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Rodriguez, Erik Ali Tirrell David A. Dougherty Dennis A. "In Vivo Incorporation of Multiple Unnatural Amino Acids /." Diss., Pasadena, Calif. : California Institute of Technology, 2009. http://resolver.caltech.edu/CaltechETD:etd-01122009-153110.
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Wang, Jinfan. "In Vitro Kinetics of Ribosomal Incorporation of Unnatural Amino Acids." Doctoral thesis, Uppsala universitet, Struktur- och molekylärbiologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-282023.
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Ribosomal incorporation of unnatural amino acids (AAs) into peptides or proteins has found broad applications in studying translation mechanism, discovering potential therapeutics, and probing protein structure and function. However, such applications are generally limited by the low incorporation efficiencies of the unnatural AAs. With in vitro kinetics studies using a purified E. coli translation system, we found that the natural N-alkyl AA carrier, tRNAPro, could hasten the incorporation of N-methyl AAs. Also, the incorporation rate increased remarkably with increasing pH in the range of 7 to 8.5, suggesting the rate was limited by peptidyl transfer, not accommodation. Competition experiments revealed that several futile cycles of delivery and rejection of the A site N-methyl AA-tRNA were required per peptide bond formation, and the incorporation yield could be increased by using a higher Mg2+ concentration. Kinetics of ribosomal polymerization, using AA-tRNA substrates prepared from the standard N-NVOC-AA-pdCpA chemoenzymatic ligation method, clarified that the inefficiency of incorporation was due to the penultimate dC. This dC prompted faster peptidyl-tRNA drop-off, leading to loss of processivities along consecutive incorporations. Circumventing the penultimate dC by using our N-NVOC-AA-pCpA chemoenzymatic ligation or the flexizyme charging method to prepare the AA-tRNA substrates was able to improve the efficiencies of ribosomal consecutive incorporations of unnatural AAs. By studying the translation steps after aminoacylation of tRNAPyl, the favored carrier for unnatural AAs in vivo, we demonstrated surprisingly slow biphasic kinetics of tRNAPyl-mediated amber suppression in vitro. The fast phase amplitude increased with increasing EF-Tu concentration, allowing measurement of Kd of EF-Tu binding. Results revealed ~25-fold weaker EF-Tu binding affinity of the tRNAPyl body than that of E. coli tRNAPhe. The fast phase rate was ~30-fold slower than that of native substrates, and this rate was limited by the ~10-fold less efficient AA-tRNAPyl:EF-Tu:GTP ternary complex binding to the ribosome. The incorporation was so slow that termination by RF2 mis-reading of the amber codon became a significant competing reaction. The processivity was unexpectedly impaired as ~40% of the dipeptidyl-tRNAPyl could not be elongated to tripeptide. This new overall understanding opens a window of improving unnatural AA incorporation both in vitro and in vivo.
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Erickson, Sarah. "Using Unnatural Amino Acid Incorporation to Modify and Manipulate Adeno-Associated Virus:." Thesis, Boston College, 2020. http://hdl.handle.net/2345/bc-ir:108955.
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Thesis advisor: Eranthie WeerapanaAdeno-Associated Virus (AAV) has been developed into a powerful therapeutic tool - in the last ten years it has acted as a gene-delivery vehicle in several approved therapeutics and many more therapeutics on trial. Despite extensive research, gaps in our understanding of AAV’s infectious cycle still exist, and further development is needed for the creation of improved gene therapy vectors. Technology to incorporate Unnatural Amino Acids (UAAs) into the AAV capsid has recently been developed, and could aid in both furthering our understanding of AAV’s biology and in the therapeutic advancement of AAV. In this work, we demonstrate how the functionalization of the AAV capsid using UAA incorporation can advance our control over the AAV capsid and aid in probing and manipulating AAV biology. We describe our use UAA incorporation to place a bio-orthogonal reactive handle into AAV’s capsid followed by functionalization with a targeting moiety and demonstrate the unprecedented amount of control that UAA incorporation provides in the creation of a functional virus conjugate. We are able to control both the precise placement and the stoichiometry of the targeting moiety on the AAV capsid, providing a platform that, for the first time, can undergo rigorous optimization analogous to that which medicinal chemists put small molecules through. We also describe the creation of a new platform to site-specifically modify the AAV capsid using cysteine incorporation, a technique that retains the ability to site-specifically modify the capsid as UAA incorporation does, but does not require the excess machinery that UAA incorporation requires. Next we discuss the incorporation of a photocaging amino acid, NBK, into the AAV capsid. Using NBK, we were able to effectively block AAV’s primary binding interaction with Heparan Sulfate Proteoglycan (HSPG) and control the timing of AAV infection using light to chemically remove the photo-protecting group. While photocaging the HSPG interaction is only a proof of concept, it demonstrates the remarkable amount of control that UAA incorporation affords, and lends insight to what could be accomplished using the functionalities that can be placed on the AAV capsid with UAAs
Thesis (PhD) — Boston College, 2020
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Monahan, Sarah Lynn Dervan Peter B. "Site-specific incorporation of unnatural amino acids into receptors expressed in mammalian cells /." Diss., Pasadena, Calif. : California Institute of Technology, 2004. http://resolver.caltech.edu/CaltechETD:etd-05252004-153512.
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Crane, Peter. "Protein based molecular probes by unnatural amino acid incorporation." Thesis, University of Oxford, 2018. http://ora.ox.ac.uk/objects/uuid:772076fc-00f2-4ca7-bfa9-3da1ce7093cb.
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The "tag & modify" strategy for protein modification relies upon the genetic incorporation of an uncommon or unnatural amino acid into a protein backbone, followed by a chemo-selective modification to yield differentially modified proteins. This thesis describes the creation of a protein-based glycoconjugate tool for interrogating biological function. In Chapter 2, the unnatural amino acid, azidohomoalanine was genetically incorporated into a library of distance defined Np276 proteins via a selective pressure incorporation. Methods to prevent the common post translational modification N-terminal gluconylation were identified, including preliminary work on a small molecule intervention. The proteins were subsequently characterised with respect to other members of the (limited) family of pentapeptide repeat protein and the key biophysical parameters (TM, stability) with relate to it being a multivalent scaffold were investigated. In Chapter 3, An initial investigation into obtaining a selective amine modification initially via N-hydroxysuccinimide esters, led to the discovery (and characterisation) of a clear selectivity for N-terminal proline Isothiocyanate modification. The dual modification of proteins via the N-term Pro & the ubiquitous (glyco) copper(I)-catalysed azide alkyne cycloaddition was subsequently used to generate homogenously dual modified Np276 scaffolds. In Chapter 4, these proteins were then used in a FACS assay against a murine sialoadhesin - chinese hamster ovary cell line, the results showing promise for the further development of multivalent glycated probes of function.
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Tian, Meilin. "Structure-function studies of membrane proteins by site-specific incorporation of unnatural amino acids." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066166.
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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 membranairesMembrane 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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Tang, Yi Tirrell David A. "Protein engineering using unnatural amino acids : incorporation of leucine analogs into recombinant protein in vivo /." Diss., Pasadena, Calif. : California Institute of Technology, 2002. http://resolver.caltech.edu/CaltechETD:etd-08152006-084149.
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Nguyen, Duy Phuoc. "Unnatural amino acid incorporation via the orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610160.
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Shi, Zhengtao. "Structure-function studies of adenylate kinase by site-specific incorporation of both natural and unnatural amino acids /." The Ohio State University, 1994. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487854314871531.
