有鑑於基因密碼子擴展技術的廣泛應用，非典型胺基酸能被準確嵌入蛋白質特定位點之中。源自古菌Methanosarcina mazei (Mm) 和 Candidatus Methanomethylophilus alvus (Ma) 的吡咯離胺醯-tRNA合成酶 (pyrrolysyl-tRNA synthetase, PylRS)，由於其酵素活化位對突變容忍度高，經酵素工程後，常作為強而有力之化學生物學工具，可將多於200種非典型胺基酸嵌入蛋白質中。然而，此領域發展卻受限於PylRS天生的多向受質活性中心。 由於PylRS同源自苯丙氨醯-tRNA合成酶 (phenylalanyl-tRNA synthetase)，故PylRS經酵素工程後，突變株特別容易對廣泛苯丙氨 (Phe) 類似物產生活性。然而，突變株相對野生株之活性，卻是大為降低，此問題隨之導致整體蛋白質表現量下降。此外，突變株的多向受質活性也可能導致其將天然胺基酸作為受質，而產生不預期之背景表現。因此，發展具高酵素活性及可控制酵素活性的PylRS突變株，將有利於此領域的發展。

本研究的第一目標，為設計具高酵素活性及多受質廣度的新型PylRS突變株。基於結構引導的理性設計，不同以往於酵素活化位附近進行突變，我們將野生型MmPylRS N端和C端結構域上的tRNA結合域進行酵素工程，進而得到高效率突變株。接著為測試新突變點能否應用於其他突變株上，進而提高活性，我們設計了對Phe 和色胺酸 (Trp) 類似物具活性的多受質高效新型突變株。最後我們證明，在MmPylRS 和tRNA交互作用的結合域進行突變，能有效提高酵素活性，而這些突變也能提高其他具多受質廣度突變株的胺醯化效率。

本論文的第二目標，為利用前述之多受質高效新型MmPylRS突變株，來研究與疾病相關的USP酵素功能。我們將Trp 類似物用來研究Trp475 對USP30的活性影響和受質專一性，從而改造出能專一辨認K6泛素鏈的USP30突變株。此外，我們開發了一種在非變性條件下，快速合成各種特定位點的泛素-類小泛素二聚體和類小泛素化蛋白質修飾的平台。在MmPylRS突變株的幫助下，類小泛素中特定位點的離胺酸被硒代烷基半胱氨酸取代，進行氧化脫去反應的氧化硒能產生具親電中心的脫氫丙氨酸。我們將製備好的類小泛素和羧基端被修飾、具有親核性硫醇基的泛素於水相中進行麥可加成反應，合成出泛素-類小泛素二聚體。最後，我們以去泛素化酵素 USP7 處理合成的二聚體，發現 USP7 能將其鍵結水解，也發現 USP7 的羧基末端結構域對不同泛素-類小泛素二聚體的認知結合扮演重要角色。

本論文的第三目標，為開發能精準控制PylRS活性的分子開關，進而降低由於PylRS多向受質活性所導致的背景表現問題。利用蛋白片段互補技術作為發想， MaPylRS被分裂成兩個不具活性的N端和C端片段，兩個片段分別和兩個目標蛋白融合，當兩個目標蛋白進行交互作用時，分裂的兩片段也互補重組，重新恢復MaPylRS酵素功能。我們證明MaPylRS分裂酵素能被用來當作生物傳感器，在非典型胺基酸的存在下，利用含有終止密碼的報告者基因，偵測細胞凋亡途徑成員Bcl-2家族間，或介導宿主細胞與 SARS-CoV-2感染間的蛋白質-蛋白質交互作用。 最後，我們證明MaPylRS分裂酵素能和小分子化學誘導-蛋白質二聚化系統相互兼容，使基因表現能在原核和真核系統中，同時被非典型胺基酸，和另一個小分子化合物精準控制。

Genetic code expansion has been broadly used to equip proteins with non-canonical amino acids (ncAAs). Owing to remarkable mutation tolerance in the active site, pyrrolysyl-tRNA synthetases derived from archaeal Methanosarcina mazei (MmPylRS) and Candidatus Methanomethylophilus alvus (MaPylRS) have emerged as powerful tools to incorporate more than 200 chemically diverse ncAAs into proteins at designated positions. However, limitations arise from the field is unified by a single problem – the polyspecific nature of this ancient enzyme.

Diverged from an ancestral phenylalanyl-tRNA synthetase (PheRS), engineered PylRSs typically display wide substrate scope in charging phenylalanine (Phe) analogs. However, low aminoacylation activity is often observed in evolved PylRSs and this leads to significant reduction in the yield of producing proteins containing ncAAs. The other issue is the promiscuous activity of the engineered PylRSs can lead to mischarging of the suppressor tRNA with natural amino acids causing significant background translation of the target gene. Hence, developing rationale-based polyspecific PylRSs with enhanced efficiency and controllable activity would help pave the way to generate robust PylRS variants.

Therefore, the first aim was to devise designer PylRSs with prominent activity and poly-substrate selectivity. We began by employing structure-guided engineering at the interface between tRNA binding domain and cognate tRNAPyl of MmPylRS, which is away from the active site, to generate a designer PylRS with robust efficiency. We further expanded the substrate selectivity of this PylRS variant by introducing rationally designed mutations in the active site. We demonstrated that, compared to the previously evolved variant, the newly designed pocket displayed a distinct polyspecificity in charging not only Phe but also tryptophan (Trp) derivatives with high suppression efficiency.

