Thesis advisor: Abhishek Chatterjee / This dissertation explores the development and applications of bioorthogonal chemistries, particularly focusing on genetically encoded methods for site-specific protein modification. Advances in genetic code expansion (GCE), such as the development of efficient aminoacyl tRNA synthetase (aaRS) enzymes, enable the incorporation of noncanonical amino acids (ncAAs) with bioorthogonal handles into proteins, allowing precise chemical modifications with diverse applications in basic biological research and therapeutic development. Here, I present new methodologies for chemoselective modification at encoded 5-hydroxytryptophan (5HTP) residues under mild oxidative conditions. I demonstrate that such reactions allow up to three distinct modifications at a single protein residue, facilitating the creation of functional multicomponent bioconjugates. This work also explores the use of our aaRS enzymes in nascent proteome tracking, allowing cell-type-specific labeling and time-resolved tracking of newly synthesized proteins in complex biological systems. Further, I demonstrate that genetically encoded biosynthesis and proteome-wide incorporation of 5HTP allows selective enrichment from mixed bacterial and mammalian environments when using our mild oxidative conditions to label the residues. These new bioorthogonal chemistries and enzymes for their proteomic incorporation enable the creation of antibody-drug conjugates, protein-protein conjugates, and enable access to new methods of nascent proteome labelling and enrichment. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Abhishek Chatterjee / Thesis advisor: Jia Niu / Directed Evolution has emerged as an invaluable tool for advancing protein functions in both research and industry. Our lab has pioneered a directed evolution platform in mammalian cells, utilizing an AAV delivery vector to package a DNA library and linking the biomolecule of interest to AAV production. During my tenure in Prof. Chatterjee's lab, I focused on harnessing our lab’s directed evolution platform, known as Virus-Assisted Directed Evolution of tRNA (VADER), to develop highly efficient tRNAs for genetic code expansion. Additionally, I contributed to the development of the AAV-based selection platform, termed Virus-Assisted Mutational Profiling (VAMP), as a profiling tool. Through the utilization of VAMP, I conducted comprehensive profiling of tRNA and RNA polymerase III promoter sequences. This enabled me to gain insights into regions of flexibility and evolution, ultimately leading to the construction of improved constructs with enhanced activity relative to the starting sequence. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Abhishek Chatterjee / Genetic code expansion (GCE) is an enabling technology whereby noncanonical amino acids (ncAAs) can be site-specifically incorporated into proteins of interest, allowing for vast applications and an improved understanding of structure-function relationships in biology. GCE stands out as a versatile platform due to the use of a variety of engineered aminoacyl-tRNA synthetase (aaRS)/transfer RNA (tRNA) pairs, and it has endowed proteins with over 200 distinct ncAAs in both prokaryotic and eukaryotic systems. My dissertation outlines endeavors aimed at broadening the chemical diversity of α-amino side chains and substrates beyond α-amino acids in both prokaryotic and eukaryotic organisms through the utilization of GCE technology. This was achieved by creating universal GCE platforms called altered translational machinery (ATM) strains, which eliminate the limitations of orthogonality for the evolution of aaRS/tRNA pairs. This expansion enables the use of the same aaRS/tRNA pair for ncAA incorporation functionalities into multiple domains of life. Moreover, the diversity of ncAAs that can be genetically encoded in eukaryotic cells was enhanced by evolving the E. coli leucyl-tRNA synthetase (EcLeuRS)/tRNA pair using a yeast-based selection system. This advancement facilitated the incorporation of novel ncAAs into proteins within mammalian cells. Additionally, I worked toward developing a platform for introducing monomers into the genetic code beyond α-amino acids. This involved developing an aaRS evolution platform that doesn't rely on translation as a selectable readout. Finally, I worked towards the creation of polyester-polyamide oligomers with sequence control as a step towards the goal of generating sequence-defined biopolymers with new-to-nature backbone chemistries. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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Thesis advisor: Abhishek Chatterjee / The emergence of genetic code expansion (GCE) technology, which enables sitespecific incorporation of unnatural amino acids (UAAs) into proteins, has facilitated powerful new ways to probe and engineer protein structure and function. Using engineered orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs that suppress repurposed nonsense codons, a variety of structurally diverse UAAs have been incorporated into proteins in living cells. This technology offers tremendous potential for deciphering the complex biology of eukaryotes, but its scope in eukaryotic systems remains restricted due to several technical limitations. For example, development of the engineered tRNA/aaRS