Vpr蛋白在人類免疫缺乏病毒1的生命週期中扮演多重角色，例如，Vpr能夠協助預嵌入複合體（pre-integration complex）穿過核膜進入細胞核、反式激活長末端重複（long-terminal repeat）所調節的基因、誘發細胞凋亡以及引發細胞週期停滯於G2期，而這些角色使病毒對細胞的毒性及影響加劇。另外，研究指出Vpr能夠和膜脂質作用，例如，Vpr能在膜上形成陽離子選擇通道、促使膜的通透性增加，並且能有效的將DNA從膜外送入細胞。然而，我們並不清楚Vpr與膜作用的機制為何，以及此作用會受到哪些因素的影響。在過去，為了大量生產Vpr以研究其結構及特性，藉由大腸桿菌表達重組蛋白的方式，因受到細菌停滯效應的影響，產量並不理想。因此在之前的蛋白質結構研究中，主要藉由化學合成的方式製造蛋白質，並因受限其溶解度，結構是在極端的有機溶劑中鑑定。
在此研究中，我們設計了一個利用大腸桿菌表現His-tagged GB1-fused Vpr蛋白的新穎載體，顯著地提升了蛋白質的產量。藉由細菌在攝氏18度、自訂的培養基（defined growth medium）中所產出高達每升10毫克的蛋白質產量，使後續對Vpr的生物化學及生物物理性質的系統性鑑定更加容易。為了更深入了解Vpr與膜之間的作用，我們分析了Vpr在許多不同類膜構造中的整體二級結構，包括在脂疊（bicelle）、微脂體（liposome） 以及利用十二烷基膽鹼（dodecylphosphorylcholine）界面活性劑來形成的微胞（micelle）。另外，在鈣黃綠素釋出實驗與共組裝奈米圓盤實驗中，我們發現Vpr與膜之間的交互作用在含有1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)（DOPG）脂質的情況勝於只含有1,2-dioleoyl-sn-glycero-3-phosphocholine（DOPC）脂質。為了量化Vpr和膜之間的結合強度，我們更進一步的利用石墨烯場效電晶體（graphene based field effect transistor）生物感測器，測得Vpr和含有DOPG的膜之間的解離常數為9.6 ± 2.1 μM。而Vpr與只有DOPC的膜之間的作用，無法量測到顯著的變化，證明Vpr與DOPC之間的作用相對微弱。
在過去，Vpr促使細胞凋亡的現象被認為是來自於Vpr和電壓依賴性陰離子選擇性通道（voltage-dependent anion channel）之間的交互作用，為了更加了解他們的作用強度，我們利用上述生物感測器來定量。當人類電壓依賴性陰離子選擇性通道1（hVDAC-1）置於只含有DOPC脂質的膜時，我們量測到Vpr和hVDAC-1之間的解離常數為5.1 ± 0.9 μM，為其他鑑定提供了參考依據。
在細胞膜中膽固醇是脂筏的主要成分，在HIV-1的生命週期，特別是病毒組裝及出芽的過程中，扮演重要的角色。因此，我們希望進一步探討膽固醇對Vpr和膜之間的影響。首先，在鈣黃綠素釋放實驗中，發現膜的通透性會隨著膽固醇濃度增加而減少。另外，我們還使用了固態核磁共振來得知Vpr在蛋白微脂體（proteoliposome）中局部區域的化學環境。在交叉極化（cross polarization）魔術角旋轉（magic angle spinning）核磁共振的訊號中，我們發現碳13呈現出較寬的化學位移分布，表示Vpr在蛋白微脂體中感受到多樣的化學環境。在碳{磷}的旋轉回聲雙共振（rotational-echo double-resonance）實驗中，我們發現兩種不同退相特徵（dephasing feature）的共振訊號，分別對應於Vpr上的半胱胺酸跟脂質上的磷酸基之間不同的距離。儘管我們並沒有足夠證據顯示膽固醇會直接作用於Vpr，或是改變其結構，但是膽固醇的存在確實改變了Vpr在不同化學環境的分布，這顯示出Vpr跟膜之間的作用確實會受到膽固醇的調控。
此篇研究顯示，對於Vpr和膜之間的作用，膜脂質的成分是一個重要的影響因素。我們相信，藉由更深入的了解Vpr的功能以及所扮演的角色，有助於對後天免疫缺乏症候群提供新的治療方法。