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Italia, James Sebastian. "Development and Applications of Universal Genetic Code Expansion Platforms:." Thesis, Boston College, 2019. http://hdl.handle.net/2345/bc-ir:108354.
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Thesis advisor: Abhishek ChatterjeeThe 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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Qi, Xin Dervan Peter B. "Unnatural amino acid incorporation to rewrite the genetic code and RNA-peptide interactions /." Diss., Pasadena, Calif. : California Institute of Technology, 2005. http://resolver.caltech.edu/CaltechETD:etd-05272005-133323.
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Seyedsayamdost, Mohammad R. "Investigation of the mechanism of radical propagation in E. coli ribonucleotide reductase by site-specific incorporation of unnatural amino acids." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/43089.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2008.Vita.
Includes bibliographical references.
Inside the cell, ribonucleotide reductases (RNRs) are responsible for the conversion of nucleotides to 2'-deoxynucleotides, an essential step in DNA biosynthesis and repair. The E. coli RNR is the best studied RNR to date and consists of two protein subunits, a2 and P2. a2 is the site of nucleotide reduction and 02 contains a diiron tyrosyl radical (Y122*) cofactor. Each turnover requires radical propagation from the Y122* in 32 to the active site of a2 over 35 A. The mechanism of this unprecedented, long-range radical propagation step is poorly understood. Based on structural studies, a pathway of aromatic residues has been proposed to participate in this process. Site-directed mutants of these residues have been uninformative. In an effort to understand radical propagation, we have employed expressed protein ligation and suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodologies to site-specifically insert unnatural tyrosine analogues into 12 and a2, at residues believed to be involved. On the basis of results with the radical traps 3,4-dihydroxyphenylalanine (DOPA) and 3-aminotyrosine (NH2Y), which we have incorporated into 32 and a2, respectively, and a series of fluorotyrosines (FnYs, n=2, 3, 4), which we have established as probes for proton-coupled electron transfer reactions and incorporated into 12, we propose a mechanism for radical transfer in RNR. We show that binding of substrate and effector are essential for control and gating of radical propagation. We further demonstrate that three Ys, 12-Y356, a2-Y731 and a2-Y730, are redox-active and participate in hole propagation. The NH2Y. observed with NH2Y-a2s likely constitutes the first observation of a transiently oxidized intermediate during active radical propagation. In 12, Y356 participates in radical transfer by an orthogonal proton-coupled electron transfer mechanism, where long-range electron transfer is coupled to short-range, off-pathway proton transfer.
(cont) Within a2, Y731 and Y730o participate by a hydrogen atom transfer mechanism where the proton and electron originate from and arrive at the same moiety. We also establish the positions of these three Ys in the a2/32 complex and present direct evidence for the reversible nature of radical propagation.
by Mohammad R. Seyedsayamdost.
Ph.D.
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Shrestha, Prashanta. "Streamlined Extract Preparation for E. coli-Based Cell-Free Protein Synthesis and Rapid Site-Specific Incorporation of Unnatural Amino Acids in Proteins." BYU ScholarsArchive, 2012. https://scholarsarchive.byu.edu/etd/3917.
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This thesis reports the viability of E. coli cell extracts prepared using equipment that is both common to biotechnology laboratories and able to process small volume samples and expression of proteins containing unnatural amino acids (UAAs) at higher level using PCR amplified linear DNA templates (LETs) in cell-free protein synthesis (CFPS) system. E. coli-based cell extracts are a vital component of inexpensive and high-yielding CFPS reactions. However, effective preparation of E. coli cell extract is limited to high-pressure homogenizers (French press style or impinge-style) or bead mill homogenizers, which all require a significant capital investment. This work specifically assessed the following capital cost lysis techniques: (1) sonication, (2) bead vortex mixing, (3) freeze-thaw cycling, and (4) lysozyme incubation to prepare E. coli cell extract for CFPS. In this work, simple shake flask fermentation with a commercially available E. coli strain was used. Additionally, the RNA polymerase was over expressed in the E. coli cells prior to lysis which eliminated the need to add independently purified RNA polymerase to the CFPS reaction. As a result, high yielding E. coli-based cell extract was prepared using equipment requiring reduced capital investment and common to biotechnology laboratories. To our knowledge, this is the first successful prokaryote-based CFPS reaction to be carried out with extract prepared by sonication or bead vortex mixing. LETs are an attractive alternative to plasmids for site-specific incorporation of unnatural amino acids in proteins in the CFPS system because of their short preparation time and ease of production. However, major limitations associated with LETs are: (1) their degradation by RecBCD enzyme present in the cell-extract used for CFPS and (2) high CFPS energy costs. In this work, we report the optimization of LET-based CFPS for improved protein yield by inhibiting the RecBCD enzyme with small inhibitor molecules resulting in three fold increment in yield of protein containing UAA. We also assessed alternative energy sources such as glucose, fructose-1,6-bisphospate, creatine phosphate/creatine kinase, and high glutamate salt for cost reduction. This work could be important for high-throughput applications based on linear expression templates. This work demonstrates simple E. coli extract preparation and improved yield with linear expression templates for further advancements of cell-free protein synthesis system.
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Duodu, Portia. "Site-specific photo-proteolysis of proteins and peptides by incorporation of unnatural amino acid : synthesis and characterization of photo-activatable α-amino acid." Strasbourg, 2010. http://www.theses.fr/2010STRA6228.
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Les structures protéiques impliquées dans les phénomènes physiologiques comme la coagulation sanguine, l’apoptose ou la signalisation membranaire, nécessitent souvent une activation préliminaire de leurs formes pro-protéines inactives. Le présent travail de thèse s’inscrit dans le domaine de la biochimie, et porte sur le développement d’une photo-protéase comme un outil moléculaire original et efficace pour l’étude dynamique de protéines par photorégulation de leur activité. Les photo-protéases adoptent un concept identique à celui des précurseurs photolabile de biomolécules; En effet, l’irradiation d’une séquence peptidique modifiée au préalable avec un acide aminé photolabile permettra un clivage specifique de la chaîne polypeptidique au niveau du site d’incorporation de cet amino acide, libérant ainsi une séquence fonctionnelle. L’avantage majeur de cette méthodologie étant une protéolyse ciblée, contrôlée, et inoffensive pour l’organisme à la longueur d’onde utilisée (λ>300nm). L’isomère optiquement actif de l’acide aminé photosensible DMNPA a été synthétisé et ses propriétés de clivage ont été évaluées sur des peptides modèles. L’étude de la réaction photochimique a démontré une sonde UV intéressante pour une protéolyse ciblée in vivo. Dans un deuxième temps une méthode alternative permettant une “vraie protéolyse” par clivage de la liaison amide a aussi été développée. Ce système moléculaire de pointe est très adapté aux méthodes d’incorporation d’acide aminé dans des protéines notamment la méthode de suppression de codons stop, et devrait trouver des applications utiles dans le photo-déclenchement de processus biologiques in vitro comme in vivoProtein structures involved in post translational modifications of physiological events such as blood clotting, cell death and membrane signaling, mostly require proteolytic activation of their inactive proprotein forms. The present thesis work is a contribution to the biochemistry field, and focuses on the development of a photo-protease as an efficient molecular tool for protein dynamic studies. Photo-proteases adopt an identical concept as for “caged biomolecules”. In fact, illumination of a protein having its polypeptide backbone sequence mutated with a photoactivatable amino acid, induces cleavage of the polypeptide backbone at the specific site of incorporation of the unnatural amino acid, releasing thereof functional peptides/proteins. This methodology offers a spatio-temporal controlled proteolysis, with the use of light being harmless to most living tissues at λ>300nm. One such amino acid was conceived: DMNPA. A stereodivergent synthesis to optical isomers was established, and polypeptide backbone cleavage properties have been assessed on short peptidyl sequences. Photolysis of model peptides revealed a near-UV probe interesting for targeted proteolysis in living cells. We have also developed an original two-step processing reaction leading to efficient amide bond cleavage after UV illumination. Thus a new and efficient biomolecular tool is now accessible for site-specific proteolysis. This state of the art molecular system, is very adapted to current methods of cell delivery of bioagents, notably the nonsense codon suppression methodology, and should find useful applications in phototriggering physiological events in vitro likewise in vivo
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Broadbent, Andrew. "Aminoacyl-tRNA Synthetase Production for Unnatural Amino Acid Incorporation and Preservation of Linear Expression Templates in Cell-Free Protein Synthesis Reactions." BYU ScholarsArchive, 2016. https://scholarsarchive.byu.edu/etd/5703.