pairs for eukaryotic GCE traditionally relied on a eukaryotic cell-based directed evolution system, which are significantly less efficient relative to bacteria-based engineering platforms. The work described in this thesis establishes a new paradigm in GCE through the development of a novel class of universal tRNA/aaRS pairs, which can be used for ncAA incorporation in both E. coli and eukaryotes. We achieve this by developing engineered strains of E. coli, where one of its endogenous tRNA/aaRS pair is functionally replaced with an evolutionarily distant counterpart. The liberated pair can then be used for GCE in the resulting altered translational machinery (ATM) strain, as well as any eukaryote. Using this strategy, we have been able to genetically encode new bioconjugation chemistries, post-translational modifications, and facilitate the incorporation of multiple, distinct ncAAs into a single protein. The ATM technology holds enormous promise for significantly expanding the scope of the GCE technology in both bacteria and eukaryotes. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Abhishek Chatterjee / The study of protein structure and function has advanced significantly with the development of genetic code expansion (GCE) technology for the incorporation of noncanonical amino acids (ncAAs), revolutionizing synthetic biology by enabling the introduction of novel functionalities into proteins. Within eukaryotic systems, these advancements have paved the way for deeper investigations into complex protein functions critical to human biology and have spurred the development of innovative biotherapeutic solutions.The work described within this dissertation has aimed to further advance various applications of mammalian GCE. This includes the construction of next-generation homogenous antibody-drug conjugates (ADCs) both using a genetically encoded photocaged cysteine and with a dual incorporation system for the construction of a dual-drug conjugate. Multiple new platforms were developed for the incorporation of two or even three ncAAs within a single protein, utilizing a novel aaRS/tRNA pair and evolved hyper-efficient tRNAs. GCE-enabled precise protein modification was also utilized to spectroscopically study the conformational dynamics of dimeric EGFR. Additionally, platforms were established for the precise installation of post-translational modification (PTM) mimics within mammalian proteins, allowing for their programmed activation. Finally, an innovative strategy for the study of protein-protein interactions using genetically encoded photocrosslinkers was developed. Collectively, these efforts have contributed to the development of novel tools for studying protein function in mammalian cells and advancing the creation of new biotherapeutics through GCE technology. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Les protéines membranaires comme les récepteurs, les canaux ioniques et les transporteurs possèdent des rôles cruciaux dans les processus biologiques tels que la signalisation physiologique et les fonctions cellulaires. La description dynamique et fonctionnelle des structures protéiques est fondamentale pour comprendre la plupart des processus concernant les macromolécules biologiques. L'incorporation, dans des protéines, d'acides aminés non naturels (Uaas) possédant des propriétés physiques ou chimiques spécifiques fournit un puissant outil pour définir la structure et la dynamique de protéines complexes. Ces sondes permettent le suivi et la détection en temps réel de la conformation des récepteurs et des complexes de signalisation. Les approches d'expansion du code génétique ont permis l'incorporation d'Uaas servant de sondes dans des protéines avec une précision moléculaire. L'expansion héréditaire du code génétique peut permettre d'étudier la biologie des protéines de manière systémique.Avec cette stratégie, des Uaas capables de photopontage ont été utilisés pour étudier la relation structure/fonction des Protéines G Couplées aux Récepteurs (GPCR), telles que l'identification de la liaison du ligand ou des interactions protéine-protéine, en détectant les changements dynamiques avec les Uaas spectroscopiques et l'étiquetage bioorthogonal. Sur la base d'applications relativement bien établies d'Uaa dans les GPCR, ici, les analyses fonctionnelles sont combinées à l'incorporation génétique d'un Uaa photosensible spécifique au site, p-azido-L-phénylalanine (AzF) dans d'autres protéines membranaires, pour détecter la protéine, les changements conformationnels et les interactions protéiques. Contrairement à d’autres molécules photosensibles qui permettent aux protéines de répondre à la lumière, l'insertion des Uaas directement dans la chaine d’acides aminés offre des possibilités uniques pour le photo-contrôle de la protéine. Les aspects dynamiques de l'allostérie sont plus difficiles à visualiser que les modèles structuraux statiques. Une stratégie photochimique est présentée pour caractériser la dynamique des mécanismes allostériques des récepteurs NMDA neuronaux (NMDAR). Ces récepteurs appartiennent à la famille des canaux ioniques activés par le glutamate et portent la transmission synaptique excitatrice rapide associée à l'apprentissage et à la mémoire. En combinant le balayage AzF et un test fonctionnel résistant à la lumière, nous avons pu apporter des