Vpr protein plays multiple roles in the human immunodeficiency virus type 1 (HIV-1) life cycle, such as assisting the nuclear import of pre-integration complex (PIC), transactivating the long-terminal repeat (LTR) directed gene expression, inducing the apoptosis and triggering the G2 cell cycle arrest, which exacerbate virulence and cell toxicity. Moreover, Vpr can interact with the membrane to form a cation-selective channel, increase membrane permeability and efficiently transfect cells with associated DNA. However, how Vpr interacts with the membrane and what factors affect the interaction remain unclear. Hampered by the bacteriostatic effect leading to the low yield of the recombinant protein by E. coli expression, only synthesized proteins and peptide fragments in the extreme conditions have been used for structural studies.
In this work, we designed a novel E. coli expression construct encoding His-tagged GB1-fused Vpr, giving significant improvement in yield. Up to ~10 mg/L of protein production expressed by the cells at 18°C in a defined growth medium, offered an opportunity to systematically characterize the biochemical and biophysical properties of Vpr. To gain insight into the role of interaction between Vpr and membrane, we have analyzed the global secondary structure of Vpr in various membrane mimics, including dodecylphosphorylcholine (DPC) detergent micelles, bicelles and liposomes. In addition, we have demonstrated the effect of lipid composition on the Vpr-membrane interaction using calcein release assay and nanodisc co-assembly assay. The interaction between Vpr and the membrane containing 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) was found to be much stronger than the interaction between Vpr and the membrane containing solely 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We further used a graphene field-effect transistor (G-FET) biosensor, with the modification of a supported lipid bilayer (SLB), to quantify the interaction of Vpr and the membrane containing DOPG lipids with the dissociation constant determined to be Kd = 9.6 ± 2.1 μM. In contrast, the interaction between Vpr and the membrane containing solely DOPC lipids is too weak to be quantified by SLB/G-FET. As the interaction of Vpr and voltage-dependent anion channel (VDAC) was thought to be responsible for the apoptosis triggered by Vpr, we further used a SLB/G-FET biosensor to characterize their interaction. The dissociation constant, describing the affinity between Vpr and human voltage-dependent anion channel 1 (hVDAC-1) embedded in the membrane containing solely DOPC lipid, was determined to be Kd = 5.1 ± 0.9 μM. This data can serve as a reference for other studies. Cholesterol, a major component in the lipid raft within plasma membrane, plays important roles in the HIV-1 life cycle, especially in the process of virus assembly and budding. Therefore, we further explored the effect of cholesterol on the Vpr-membrane interaction. Using calcein release assay, we first observed that the membrane permeability was reduced in response to the increasing of cholesterol concentration. To gain more insight into the Vpr-membrane complex, solid-state NMR (ssNMR) was used to characterize Vpr proteoliposome in order to probe the local chemical environments of Vpr. The 13C CPMAS NMR signal, exhibiting broad chemical shift distribution, revealed that Vpr experienced multiple chemical environments in the proteoliposome. The 13C{31P} rotational-echo double-resonance (REDOR) experiment demonstrated two resonance peaks were with distinct difference in terms of dephasing feature, corresponding to the existence of the two different distances between the cysteine residue and the phosphate group on the lipid. The presence of cholesterol altered the distribution of Vpr in these two environments, suggesting that the Vpr-membrane interaction could indeed be modulated by cholesterol, albeit no evidence supporting that cholesterol binds to the protein or changes the conformation of Vpr. These studies revealed that the lipid composition of membrane is an important factor in the Vpr-membrane interaction. We believe that more insights into the functional roles of Vpr will contribute to the new treatment of acquired immune deficiency syndrome (AIDS).

Abstract (Chinese) /page ii
Abstract (English) / page v
Acknowledgements /page viii
Abbreviations /page xii
List of chemicals and instruments /page xv
List of scheme and figures /page xvii
List of tables /page xx
Table of contents /page xxi
Chapter 1 Background and motivations /page 1
1.1 Background
1.1.1 The importance of Vpr in HIV-1 /page 1
1.1.2 The roles of Vpr in pre-integration complex /page 4
1.1.3 The roles of Vpr in nuclear import /page 4
1.1.4 The roles of Vpr in LTR transactivation /page 6
1.1.5 The roles of Vpr in apoptosis /page 7
1.1.6 The roles of Vpr in G2 cell cycle arrest /page 8
1.1.7 The structure and molecular properties of Vpr /page 10
1.1.8 The Vpr-membrane interaction /page 12
1.1.9 The potential role of cholesterol in the Vpr-membrane interaction /page 13
1.2 Motivations /page 16
Chapter 2 Vpr gene construct, protein expression and purification /page 17
2.1 Challenges for the Vpr protein production /page 17
2.2 Materials and methods /page 18
2.2.1 A construct for His-tagged GB1-fused Vpr expression /page 18
2.2.2 A construct for R77Q His-tagged GB1-fused Vpr expression /page 21
2.2.3 His-tagged GB1-fused Vpr protein expression /page 24
2.2.4 His-tagged GB1-fused Vpr protein purification /page 27
2.3 Results and discussions /page 28
2.3.1 The production of soluble GB1-fused Vpr /page 28
2.3.2 Characterization of soluble GB1-fused Vpr aggregation /page 31
2.3.3 Characterization of the protein expression /page 32
2.3.4 Production of Vpr protein by TEV protease cleavage /page 34
2.4 Conclusions /page 40


