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The Development Of Intrinsically Fluorescent Unnatural Amino Acids For In Vivo Incorporation Into Proteins
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Book Synopsis The Development of Intrinsically Fluorescent Unnatural Amino Acids for in Vivo Incorporation Into Proteins by : Itthipol Sungwienwong
Download or read book The Development of Intrinsically Fluorescent Unnatural Amino Acids for in Vivo Incorporation Into Proteins written by Itthipol Sungwienwong and published by . This book was released on 2018 with total page 306 pages. Available in PDF, EPUB and Kindle. Book excerpt: The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein dynamics either alone or as part of a Förster resonance energy transfer (FRET) or photo-induced electron transfer (eT) probe pair. We have previously reported the genetic incorporation of Acd by an aminoacyl tRNA synthetase (RS). However, this RS, developed from a library of permissive RSs, also incorporates N-phenyl-amino-phenylalanine (Npf), a trace byproduct of one Acd synthetic route. We have performed negative selections in the presence of Npf and analyzed the selectivity of the resulting AcdRSs by in vivo protein expression and detailed kinetic analyses of the purified RSs. We find that selection conferred a ~50-fold increase in selectivity for Acd over Npf, eliminating incorporation of Npf contaminants, and allowing one to use a high yielding Acd synthetic route for improved overall expression of Acd-containing proteins. More generally, our report also provides a cautionary tale on the use of permissive RSs, as well as a strategy for improving selectivity for the target amino acid. In spite of its utility for studying proteins by fluorescence spectroscopy, Acd can potentially be improved by making it longer wavelength or brighter. We reported the synthesis of Acd core derivatives and their photophysical characterization. We also performed ab initio calculations of the absorption and emission spectra of Acd derivatives, which agree well with experimental measurements. The amino acid aminoacridonylalanine (Aad) was synthesized in forms appropriate for genetic incorporation and peptide synthesis. We show that Aad is a superior FRET acceptor to Acd in a peptide cleavage assay, and that Aad can be activated by an aminoacyl tRNA synthetase for genetic incorporation. Together, these results show that we can use computation to design enhanced Acd derivatives which can be used in peptides and proteins. Finally, the Aad synthesis has been improved and it will be further tested in vivo incorporations into proteins, and alkylated Aad core analogs show improved brightness making their use as amino acids promising.
Download or read book Non-Natural Amino Acids written by and published by Academic Press. This book was released on 2009-07-24 with total page 334 pages. Available in PDF, EPUB and Kindle. Book excerpt: By combining the tools of organic chemistry with those of physical biochemistry and cell biology, Non-Natural Amino Acids aims to provide fundamental insights into how proteins work within the context of complex biological systems of biomedical interest. The critically acclaimed laboratory standard for 40 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. With more than 400 volumes published, each Methods in Enzymology volume presents material that is relevant in today's labs -- truly an essential publication for researchers in all fields of life sciences. Demonstrates how the tools and principles of chemistry combined with the molecules and processes of living cells can be combined to create molecules with new properties and functions found neither in nature nor in the test tube Presents new insights into the molecular mechanisms of complex biological and chemical systems that can be gained by studying the structure and function of non-natural molecules Provides a "one-stop shop" for tried and tested essential techniques, eliminating the need to wade through untested or unreliable methods
Book Synopsis In Vivo Incorporation of Unnatural Amino Acids Into Proteins by :
Download or read book In Vivo Incorporation of Unnatural Amino Acids Into Proteins written by and published by . This book was released on 2002 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: A method for the site-specific incorporation of unnatural amino acids into proteins in vivo would significantly facilitate studies of the cellular function of proteins, as well as make possible the biosynthesis of unnatural polymers and proteins with novel structures and activities. Our approach consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pair that are not catalytically competent with all the endogenous Escherichia coli tRNAs an aminoacyl-tRNA synthetases, followed by directed evolution of such orthogonal aminoacyl-tRNA synthetases to alter their amino acid specificities. A new orthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair in E. coli has been derived from the Saccharomyces cerevisiae tRNA (sub Asp) and aspartyl-tRNA syathetase, and the in vitro and in vivo characteristics of this pair were determined. In order to achieve a high specificity for the amino acid, a direct selection for site-specific incorporation of unnatural amino acids into a reporter epitope displayed on the surface of M13 phage has been developed and characterized. Under simulated selection conditions, phage particles displaying aspartate were enriched over 300-fold from a pool of phage displaying asparagine using monoclonal antibodies raised against the aspartate-containing epitope. The direct phage selection offers very high specificity for the amino acid of interest, Which cannot be achieved by conventional methods.