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Proteins—polymers of amino acids—are a major class of biomolecules whose myriad functions facilitate many crucial biological processes. Accordingly, human control over these biological processes depends upon the ability to study, produce, and modify proteins. One innovative tool for accomplishing these aims is cell-free protein synthesis (CFPS). This technique, rather than using living cells to make protein, simply extracts the cells' natural protein-making machinery and then uses it to produce protein in vitro. Because living cells are no longer involved, scientists can freely adapt the protein production environment in ways not otherwise possible. However, improved versatility and yield of CFPS protein production is still the subject of considerable research. This work focuses on two ideas for furthering that research.The first idea is the adaptation of CFPS to make proteins containing unnatural amino acids. Unnatural amino acids are not found in natural biological proteins; they are synthesized artificially to possess useful properties which are then conferred upon any protein made with them. However, current methods for incorporating unnatural amino acids do not allow incorporation of more than one type of unnatural amino acid into a single protein. This work helps lay the groundwork for the incorporation of different unnatural amino acid types into proteins. It does this by using modified aminoacyl-tRNA synthetases (aaRSs), which are key components in CFPS, to be compatible with unnatural amino acids. The second idea is the preservation of DNA templates from enzyme degradation in CFPS. Among the advantages of CFPS is the option of using linear expression templates (LETs) in place of plasmids as the DNA template for protein production. Because LETs can be produced more quickly than plasmids can, using LETs greatly reduces the time required to obtain a DNA template for protein production. This renders CFPS a better candidate for high-throughput testing of proteins. However, LETs are more susceptible to enzyme-mediated degradation than plasmids are, which means that LET-based CFPS protein yields are lower than plasmid-based CFPS yields. This work explores the possibility of increasing the protein yield of LET-based CFPS by addition of sacrificial DNA, DNA which is not used as a protein-making template but which is degraded by the enzymes in place of the LETs.
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Mayo, Daniel J. "Synthetic methodologies for labeling membrane proteins and studies utilizing electron paramagnetic resonance in biologically relevant lipid architectures." Miami University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=miami1343434201.
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Davenport, Eric Parker. "Fluorescent Probes to Investigate Homologous Recombination Dynamics." DigitalCommons@USU, 2016. https://digitalcommons.usu.edu/etd/5007.
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There are multiple mechanisms by which DNA can become damaged. Such damage must be repaired for the cell to avoid ill-health consequences. Homologous recombination (HR) is a means of repairing one specific type of damage, a double-strand break (DSB). This complex pathway includes the Rad51-DNA nucleoprotein filament as its primary machinery. Current methodology for studying HR proteins includes the use of fluorescently labeled DNA to probe for HR dynamics. This technique limits the number of proteins that can be involved in experimentation, and often only works as an end reporter. The work here aims at improving upon standard techniques by creating two fluorescent protein probes. The first probe was developed by directly attaching a fluorophore to Saccharomyces cerevisiae Rad51 with the use of click chemistry and the incorporation of unnatural amino acids. This probe could function as a primary reporter on the formation and dissociation of the Rad51-DNA filament in the presence of pro- and anti- HR mediator proteins. The second probe was created by labeling the exterior cysteine residues of Plasmodium falciparum single strand DNA binding protein (SSB) with a fluorophore via maleimide chemistry. This probe acts as a secondary reporter for HR dynamics by signaling for when free single stranded DNA (ssDNA) is available.
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El, Marrouni El Ghazaoui Abdellatif. "Synthesis of unusual alpha-amino acids and study of the effect of their incorporation into antimicrobial peptides. Total synthesis of biactive marine natural products and analogues thereof." Doctoral thesis, Universitat de Girona, 2012. http://hdl.handle.net/10803/80815.
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The principle theme of this thesis was the synthesis of bioactive compounds. To this end, this work was focus on two main projects. The first one, which was carried out in the Department of Chemistry of the University of Girona under the supervision of Dr Montserrat Heras, concerned the synthesis of new unnatural amino acids bearing a pyrimidine ring within their side chain for incorporation into the antimicrobial peptide BP100 following a rational design in order to improve its biological profile. On the other hand, the second chapter of this thesis was developed in collaboration with the Laboratoire de Chimie Organique (ESPCI-ParisTech, Paris, France) under the guidance of Pr Janine Cossy and Dr Arseniyadis. This chapter was centered on the total synthesis of three marine natural products with complex structures and interesting biological activities: acremolide B, (–) bitungolide F and lyngbouilloside.Aquesta tesi s'ha centrat en la preparació de nous compostos bioactius seguint dues estratègies diferents. El primer projecte es va portar a terme sota la supervisió de la Dra. Montserrat Heras del grup LIPPSO del Departament de Química i ha permés el desenvolupament de noves metodologies per la síntesi de nous aminoàcids no naturals. i el seu ús en la preparació d'anàlegs del pèptid antimicrobià BP100 amb l'objectiu de millorar-ne les propietats biològiques. El segon projecte és fruit de la col•laboració amb la Prof. Janine Cossy i el Dr. Stellios Arseniyadis del "Laboratoire de Chimie Organique" de l'Ecole Superieur de Physique et Chimie Industrielles (ESPCI-ParisTech, Paris, França). I ha permés posar a punt tres estratègies sintètiques convergents i versàtils per l’obtenció de tres productes naturals de gran complexitat estructural i interessants activitats biològiques – l'acremolide B, la bitungolide F i la lyngbouilloside – aïllats recentment del fons marí de diferents punts del món.
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Mayer, Susanne Veronika [Verfasser], Kathrin [Akademischer Betreuer] Lang, Kathrin [Gutachter] Lang, and Stephan [Gutachter] Hacker. "Expanding the chemical biology toolbox: Site-specific incorporation of unnatural amino acids and bioorthogonal protein labeling to study structure and function of proteins / Susanne Veronika Mayer ; Gutachter: Kathrin Lang, Stephan Hacker ; Betreuer: Kathrin Lang." München : Universitätsbibliothek der TU München, 2019. http://d-nb.info/1215293305/34.
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Joel, Smita. "ENGINEERING PROTEINS WITH UNIQUE CHARACTERISTICS FOR DIAGNOSTICS AND BIOSENSORS." UKnowledge, 2011. http://uknowledge.uky.edu/gradschool_diss/180.