éléments permettant de mieux comprendre la dynamique des interfaces NTD (N-Terminal Domain des NMDAR) ainsi qu’un nouveau mécanisme de régulation allostérique, améliorant notre compréhension de la base structurale du mécanisme d’activation et de modulation des récepteurs NMDA.Outre l'incorporation de l’Uaa photopontant AzF dans les récepteurs neuronaux pour détecter l'effet fonctionnel, AzF a été appliqué pour piéger des interactions faibles et transitoires entre protéines dans un transporteur d'acides aminés LAT3, impliqué dans le cancer de la prostate. Les techniques de dépistage ont été établies en appliquant un photo-cross-linker positionné dans la protéine pour examiner les interactions entre LAT3 et les interacteurs inconnus et fournir des indices d'identification des partenaires de liaison.Dans l'ensemble, ce travail dévoile de nouvelles informations sur la modulation allostérique de l'activité du récepteur NMDA et sur les interactions protéines-protéines.. Les résultats pourraient fournir de nouvelles informations structurales et fonctionnelles et guider le dépistage de composés thérapeutiques pour des maladies associées au dysfonctionnement de ces protéines membranaires. / Membrane proteins including receptors, channels and transporters play crucial roles in biological processes such as physiological signaling and cellular functions. Description of dynamic structures and functions of proteins is fundamental to understand most processes involving biological macromolecules. The incorporation of unnatural amino acids (Uaas) containing distinct physical or chemical properties into proteins provides a powerful tool to define the challenging protein structure and dynamics. These probes allow monitoring and real-time detection of receptor conformational changes and signaling complexes. The genetic code expansion approaches have enabled the incorporation of Uaas serving as probes into proteins with molecular precision. Heritable expansion of the genetic code may allow protein biology to be investigated in a system-wide manner.With this strategy, photocrosslinking Uaas have been used to study GPCR structure/function relationship, such as identifying GPCR-ligand binding or protein-protein interactions, detecting dynamic changes with spectroscopic Uaas and bioorthogonal labeling. Based on relatively well-established applications of Uaa in GPCRs, here, functional assays are combined with the site-specific genetic incorporation of a photo-sensitive Uaa, p-azido-L-phenylalanine (AzF) into other membrane proteins, to probe protein conformational changes and protein interactions. Unlike photo-sensitive ligands that enable proteins in response to light, the site-specific insertion of light-sensitive Uaas facilitates directly light-sensitive proteins. Dynamic aspects of allostery are more challenging to visualize than static structural models. A photochemical strategy was presented to characterize dynamic allostery of neuronal NMDA receptors (NMDARs), which belong to the ionotropic glutamate receptor channel family and mediate the fast excitatory synaptic transmission associated with learning and memory. By combining AzF scanning and a robust light-induced functional assay the dynamics of NMDAR N-terminal domain (NTD) interfaces and novel allosteric regulation mechanism were uncovered, improving our understanding of the structural basis of NMDAR gating and modulation mechanism.Besides incorporation of photo-cross-linker AzF into neuronal receptors to detect the functional effect, AzF was used to trap transient and weak protein-protein interactions in an amino acid transporter LAT3, which is critical in prostate cancer. Screening technique was established by applying genetically encoded photo-cross-linker to examine interactions between LAT3 and unknown interactors and provide clues to identify the binding partners.Overall, the work reveals new informations about the allosteric modulation of channel activity and proteins interactions. These light-sensitive proteins facilitated by site-specific insertion of light-sensitive Uaas enable profiling diversity of proteins. The results will provide novel structural and functional information and may guide screening of therapeutic compounds for diseases associated with malfunctioning of these membrane proteins.

The ability of incorporation non-canonical amino acids (ncAAs) using translation offers researchers the ability of extend the functionality of proteins and peptides for many applications including synthetic biology, biophysical and structural studies, and discovery of novel ligands. Here we describe the three projects where the addition of ncAAs to in vitro translation systems creates useful chemical biology techniques. In the first, a fluorinated histidine derivative is used to create a novel affinity tag that allows for the selective purification of peptides from a complex mixture of proteins. In the second, the high promiscuity of an editing-deficient valine-tRNA synthetase (ValRS T222P) is used to demonstrate ribosomal translation of 13 ncAAs including those with novel side chains, α,α disubstitutions, and cyclic β amino acids. Lastly, a couple of these amino acids are integrated into the powerful ligand discovery tool of mRNA display for the discovery of helical peptide ligands.

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