The second aim was to demonstrate the use of designer PylRSs for studying disease-related enzyme functions in the USP family. Trp analogs were used to study the activity and substrate selectivity of W475 in the USP30 active site. We evolved a USP30 variant that specifically recognizes a K6 ubiquitin (Ub) linkage. In parallel, we developed a universal platform for generating site-specific SUMO or Ub modified protein using a designer PylRS-based system. The C-terminal tagged Ub was used to selectively react with SUMO2 containing dehydroalanines, yielding eight SUMO2 variants ubiquitinated at distinct lysine (Lys) positions. Finally, we revealed the important role of the C-terminal domain of USP7 in regulating the activity and selectivity for the Ub-tagged SUMO2 dimers.

The final aim was to develop a designer PylRS that can be activated on demand to precisely control the activity of enzyme to prevent background translation. To do this, MaPylRS was split into fragments that were inactive, but which could be reactivated when they were brought into proximity. These fragments were initially tagged with proteins that interact with each other. The protein-protein interactions (PPIs) restored the activity of the split PylRS which can be detected, in the presence of a ncAA, using diverse reporter genes that contain an in-frame stop codon. Further, it was shown that the split PylRS can serve as versatile biosensors for detecting diverse PPIs, including therapeutically relevant interactions such as those in the Bcl-2 family and interactions that mediate infection of SARS-CoV-2. Finally, we demonstrated that the split PylRS works effectively with a small molecule-dependent dimerization system to activate chemically-induced stop codon suppression in bacteria and eukaryote.

TABLE OF CONTENTS

摘要 I
ABSTRACT III
ACKNOWLEDGEMENTS V
DEDICATION VII
CONTRIBUTORS AND FUNDING SOURCES VIII
TABLE OF CONTENTS X
LIST OF FIGURES XV
LIST OF TABLES XVIII
ABBREVIATIONS XIX

CHAPTER I INTRODUCTION 22
I.1 An Overview of Genetic Code Expansion 22
I.2 Genetic Code Expansion in Nature 28
I.3 Non-canonical Amino Acids Incorporation in vitro and in vivo 33
I.4 A Facile Tool in Genetic Code Expansion: Pyrrolysyl-tRNA Synthetase 38
I.5 Applications of Genetic Code Expansion 42
I.6 Specific Aims 45

CHAPTER II DESIGN PYRROLYSYL-TRNA SYNTHETASES TO ENHANCE SUPPRESSION EFFICIENCY AND EXPAND THE SUBSTRATE SCOPE† 48
II.1 Introduction 48
II.2 Experimental Details 52
II.2.1 General 52
II.2.2 Primer list 55
II.2.3 Protein purification 58
II.2.4 Western blot analysis 61
II.2.5 ESI-MS characterization 62
II.2.6 MALDI-TOF-MS/MS characterization 62
II.2.7 X-crystal structural analysis and modeling 63
II.2.8 In vivo amber suppression assay 63
II.2.9 Kinetic analysis 65
II.3 Results and Discussion 66
II.3.1 Substrate specificity of MmPylRS and MaPylRS 66
II.3.2 Rational design of an efficient MmPylRS 68
II.3.3 Substrate specificity of designer MmPylRS variants 75
II.3.4 Characterization of sfGFP containing ncAAs 84
II.3.5 Rational design of a polyspecific MmPylRS 90
II.3.6 Substrate specificity of polyspecific MmPylRS variants 91
II.3.7 Kinetic analysis of FOWRS2 with L- and D-Phe analogs 97
II.4 Conclusion 100

CHAPTER III DESIGN PYRROLYSYL-TRNA SYNTHETASES TO INVESTIGATE DISEASES-ASSOCIATED ENZYME FUNCTIONS† 103
III.1 Introduction 103
III.2 Experimental Details 108
III.2.1 General 108
III.2.2 Primer list 110
III.2.3 Protein purification 113
III.2.4 LC-ESI-MS/MS analysis 120
III.2.5 Photo-physical analysis 120
III.2.6 Di-ubiquitin cleavage assay 121
III.2.7 Ubiquitin-AMC cleavage assay 121
III.2.8 Western blotting 122
III.2.9 Protein chemistry 122
III.3 Results and Discussion 126
III.3.1 Photo-physical analysis of FOWRS2 encoded Trp analogs 126
III.3.2 Unraveling the role of W475 in USP30 active site by FOWRS2 129
III.3.3 Determining activity and substrate selectivity of USP30 variants 132
III.3.4 Preparing site-specific SUMO-modified sfGFP by N-ZRS 135
III.3.5 Preparing site-specific Ub-modified SUMO2 by N-ZRS 138
III.3.6 Unraveling substrate selectivity of USP7 by Ub-SUMO2 dimers 142
III.4 Conclusion 146

CHAPTER IV SPLIT PYRROLYSYL-TRNA SYNTHETASES FOR CHEMICALLY-INDUCED GENETIC CODE EXPANSION 148
IV.1 Introduction 148
IV.2 Experimental Details 151
IV.2.1 General 151
IV.2.2 sfGFP expression assay 151
IV.2.3 Chloramphenicol resistance assay 152
IV.2.4 SEAP expression assay 152
IV.2.5 Plasmid list 153
IV.3 Results and Discussion 158
IV.3.1 Design and validation of a split PylRS 158
IV.3.2 Design and validation of a split TyrRS 164
IV.3.3 Detecting therapeutically relevant PPIs using split PylRS 168
IV.3.4 Small-molecule-induced stop codon suppression using split aaRSs 171
IV.3.5 Controlling TAG and TAA stop codon suppression using split aaRSs 174
IV.3.6 Controlling gene expression in mammalian cells using small-molecule-induced stop codon suppression 176
IV.4 Conclusion 179

CHAPTER V DISCUSSION AND CONCLUDING REMARKS 182
REFERENCES 190
APPENDIX 224

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