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Proteins possess a broad range of structural and functional properties and, therefore, can be employed in a variety of biomedical applications. While a good number of protein-based biosensing systems and biosensors for target analytes have been developed, the search for versatile, highly sensitive and selective sensors with long term stability able to provide fast detection of target analytes continues to be a challenge. To that end, we now report the design and development of modified proteins with tailored characteristics and their further utilization in the development of biosensing systems.
We take advantage of binding proteins that undergo a change in conformation upon binding to their respective target ligand analytes for the development of highly selective biosensing systems. The first class of binding proteins that was explored for this purpose was antibodies. A non-canonical site in the variable region of a monoclonal antibody was tagged with a fluorescent probe to sense the binding of analyte to its corresponding antigen-binding site. The strategy employed for designing antibodysensing molecules is universal as it can be employed for sensing any biomolecule of interest provided that there is an available antibody against the target ligand analyte.
In a second strategy, we utilized designer glucose recognition proteins (GRPs) that were prepared by incorporation of unnatural amino acids in the glucose/galactose binding protein (GBP) of Escherichia coli and its truncated fragments. By taking advantage of the global incorporation method, we were able to fine-tune the binding affinity and thermal stability of the proteins, thus, allowing for the development of a reagentless fluorescence based fiber optic glucose biosensor capable of monitoring glucose in the hypoglycemic, normal, and hyperglycemic range, as well as in the hypothermic and hyperthermic temperature range.
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Kirov, Miroslav [Verfasser], Lutz [Akademischer Betreuer] Schmitt, and Ulrich [Akademischer Betreuer] Schulte. "The incorporation of an unnatural amino acid to study the nucleotide binding domain of the ABC transporter HlyB from Escherichia coli / Miroslav Kirov. Gutachter: Ulrich Schulte. Betreuer: Lutz Schmitt." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2014. http://d-nb.info/1051076803/34.
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Reeve, P. "Functionalising unnatural amino acids." Thesis, University of Sheffield, 2017. http://etheses.whiterose.ac.uk/19650/.
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Middleton, Richard John. "Synthesis of unnatural [alpha] - amino acids." Thesis, Imperial College London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338866.
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Fryer, Andrew Mark. "Syntheses of natural and unnatural kainoid amino acids." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393580.
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Reddington, Samuel C. "Introducing novel protein functionality using unnatural amino acids." Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/56226/.
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This thesis examines the tolerance and effects of unnatural amino acid (Uaa) incorporation into proteins in Escherichia coli using an expanded genetic code. Uaa incorporation was used to alter or install new properties in the target proteins, superfolder Green Fluorescent Protein (sfGFP) and cytochrome b562. Chapter 3 deals with the technical aspects of Uaa incorporation including orthogonality of the machinery and yield. Substitution of residues in sfGFP for unnatural analogues of tyrosine was shown to be a valuable way of altering the properties of the protein. Variants were generated with red-shifted fluorescence and altered excitation spectra. The majority of this work focused on the Uaa, p-azido-L-phenylalanine (azPhe) as it has a number of properties that would be desirable for use in proteins such as photoreactivity and selective reactivity with alkynes. By incorporating azPhe into key residues of sfGFP, variants were created that could be controlled using light (Chapter 4). Light-dependant fluorescence activation, deactivation and switching were demonstrated in vitro and in live cells. The molecular basis for these changes was investigated by a combination of spectroscopy and X-ray crystallography in Chapter 5. The photoreactivity of azPhe was exploited for a different purpose in Chapter 6. Proteins were used as an alternative to synthetic cages for studying low temperature phenyl azide photochemistry. Here, two radicals (anilino and triplet phenyl nitrene) were successfully caged and detected on photolysis, with the radical observed dependant on the protein environment. Finally, in Chapter 7 the selective reactivity of azPhe was used to create proteins capable of site-specific modification (via Click chemistry). The position of azPhe on the protein surface was shown to have a significant effect on reaction yield and kinetics. Modification was used to install proteins with novel properties such as red-shifted fluorescence emission and the ability to bind to non-biological materials like graphene.
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MAZZOLENI, ELISA. "Exploration of new techniques for purification and chemo-selective conjugation of bioreagents for immunodiagnostic applications." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/68468.
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Antigene e anticorpo sono i due reagenti chiave di un saggio immunodiagnostico. L’indagine di nuove tecniche e il miglioramento di processi quali la purificazione e la marcatura sito-specifica di antigeni e anticorpi possono promuovere lo sviluppo di nuovi reagenti più efficienti capaci di migliorare la performance dei saggi immunodiagnostici.
La prima parte di questa tesi è stata focalizzata sull’esplorazione di tecniche biotecnologiche innovative nella produzione di antigeni al fine di migliorare i saggi per la rilevazione degli anticorpi IgM e IgG specifici per il virus Epstein-Barr. Il virus EBV è causa della mononucleosi infettiva ed è associato ad un crescente numero di tumori; per questa motivo è importante sviluppare saggi diagnostici per la rilevazione di EBV ad alta specificità e sensibilità. La proteina VCA p18 è uno degli antigeni più importanti per la diagnosi di EBV. I saggi attuali Diasorin LIAISON EBV VCA IgM and IgG si basano su un singolo antigene corrispondente alla regione C-terminale immunodominante della proteina p18, immobilizzata su fase solida. I vari metodi esplorati in questa tesi hanno permesso di ottenere diverse varianti dell’antigene p18 con lo scopo di migliorare le prestazioni dei saggi EBV VCA IgM e IgG a diversi livelli: 1_produzione dell’antigene p18; 2_immobilizzazione dell’antigene p18 su fase solida; 3_formato di saggio. 1_La lunghezza della regione C-terminale della proteina p18 (57aa), risulta essere considerevole per il processo sintetico ma, allo stesso tempo, troppo piccola per essere prodotta in modo efficiente per via ricombinante. Per superare questo problema, abbiamo esplorato il sistema Elastin Like Polypeptides (ELP)-Inteina basato sull’uso di una proteina capace di effettuare auto-cleavage (inteina) e un tag responsivo alla temperatura (ELP). Questa tecnica si è rivelata un eccellente sistema per la produzione del peptide p18. 2_Lo sviluppo di diverse varianti dell’antigene p18 ha permesso di esplorare varie tecniche per l’immobilizzazione dello stesso antigene su fase solida: coating covalente diretto, attraverso il complesso streptavidina-biotina e attraverso l’uso dei peptidi leucine zipper (o velcro). L’immobilizzazione del peptide p18 su fase solida attraverso questi vari metodi è avvenuta con successo e l’attività immunochimica dell’antigene, immobilizzato con queste tecniche innovative, è comparabile o migliore rispetto a quella del peptide sintetico utilizzato nei saggi attuali. 3_Nonostante il saggio Diasorin LIAISON EBV VCA IgM abbia una buona performance analitica, al fine di ottenere un aumento di specificità, è stato esplorato un nuovo tipo di formato di saggio. Sfortunatamente i risultati indicano che questo diverso tipo di formato raggiunge un livello di specificità minore rispetto a quello del saggio attuale.
