmembrane ion potential is absent, pH change is not sufficient to induce the conformational changes required for the calcium transportation. Several previously unidentified conformations are reported, revealing distinct differences in structure as opposed to those crystalized in detergents. Our findings suggest that BsYetJ operates as a pH-dependent, voltage-gated calcium channel. In Chapter 3, we report the use of spin-labeled ND to improve the structural determination of MP by ESR. The spin-labeled ND show a narrow and homogeneous distance distribution, allowing a distinct improvement in the spectroscopic resolution. With this approach, the determination of MP structures can be studied at high resolution in NDs.

摘要 i
Abstract ii
謝誌 iii
Abbreviations ix
CHAPTER 1 General Introduction 1
1.1 Nanodiscs (NDs) 1
1.2 Apoptosis and the TMBIM protein family 2
1.3 Site-directed spin labeling (SDSL) technique and electron spin resonance (ESR) 6
1.4 Double electron-electron resonance (DEER) 9
References 13
CHAPTER 2 Structure and Regulation of the TMBIM6 Calcium-leak Channel in Lipid Nanodiscs 17
2.1 Chapter abstract 17
2.2 Introduction 18
2.3 Methods 20
2.3.1 Expression, purification, and spin-labeling of BsYetJ 20
2.3.2 Expression and purification of membrane scaffold protein 21
2.3.3 Preparation of BsYetJ in lipid nanodiscs 22
2.3.4 Preparation of nanodiscs containing spin-labeled lipids 23
2.3.5 DEER and cw-ESR measurements 23
2.3.6 Proteoliposome reconstitution and calcium flux assay 24
2.3.7 Determination of DEER-derived structural models of BsYetJ 25
2.4 Results 27
2.4.1 Determination of BsYetJ apo conformation in nanodiscs 27
2.4.2 Dynamic equilibrium between ensembles of two TM7 conformations. 31
2.4.3 Structure of BsYetJ in holo state 34
2.4.4 Study of the highly conserved residue D171 36
2.4.5 Measurements of the calcium flux activity 36
2.4.6 DEER-derived structural models 39
2.5 Discussion 42
2.5.1 BsYetJ in detergent versus lipid nanodisc are structurally different 42
2.5.2 Only in apo (not holo) state is BsYetJ in nanodiscs sensitive to pH 42
2.5.3 Both pH and transmembrane ion potential are critical 43
2.6 Conclusions 44
2.7 Supplementary figures 45
References 57
CHAPTER 3 Doubly Spin-labeled Nanodiscs to Improve Structural Determination of Membrane Proteins by ESR 62
3.1 Chapter abstract 62
3.2 Introduction 63
3.3 Methods 65
3.3.1 Expression, purification, and spin-labeling of BsYetJ 65
3.3.2 Expression, purification, and spin-labeling of MSP 66
3.3.3 Preparation of nanodiscs samples 68
3.3.4 Sample preparation for DEER measurements 69
3.3.5 Separation of the DEER signal contributions from ND+ and MP+ samples 70
3.4 Results and discussion 73
3.4.1 Doubly spin-labeled MSP mutants form stable nanodiscs 73
3.4.2 Changes in ND geometry associated with MP incorporation can be detected 75
3.4.3 ND+ enhances the overall spin signals, hence improving the distance resolution 77
3.4.4 Two conformations of BsYetJ in ND are clearly revealed 77
3.4.5 Structure of BsYetJ in ND is studied at an improved resolution 81
3.5 Conclusions 84
3.6 Supplementary figures 85
References 88

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