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A method for the genetically encoded incorporation of FRET pairs into proteinsLammers, Christoph 15 July 2014 (has links)
No description available.
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Development and Applications of Universal Genetic Code Expansion Platforms:Italia, James Sebastian January 2019 (has links)
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.
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Structural Bioinformatics to Understand the Origin of the Genetic Code: Structural Motif Detection in Aminoacyl-tRNA SynthetasesKaiser, Florian 23 October 2018 (has links)
One of the most profound open questions in biology is how the genetic code developed. The blueprints for proteins are encoded by triplets of nucleic acids, which in turn require proteins for interpretation and replication. The mere existence of this self-referencing system is a chicken-and-egg dilemma. Aminoacyl-tRNA synthetases are key players in the transfer of genetic information and reflect the earliest episode of life. These enzymes are responsible for loading tRNA molecules with the correct amino acid. Two protein superfamilies of aminoacyl-tRNA synthetases emerged, each responsible for ten amino acids. Despite sequence and structure similarity, the delicate balance between these superfamilies is manifested in two structural motifs, which were identified in the context of this thesis: the Backbone Brackets and the Arginine Tweezers. Both motifs realize constant ligand recognition and can be found in almost all protein structures of aminoacyl-tRNA synthetases.
In this thesis, I thoroughly characterized Backbone Brackets and Arginine Tweezers. The specific characteristics of these motifs require high-precision methods for their detection and analysis. However, existing algorithms do not feature an adequate computational representation of structural motifs at the atom level and the support of isofunctional residue mutations. In order to address these limitations, I designed the Fit3D algorithm for template-based and template-free detection of structural motifs. I show that proper computational representation of structural motifs is crucial and improves accuracy up to 26% for a benchmark dataset. Fit3D is a general-purpose tool for structural motif detection in high-resolution protein structure data. In conjunction with the accelerating progress in experimental methods, the demand for such tools will increase rapidly over the next years.
I applied Fit3D to structures of aminoacyl-tRNA synthetases to investigate whether Backbone Brackets and Arginine Tweezers are universal building blocks for ligand recognition, and to quantify structural changes upon ligand binding. While the Arginine Tweezers motif is exclusively found in aminoacyl-tRNA synthetases and paralogs, the Backbone Brackets seem to be a general pattern to recognize functional groups of certain ligands. The results show subtle differences in side chain orientation for one structural motif and a backbone shift for the other. This suggests a structural rearrangement to be a general mechanism in some aminoacyl-tRNA synthetases. The detailed level of these analyses would not have been possible without high-precision structural motif detection with Fit3D.
The results emphasize the importance of structural motifs, which consist of only a few residues, for the global function of the enzyme. Furthermore, the stunning conservation of the structural motifs located in the core domains of aminoacyl-tRNA synthetases suggests their presence in the earliest predecessors of these enzymes. Both motifs might have played a fundamental role in shaping the genetic code as we know it.
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Structure-function studies of membrane proteins by site-specific incorporation of unnatural amino acids / Etudes structure-fonction de protéines membranaires par incorporation spécifique d'acides aminés non naturelsTian, Meilin 20 June 2017 (has links)
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.
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In vitro genetic code expansion and selected applicationsIqbal, Emil S 01 January 2018 (has links)
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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Investigation of Nucleosome Dynamics by Genetic Code ExpansionHahn, Liljan 10 March 2015 (has links)
No description available.
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Biofyzikální charakterizace proteinových knihoven z různých repertoárů aminokyselin / Biophysical characterization of protein libraries composed of different amino acid repertoiresNeuwirthová, Tereza January 2020 (has links)
This study is part of a project which aims to understand evolution of genetic code together with structural and functional analysis of prebiotic proteins. The repertoire of amino acids in the first proteins was probably developing in time and it influenced the development of structure and function of today's proteins. First amino acid alphabet was apparently only half of the size of present alphabet, which contains twenty amino acids. These ten amino acids were probably prebiotically available from endogenous and exogenous sources. This work includes cell-free expression and purification of two randomized protein libraries (containing approximately 1011 variants) with various amino acid composition and following comparison of their propensity to form secondary (using circular dichroism) and tertiary (using proteolytical analysis of sequences) structures. First library contains only ten probably prebiotically available amino acids; second library contains all twenty amino acids in today's genetic code. This project could help us understand benefits of genetic code expansion in terms of developing structure in protein sequences. The whole research could theoretically contribute a few basic questions not only in the fields of protein evolution but also in areas of synthetic biology or protein...
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STUDIES OF THE PYRROLYSYL-TRNA SYNTHETASEJiang, Ruisheng 23 July 2013 (has links)
No description available.
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A novel aminoacyl-tRNA synthetase and its amino acid, pyrrolysine, the 22nd genetically encoded amino acidLarue, Ross C. January 2009 (has links)
No description available.
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Méthodes et algorithmes pour l’amélioration de l’inférence de l’histoire évolutive des génomesNoutahi, Finagnon Marc-Rolland Emmanuel 07 1900 (has links)
No description available.
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