La seconda parte di questa tesi è stata focalizzata sull’esplorazione di un metodo innovativo per la marcatura sito specifica degli anticorpi. Uno degli approcci più promettenti è basato sulla generazione di gruppi tiolo liberi attraverso la riduzione parziale e selettiva dei ponti disolfuro inter-catena presenti a livello della “hinge region” e la loro reazione con molecole marcanti caratterizzate dal possedere gruppi funzionali reattivi verso i gruppi sulfidrilici. Questa tecnologia è stata utilizzata per la biotinilazione di due diversi anticorpi usati attualmente per il rilevamento della proteina virale p24 di HIV e per l’antigene FGF23. I risultati suggeriscono che la biotinilazione sito-specifica rispetto a quella classica random promuove un miglioramento dell'attività immunochimica degli anticorpi con una conseguente ottimizzazione della performance dei saggi immunodiagnostici.Antigen and antibody are the two key reagents for an immunodiagnostic assay. Investigation of new techniques and improvement of processes such as purification and site-specific labeling of antigen and antibody molecules can promote the development of new more powerful bioreagents able of improving the performance of immunodiagnostic assays. The first part of this thesis aimed to explore innovative biotechnology techniques in antigen production for the improvement of immunoassays that allow the detection of antibodies directed against the Epstein-Barr virus. EBV virus is the causative agent of infectious mononucleosis and it is considered to be associated with a still increasing number of tumors; for this reason it is important to develop diagnostic assays for EBV detection with high specificity and sensitivity. The viral capsid protein VCA p18 is one of the most important antigens for the diagnosis of EBV. The current Diasorin LIAISON EBV VCA IgM and IgG assays rely on a single antigen, consisting in a synthetic peptide corresponding to the immunodominant C-terminal portion of the p18 protein, which is immobilized on solid phase (indirect format). The several methods explored in this thesis have allowed to obtain different variants of the p18 antigen with the aim to improve the performance of DiaSorin LIAISON EBV VCA IgM and IgG assays at different levels: 1_production of p18 antigen; 2_immobilization of p18 antigen on solid phase; 3_immunoassay format. 1_ The length of the immunodominant C-terminal portion of the p18 protein (57aa) appears to be considerable for the synthetic route but, at the same time, too small to be effectively produced in a recombinant fashion. To overcome this problem, we explored the Elastin Like Polypeptides (ELP)-Intein system, a method based on the use of a self-cleavable protein (the intein) and a temperature responsive tag (ELP). This technique has proved to be an excellent system for the preparation of the p18 peptide. 2_The development of different variants of p18 antigen has enabled to explore different techniques for the immobilization of the same antigen on solid phase: direct covalent coating, through streptavidin-biotin complex and through an innovative procedure based on the use of leucine zipper (or “velcro”) peptides. The immobilization of the p18 peptide on solid phase through these different methods occurred successfully and the immunochemical activity of the antigen, immobilized with these innovative techniques, is comparable or better than that of the synthetic peptide used in the current immunoassays. 3_Despite the DiaSorin LIAISON EBV VCA IgM immonoassay has a good analytical performance, in order to obtain an increase of specificity, a new assay format was explored. Unfortunately, the results indicate that this different type of format reaches a lower level of specificity than that of current assay. The second part of this thesis aimed to explore an innovative method for the site-specific labeling of antibodies. One the most promising approach is based on the generation of free thiol groups by selective partial reduction of the interchain disulfide bridges present at the level of the “hinge region” and their reaction to labels carrying sulfhydryl-reactive chemical groups. This technology was used for the biotinylation of two different antibodies currently used in immunoassays for the HIV p24 viral protein and for FGF23 antigen detection. The results suggest that the site-specific biotinylation compared to the random traditional biotinylation promotes a great improvement of antibodies immunochemical activity with a consequent optimization of immunodiagnostic assays performance.
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Bilgiçer, Zihni Basar. "Protein design using unnatural amino acids with fluorinated side chains /." Thesis, Connect to Dissertations & Theses @ Tufts University, 2005.
Find full textAbstract:
Thesis (Ph.D.)--Tufts University, 2005.Adviser: Krishna Kumar. Submitted to the Dept. of Chemistry. Includes bibliographical references. Access restricted to members of the Tufts University community. Also available via the World Wide Web;
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Valancogne, Ingrid. "Synthesis of unnatural amino acids and dipeptides for potential catalysts." Thesis, University of Newcastle Upon Tyne, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300361.
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Bhushan, Bhaskar. "Unnatural amino acids as metal-mediated probes of biological function." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:4b1cbed6-1151-4b9f-ad97-aa5765cc9384.
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Conjugation reactions on proteins have been used to access various post-translational modifications, for targeted delivery of drugs, for microscopy, and in studying receptor-ligand interactions. However, the ability to modify native proteins is constrained by the reactive functionalities of naturally occurring amino acids. This has driven research into the incorporation of unnatural amino acids (UAAs) into proteins. Research in this area has been motivated both by the possibility of increasing the breadth of chemical techniques for protein modification by introducing novel 'bio-orthogonal' reactive groups via UAA incorporation, as well as generating well-defined conjugates by the site-selective incorporation of these UAAs into proteins. The objective of this thesis is to both expand the diversity of UAAs for access to new metal-mediated reactions on proteins, as well as to utilise these reactions to reveal functional information about a range of biological systems. A brief introduction into current protein conjugation and UAA incorporation methods will be made in Chapter 1. In Chapter 2, the genetic incorporation of alkene-bearing UAAs into recombinant proteins expressed in both bacterial and mammalian systems is discussed. This technique is demonstrated to enable Ru-catalysed olefin cross-metathesis (CM) reactions on the resultant proteins. This work builds upon previously established methods to chemically incorporate CM handles onto proteins. The rational design of UAAs, as well as assays and modelling studies to screen them for recognition by the cellular incorporation machinery are discussed in detail. The expression of a range of alkene-tagged recombinant proteins, their complete characterisation, as well as the development of a more general protocol for on-protein CM is elucidated. In Chapter 3, the utility of UAA incorporation to probe mammalian cell translation systems is examined. Incorporation of an azide-bearing UAA, in addition to heavy stable-isotope labelled amino acids is used to uncover a previously unreported system of protein synthesis in mammalian cell nuclei, along with rapid metabolic degradation of the synthesised peptides. Various orthogonal methods for the detection of this system as well as possible reasons for its conservation are discussed. In Chapter 4, UAA incorporation and metal-mediated bioconjugation reactions are utilised in the development of a novel and generally applicable proteomics technique. This technique is used to determine quantitative changes in cell proteomes in response to external stimuli, and may be applied to systems to which traditional proteomics techniques cannot, such as ex vivo primary cells. Finally, in Chapter 5, further applications of UAA incorporation are discussed. Preliminary results are reported in efforts to use UAAs in the vibrational Raman microscopic imaging of biological systems, in generating HIV vaccines, and inducing T-cell stimulation.
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Drummond, Lorna J. "New methodology for the stereoselective synthesis of unnatural alpha-amino acids." Thesis, University of Glasgow, 2011. http://theses.gla.ac.uk/2827/.
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New general methodology for the stereoselective synthesis of unnatural alpha-amino acids has been developed. Early work focussed on investigating methods for the generation of chiral allylic alcohols using cross-metathesis of a simple enone with various terminal alkenes, followed by an asymmetric ketone reduction. The allylic alcohols were then converted to protected allylic amines via Overman rearrangement chemistry. Oxidative alkene cleavage and hydrolysis of these intermediates generated a range of alpha-amino acid targets. Attempts were also made to apply the developed methodology to the synthesis of a simple alpha,alpha-disubstituted alpha-amino acid target. The Overman rearrangement was also applied to the generation of a late-stage intermediate which could be used in the synthesis of (2S,3S)-capreomycidine, a component of a number of peptides which exhibit antibacterial activity.