Chapter 3 Structural and functional characterizations of Vpr protein in membrane mimic systems /page 41
3.1 Challenges for studying the Vpr-membrane interaction /page 41
3.2 Materials and methods /page 43
3.2.1 The solubilization of Vpr in DPC detergent micelles /page 43
3.2.2 The incorporation of Vpr into liposomes /page 44
3.2.3 The secondary structure characterization by CD spectroscopy /page 46
3.2.4 Characterization of the Vpr-membrane interaction by SPR /page 49
3.2.5 Characterization of the Vpr-membrane interaction by MST /page 51
3.2.6 Characterization of the Vpr-membrane interaction by nanodiscs co-assembly assay /page 53
3.2.7 Characterization of the Vpr-membrane interaction by calcein release assay /page 55
3.2.8 Characterization of the Vpr-membrane interaction by graphene based field effect transistor (G-FET) biosensor /page 58
Device fabrication of G-FET /page 58
Preparation of liposomes/proteoliposomes /page 59
Preparation of SLB/G-FETs /page 60
Electrical measurements /page 61
3.3 Results and discussions /page 64
3.3.1 The secondary structure characterization of Vpr protein in DPC detergent micelles /page 64
3.3.2 Tm measurement of Vpr protein /page 66
3.3.3 The secondary structure characterization of Vpr protein in bicelles /page 68
3.3.4 The secondary structure characterization of Vpr protein in proteoliposomes /page 70
3.3.5 The Vpr-membrane interaction was lipid composition dependent characterized by calcein release assay /page 73
3.3.6 The Vpr-membrane interaction was lipid composition dependent characterized by lipid nanodisc co-assembly with Vpr /page 77
3.3.7 The Vpr-membrane interaction was lipid composition dependent characterized by G-FET biosensor /page 80
3.3.8 The characterization of Vpr-hVDAC-1 interaction by G-FET biosensor /page 87
3.4 Conclusions /page 89



Chapter 4 Cholesterol modulates the Vpr-membrane interaction /page 90
4.1 Introduction /page 90
4.1.1 Probing the residue specific membrane location /page 90
4.1.2 Challenges for ssNMR analysis of the Vpr proteoliposomes /page 91
4.2 Materials and methods /page 98
4.2.1 Vpr protein production and purification /page 98
4.2.2 Sample preparation of [U-2H,13C,15N] labeled Vpr in [2H25, 98%] DPC detergent micelles for solution NMR analysis /page 101
4.2.3 Sample preparation of the Vpr proteoliposomes /page 101
4.2.4 Calcein release assay /page 102
4.2.5 CD spectroscopic characterization /page 104
4.2.6 Solution NMR spectroscopic characterization /page 104
4.2.7 Solid-state NMR spectroscopic characterization /page 106
4.3 Results and discussions /page 108
4.3.1 The secondary structure characterization of the Vpr proteoliposomes in the presence of cholesterol /page 108
4.3.2 The [1H,15N] TROSY-HSQC spectra of WT Vpr in DPC detergent micelles /page 110
4.3.3 Sequence specific resonance assignment of Vpr protein by solution NMR spectroscopy /page 112
4.3.4 Titration of cholesterol to Vpr protein characterized by solution NMR spectroscopy /page 116
4.3.5 Cholesterol effect of the Vpr-membrane interaction characterized by calcein release assay /page 118
4.3.6 Cholesterol effect of the Vpr-membrane interaction studied by ssNMR spectroscopy /page 121
4.4 Possible mechanistic explanation /page 127
4.5 Conclusions /page 128
Chapter 5 Biological importance and outlook /page 129
5.1 Significance of the study /page 129
5.2 Biological importance /page 130
5.3 Outlook /page 132
References /page 135
Appendix /page 151

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