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Li, Shuwei Dervan Peter B. Roberts Richard W. "In vitro selection of mRNA-display libraries containing unnatural amino acids /." Diss., Pasadena, Calif. : California Institute of Technology, 2003. http://resolver.caltech.edu/CaltechETD:etd-05182003-185245.
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Halonski, John. "Utilization of Unnatural Amino Acids to Modulate Protein Structure and Function." W&M ScholarWorks, 2018. https://scholarworks.wm.edu/etd/1530192786.
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Proteins are capable of an astounding array of functions using only the 20 canonical amino acids; however, the ability to add new functional groups to the genetic code through the utilization of unnatural amino acids (UAAs) has greatly expanded our ability to study and manipulate proteins. By expanding the diversity of functional groups within proteins, a wide variety of applications in industry as well as in fields such as diagnostics, biochemistry, and materials science are now possible. These applications have further been expanded through the development and optimization of bioorthogonal reactions which can occur under physiological conditions with a high degree of specificity, allowing modulation of the structure and function of proteins within their natural state. Several applications of UAA technology involving bioorthogonal reactions will be explored in this thesis. Optimization of a previously developed bioorthogonal Glaser-Hay reaction between a protein and a fluorophore will be discussed. A further application of the Glaser-Hay reaction involving natural product synthesis will also be explored. The utilization of UAA technology to form trivalent conjugates containing multiple functionalities will be described. Furthermore, the development and optimization of organic reactions leading to the formation of trivalent structures will be explored with the intention of translating these reactions to biological systems. The ability to site-specifically immobilize a hyperthermophilic carboxylesterase enzyme onto a stabilizing resin will also be discussed and the benefits of protein immobilization will be demonstrated. Finally, the synthesis and development of novel TMS and aldehyde UAAs will be described and their applications will be explored. The applications highlighted in each chapter demonstrate some of the numerous possibilities that can be explored through modulation of the building blocks of proteins.
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Gao, Lin. "The development of mRNA display : synthesis of peptides containing unnatural amino acids." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/8883.
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Currently, one of main challenges in drug discovery is the generation of diverse compound libraries that can be easily screened to identify potential inhibitors of therapeutic targets. mRNA display is a technique to create a vast library of unique peptide molecules that can be easily screened for this purpose. mRNA display generates peptides that are covalently linked to their encoding mRNA templates. The utilization of a reconstituted translation system makes it possible to incorporate unnatural amino acids with various structures into peptides. In this project, mRNA display and an E. coli-based reconstituted translation system are combined to create peptides comprised of unnatural amino acids that are covalently attached to their encoding nucleic acid. The incorporation of N-methyl or cyclic unnatural amino acids into peptides are believed to contribute to their resistance to proteolytic degradation. My project has two main objectives. The first one is to use the mRNA display and reconstituted translation system to assemble hexa-peptides with N-methyl or cyclic unnatural amino acids. The compatibilities of these amino acids with the translation system are individually tested. The second objective is to optimize the DNA linker length for an increase in full-length translation product from what is achieved with a standard linker. These efforts will enable the synthesis of peptide-like libraries using this drug discovery platform.
N-Methyl or cyclic amino acid-pdCpA conjugates were synthesized as building blocks of E. coli reconstituted translation. The following unnatural amino acids, N-Me-L-valine, L-azetidine-2-carboxylic acid, N-Me-L-phenylalanine, L-homoproline, L-octahydro- indole-2-carboxylic acid, N-Me-L-aminohexanoic acid, and L-proline, were chemically acylated to tRNAGCC and translated into hexa-peptide with biocytin as the last amino acid. A full-length translation product of each amino acid was observed using the streptavidin-binding assay. The successful incorporation of N-methyl or cyclic amino acids into a peptide were indirectly shown by some resistance to proteinase K treatment as compared to control (natural L-alanine). The DNA linker was then optimized to increase the portion of full-length translation product in the translation mixture. A DNA linker bearing a 26mer chain of polyadenosine provided the highest observed percentage of translated full-length material.
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Klippenstein, Viktoria [Verfasser]. "Photoinactivation of glutamate receptors by genetically encoded unnatural amino acids / Viktoria Klippenstein." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2015. http://d-nb.info/1071088998/34.
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Mata, David Garcia. "Understanding Protein Structure And Function Using Rational Design And Unnatural Amino Acids." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1338392020.
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Ueda, Takehiko. "Photoregulation of Engineered Proteins by Incorporation of Nonnatural Amino Acids." Kyoto University, 1995. http://hdl.handle.net/2433/160777.
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本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものであるKyoto University (京都大学)
0048
新制・課程博士
博士(工学)
甲第6139号
工博第1466号
新制||工||1004(附属図書館)
UT51-95-V427
京都大学大学院工学研究科高分子化学専攻
(主査)教授 今西 幸男, 教授 砂本 順三, 教授 田中 渥夫
学位規則第4条第1項該当
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Kwon, Inchan Tirrell David A. "Protein engineering via site-specific incorporation of nonnatural amino acids /." Diss., Pasadena, Calif. : Caltech, 2007. http://resolver.caltech.edu/CaltechETD:etd-01222007-010333.
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Liu, Zhihua. "THE DESIGN AND SYNTHESIS OF NOVEL UNNATURAL AMINO ACIDS AND THE DESIGN AND SYNTHESIS OF PEPTIDES & PEPTIDOMIMETICS CONTAINING UNNATURAL AMINO ACIDS FOR THE STUDY OF G-PROTEIN COUPLED RECEPTORS." Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/204274.
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Nature has gifted peptides as important modulators in the human body, but these types of molecules often have not been favored when we were looking for therapeutic agents. The poor bioavailability, fast degradation and until recent high manufacturing costs of some bioactive peptides lowered their potential usage in the health industry. Under these circumstances, unnatural amino acids were developed as indispensible tools providing enormous support to peptide science. By incorporating proper unnatural amino acids into a peptide or protein, we now can significantly improve peptide's or protein's half-life, cell permeability, bio-distribution, etc. In addition, their potency and receptor/acceptor selectivity could also be enhanced. Site-specific modifications of peptides and proteins under physiological conditions with the use of unnatural amino acids also have been made easier with the advance of biotechnology. Therefore, my research described in this dissertation contributes to the efforts in the development of novel unnatural amino acids. In particular, I have focused on novel methods in the synthesis of anti beta-functionalized gamma,delta-unsaturated amino acids. These amino acids have special interests in peptide chemistry: they can provide conformational constraints to the peptide 3D structures; the beta-functionalization allows the introduction of pharmaceutically interesting side chain groups; and the terminal double bond which is orthogonal to peptide synthesis provides access to further chemical modifications. Two general methodologies for the synthesis of both racemic and optically active anti beta-functionalized gamma,delta--unsaturated amino acids were developed by using the thio-Claisen rearrangement (TCR) reaction. Excellent diastereoselectivies and enantioselectivities were obtained when C2-symmetric chiral auxiliaries were selected to control the stereochemistry outcome. The mechanism and the scope of the TCR reaction were also studied, showing unique advantages in the preparation of these biological interesting amino acids.Another effort of developing angiotensin II type 1 (AT1) receptor biased peptide ligands is also documented in this dissertation. The AT1 receptor is a 7-transmembrane G-protein coupled receptor, which recent researches have shown could be activated through a beta-arrestins only, but G-protein independent, pathway. We synthesized 12 analogs of Sar1,Ile4,Ile8-AngII (SII), and tested them in biological assays, and obtained valuable information for further "perfect" biased ligands design.
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Fowler, Lindsay S. "Synthesis of unnatural enone-containing α-amino acids : precursors to chiral N-heterocycles." Thesis, University of Glasgow, 2011. http://theses.gla.ac.uk/2524/.
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A fast and efficient synthetic route was developed for the synthesis of a novel class of enone-containing alpha-amino acid. An amino acid-derived beta-ketophosphonate ester was subjected to Horner-Wadsworth-Emmons conditions using a variety of aldehydes to produce a diverse library of alpha,beta-unsaturated amino acids. E-Configured enone-containing amino acids were also deprotected using a two-stage approach to give the parent alpha-amino acids. A minor modification to the route enabled the synthesis of Z-configured enones via the Still-Gennari reaction. A small library of Z-enones was produced using various aldehydes. Enone-functionalised alpha-amino acids were employed as substrates for an intramolecular cyclisation reaction to generate 6-substituted-4-oxopipecolic acids. A diastereoselective one-pot reductive amination/cyclisation strategy was developed to gain access to the anti-diastereomer of the chiral N-heterocycles. A small selection of 6-substituted-4-oxopipecolic acids was synthesised. 6-Substituted-4-oxopipecolic acids were also reduced diastereoselectively to generate 4- hydroxypipecolic acid analogues.
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Ieong, Ka-Weng. "Rate and Accuracy of Bacterial Protein Synthesis with Natural and Unnatural Amino Acids." Doctoral thesis, Uppsala universitet, Struktur- och molekylärbiologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-235534.
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This thesis addresses different questions regarding the rate, efficiency, and accuracy of peptide bond formation with natural as well as unnatural amino acids: Which step is rate-limiting during peptide bond formation? How does the accuracy vary with different transfer RNAs (tRNAs) and codons and how is it relevant to the living cells? Does proofreading selection of codon reading occur in a single- or multi-step manner as theoretically suggested? How does the E. coli translation system discriminate unnatural amino acids? Based on that, how to improve the incorporation efficiencies of unnatural amino acids? Based on the study on pH dependence of peptide bond formation, we show that the rate of the chemistry of peptidyl transfer to aminoacyl-tRNA (AA-tRNA) Gly-tRNAGly or Pro-tRNAPro limits the rate of peptide bond formation at physiological pH 7.5, and this could possibly be true for peptidyl transfer to all natural AA-tRNAs at physiological condition. By studying the efficiency-accuracy trade-off for codon reading by seven AA-tRNA containing ternary complexes, we observe a large variation on the accuracy of initial codon selection and identify several error hot-spots. The maximal accuracy varied 400-fold from 200 to 84000 depending on the tRNA identity, the type and position of the mismatches. We also propose a proofreading mechanism that contains two irreversible steps in sequence. This could be highly relevant to the living cells in relation to maintaining both high accuracy and high efficiency in protein synthesis. Finally, we show that peptide bond formation with small and large non-N-alkylated L- unnatural amino acids proceed at rates similar to those with natural amino acids Phe and Ala on the ribosome. Interestingly, the large side chain of the bulky unnatural amino acid only weakens its binding for elongation factor Tu (EF-Tu) but not slows down peptidyl transfer on the ribosome. Our results also suggest that the efficiency of unnatural amino acid incorporation could be improved in general by increasing EF-Tu concentration, lowering the reaction temperature and / or using tRNA bodies with optimal affinities for EF-Tu in the translation system.
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Deboves, Herve Jean Claude. "The application of organometallic chemistry to the synthesis of new unnatural amino acids." Thesis, University of Newcastle Upon Tyne, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324929.
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LOCARNO, SILVIA ALICE. "UNNATURAL AMINO ACIDS AS SYNTHETIC TOOLS FOR THE PREPARATION OF COMPLEX MOLECULAR ARCHITECTURES." Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/607175.
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Self-assembly is the process by which an organized structure spontaneously is formed from individual components, as a result of specific, local interactions between the units.
In recent years, peptide-based self-assembled structures have emerged as a powerful approach for developing soft and hybrid materials due to their biocompatibility, biodegradability and easily tuning properties of the final structure.
The introduction of non-natural amino acids into a peptide backbone imparts additional features such as reduced conformational flexibility, high tendency to adopt a well-defined secondary structure and enhanced metabolic stability.
My PhD thesis is focused on the synthesis of ultrashort peptides containing non-natural scaffolds which are able to self-assemble and form supramolecular structures. The thesis is divided into three chapters, each one reports a project regarding the synthesis and chemical characterization of scaffolds which can be inserted in peptide sequences and exploited for developing soft or hybrid materials by self-assembly.
The first chapter concerns the supramolecular assemblies of spherical shape obtained from different peptides containing Cα,α-tetrasubstituted amino acids. Firstly, the results on a pentapeptide containing norbornene amino acid is reported (work published on RSC Advances). Then, the simplification of this structure is reported until obtaining a pentapeptide containing Ala-Aib motif only. The ability of this peptide to form aggregates that can be exploited for the encapsulation of hydrophobic molecules has been studied. As a proof of concept the well-known curcumin molecule has been used. The interaction of the drug molecules with peptide aggregates has been studied using the absorption and the intrinsic fluorescence emission of curcumin.
Starting from this peptide, it is also possible to develop peptide-ligands which stabilize gold nanoparticles in aqueous solution. The stability of nanoparticles has been studied using DLS and UV-vis spectroscopy.
The second chapter reports on a β2,3-diarylamino acid, developed in our group, which is non-natural analogous of the dipeptide Phe-Phe. This amino acid has been used to develop hybrid antifouling film which has been characterized using different techniques (contact angles values, QCM-D). The third chapter concerns the preparation of an original heterocyclic scaffold to be inserted into a peptide backbone and its exploitation for the formation of soft materials by the way of an “induced assembly”. The electrospinning technique allowed the formation of electrospun nanofibers from these non-natural peptide-based small molecules and their characterization with several techniques (SEM, AFM, FT-IR, Raman) is reported.
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Brandt, Gabriel Shaw Rees Douglas C. Dougherty Dennis A. "Site-specific incorporation of synthetic amino acids into functioning ion channels /." Diss., Pasadena, Calif. : California Institute of Technology, 2003. http://resolver.caltech.edu/CaltechETD:etd-01202003-221429.
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Millward, Steven Wesley Roberts Richard W. Dervan Peter B. "The design, synthesis, and evolution of macrocyclic mRNA display libraries containing unnatural amino acids /." Diss., Pasadena, Calif. : California Institute of Technology, 2007. http://resolver.caltech.edu/CaltechETD:etd-05142007-161158.
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Streichert, Katharina [Verfasser]. "Co- and posttranslational engineering of the therapeutic glycoprotein erythropoietin with unnatural amino acids / Katharina Streichert." Konstanz : Bibliothek der Universität Konstanz, 2016. http://d-nb.info/1115727133/34.
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Rowles, Ian. "Development of novel ammonia-lyase biocatalysts for the production of unnatural amino acids for industry." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.498954.
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Nguyen, Thi D. T. "Antilarval substituted phenols, distribution of tricyclic pyrones in mice, and synthesis of unnatural amino acids." Diss., Kansas State University, 2014. http://hdl.handle.net/2097/18199.
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Doctor of PhilosophyDepartment of Chemistry
Duy H. Hua
Three research projects were carried out and they are described below. The synthesis of substituted phenolic compounds including halogenated di- and trihydroxybenzenes, aminophenols, and substituted di-tert-butylphenols are described. Redox potentials of the synthesized molecules along with various known laccase substrates were measured, and an inverse relationship between the oxidation potential and the efficiency of oxidation by laccase of halogenated hydroxybenzenes and aminophenols is demonstrated. The synthesized substituted phenols were found to be substrates but not inhibitors of laccase. We discovered a new class of di-tert-butylphenols compounds that inhibits the growth of mosquito larvae at low concentrations. Compound 17, 2,4-di-tert-butyl-6-(3-methyl-2-butenyl) phenol caused greater than 98% mortality of third-instar larvae of Anopheles gambiae in the concentration of 0.18 µM. These compounds do not inhibit laccases. It appears that they affect a new target of the mosquito that is different from those of currently existing pesticides. Two anti-Alzheimer molecules, CP2 and TP70, discovered in our laboratory were studied for their pharmacokinetics and distribution. The distribution of CP2 and TP70 in mouse brain region and various tissues of mice were examined. HPLC analysis revealed that CP2 treatment in primary neurons accumulates in mitochondria fraction. Similarly, the amount of CP2 in the brain tissue from wild type and APP/PS1 mice treated with 25 mg/kg/daily for 2 months also have the highest concentration in the mitochondria fractions in the hippocampus. The results show that CP2 and TP70 can penetrate the blood brain barrier and accumulate in the tissue in significant amounts. Pharmacokinetics and bioavailability of compound TP70 were determined. Area under the curve and bioavailability value F were calculated, and data show that TP70 has a good PK profile and bioavailability. For the preparation of a novel tripeptidyl norovirus 3C-like protease (3CL[superscript]pro) inhibitor, the P3 unnatural amino acid, (S)-3-hydroxyphenylalanine was synthesized. The P3 is designed to increase the polarity with the addition of the alcohol group. After combining the P3 unnatural amino acid with the P1 and P2 to form the novel tripeptidyl compound, a study comparing the relations between the structure and its activity (SAR) will confirm whether prediction is correct in our pursuit for an antiviral therapeutic drug in the form of a protease inhibitor.
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Wynn, Jessica Elaine. "Functionalizing Branched Peptides with Unnatural Amino Acids Toward Targeting HIV-1 RRE RNA and Microbials." Diss., Virginia Tech, 2016. http://hdl.handle.net/10919/82227.
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The interaction of the protein Rev with Rev Response Element (RRE) RNA is critical to the HIV-1 life cycle as this complex is required for the export of singly-spliced and unspliced mRNAs from the nucleus to the cytoplasm. Disruption of this interaction is considered to be a powerful strategy towards the development of HIV-1 therapeutics. Therefore, we have developed several branched peptide libraries containing unnatural amino acids to target the high-affinity binding site of RRE RNA (RRE IIB), with the idea that branching in peptides can provide multivalent contacts with folded RNA structures and boost binding affinity and selectivity for the target. Unnatural amino acids were incorporated into the library design to encourage non-canonical interactions with the RNA and to improve proteolytic stability.
The on-bead high-throughput screening of our first branched peptide library (46,656 sequences) against HIV-1 RRE RNA generated hit peptides with binding affinities in the low micromolar range. We demonstrated that branching in the peptide is required for efficient binding and selectivity towards the RNA, and that the peptides bind a large surface area of RRE IIB. Introduction of boronic acids into branched peptides boosted selectivity of the peptides for RRE IIB, and proved to be a novel and tunable mode of binding towards RNA. Additionally, we revealed that these branched peptide boronic acids (BPBAs) were cell permeable and non-toxic. One BPBA (BPBA3) bound RRE IIB selectively and was able to inhibit HIV-1 replication in vitro, revealing enzymatic cleavage of the RNA upon binding.
A second generation BPBA library that introduced acridinyl lysine as an intercalator
(4,096 sequences) was screened against RRE IIB. Several hit compounds bound in the low nanomolar regime, and a significant number of compounds inhibited HIV-1 replication in vitro. These BPBAs were also found to severely inhibit the microbial growth of bacteria and fungus, with MICs as low as 1 µg/mL against Staphylococcus aureus, Candida albicans, and Escherichia coli. These compounds were also found to significantly inhibit biofilm formation and growth, and were non-hemolytic.
High-throughput screening of a third generation BPBA library containing all unnatural amino acids (46,656 sequences) revealed several hits that bound RRE IIB RNA in the nanomolar range. Sequence motifs present in the hit peptides suggested that the location and composition of amino acids within the branched peptide structure were important for recognizing the RNA target. In particular, lead compounds 2C5 and 4B3 demonstrated selectivity towards RRE, and footprinting experiments combined with SHAPE experiments revealed different interactions of the peptides with the RNA Toxicity assays revealed no impact on cell viability for the majority of hit sequences tested up to 100 µM, and several compounds also demonstrated inhibition of HIV-1 replication.Ph. D.
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Knight, William A. "Synthesis of unnatural amino acids for genetic encoding by the pyrrolysyl-tRNA/RNA synthetase system." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3794.
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The complexity of all biomolecules in existence today can be attributed to the variation of the amino acid repertoire. In nature, 20 canonical amino acids are translated to form these biomolecules, however, many of these amino acids have revealed posttranslational modifications (i.e. acetylation, methylation) after incorporation. Amino acids that exhibit PTM are known for their involvement in cellular processes such as DNA repair and DNA replication; these PTMs are commonly found on histones within the chromatin complex. Utilization of in vivo site-specific incorporation has recently reported functionality of post-translationally modified amino acids.1 xii Here we report the synthesis and in vivo site-specific incorporation of the histone PTM, 2-hydroxyisobutyrl lysine (Khib), with the pyrrolysyl tRNA/ RNA synthetase system. This translational machine can better serve to probe Khib for functional benefits. Additionally, this thesis focuses much of its attention on the development of unnatural amino acids (UAA) with optogenetic characteristics. These UAAs, if site-specifically incorporated, can be used to control enzymes and proteins through rapid light perturbation (365nm UV light). Furthermore, discussed is the synthesis of photo-caged threonine and photo-caged serine as potential substrates for the pyrrolysyl translational machinery.
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Lung, Feng-Di Tiffany. "Design and synthesis of receptor-selective peptide ligands, and the synthesis of unnatural amino acids." Diss., The University of Arizona, 1995. http://hdl.handle.net/10150/187116.
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The discovery of endogenous opioid peptides has greatly accelerated research in opioid chemistry and biology. Studies of the physiological and pharmalogical roles of these receptors require highly potent and receptor-selective ligands for μ, δ, and κ receptors. The major goal of this project is to design and synthesize highly potent and κ receptor-selective dynorphin A analogues with specific conformational and topographical features. Therefore, a series of linear and cyclic dynorphin A analogues with global and/or local conformational constraints have been designed, synthesized, and evaluated for their biological activities. Several leads from dynorphin A analogues have been developed, and have provided new insights into requirements for high κ receptor-selectivity and potency. The incorporation of side chain conformationally constrained amino acids can provide new insights into topographical requirements for peptide ligand-receptor binding. An efficient synthesis of 2', β-dimethyltyrosine in large quantities and a new strategy of the synthesis of optically pure isomers of β-methylphenylalanine derivatives have been developed. These unnatural amino acids can be incorporated into peptides for the development of novel peptides with high potency and enhanced receptor-selectivity.