Book Synopsis Protein Evolution in the Presence of an Unnatural Amino Acid by : Amrita Singh
Download or read book Protein Evolution in the Presence of an Unnatural Amino Acid written by Amrita Singh and published by . This book was released on 2012 with total page 430 pages. Available in PDF, EPUB and Kindle. Book excerpt: The field of protein engineering has been greatly augmented by the expansion of the genetic code using unnatural amino acids as well as the development of cell-free synthesis systems with high protein yield. Cell-free synthesis systems have improved considerably since they were first described almost 40 years ago. Residue specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an amino acid depleted cell-free protein synthesis system that can be used to study residue specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines high protein expression yields with a high level of analog substitution in the target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid-depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo format. We use this amino acid depleted cell-free synthesis system for the directed evolution of streptavidin, a protein that finds wide application in molecular biology and biotechnology. We evolve streptavidin using in vitro compartmentalization in emulsions to bind to desthiobiotin and find, at the conclusion of our experiment, that our evolved streptavidin variants are capable of binding to both biotin and desthiobiotin equally well. We also discover a set of mutations for streptavidin that are potentially powerful stabilizing mutations that we believe will be of great use to the greater research community.
Book Synopsis Unnatural Amino Acid Incorporation and Click Chemistry by :
Download or read book Unnatural Amino Acid Incorporation and Click Chemistry written by and published by GRIN Verlag. This book was released on 2018-03-19 with total page 53 pages. Available in PDF, EPUB and Kindle. Book excerpt: Seminar paper from the year 2014 in the subject Chemistry - Bio-chemistry, grade: 1,0, LMU Munich (Chemie), language: English, abstract: In the present work a modified version of yellow fluorescent protein containing an unnatural structural homologue of the natural amino acid pyrrolysine with a norbornene moiety was produced by expression in Escherichia coli. The incorporation of the unnatural amino acid was achieved by amber stop codon suppression method. A bio-othogonal click reaction was performed, binding a synthetic fluorescent dye to the modified protein. All steps towards necessary for obtaining the genetically modified organism were performed and documented. The artificial amino acid, as well as the dye used in the click reaction were synthetically prepared. The success of the project was demonstrated by LC/MS studies of the products. Fluorescence spectroscopy of click reaction product and the protein was performed, but no conclusive proof of FRET effects could as yet be made. This point remains of interest for future studies.
Book Synopsis Fluorescent Analogs of Biomolecular Building Blocks by : Marcus Wilhelmsson
Download or read book Fluorescent Analogs of Biomolecular Building Blocks written by Marcus Wilhelmsson and published by John Wiley & Sons. This book was released on 2016-04-04 with total page 474 pages. Available in PDF, EPUB and Kindle. Book excerpt: Fluorescent Analogs of Biomolecular Building Blocks focuses on the design of fluorescent probes for the four major families of macromolecular building blocks. Compiling the expertise of multiple authors, this book moves from introductory chapters to an exploration of the design, synthesis, and implementation of new fluorescent analogues of biomolecular building blocks, including examples of small-molecule fluorophores and sensors that are part of biomolecular assemblies.
Book Synopsis Site-specific Incorporation of Unnatural Amino Acid Into Protein in Vivo by : Kiket Avi Omer Makoubi
Download or read book Site-specific Incorporation of Unnatural Amino Acid Into Protein in Vivo written by Kiket Avi Omer Makoubi and published by . This book was released on 2012 with total page 506 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Site Specific Incorporation of Amino Acid Analogues Into Proteins In Vivo by :
Download or read book Site Specific Incorporation of Amino Acid Analogues Into Proteins In Vivo written by and published by . This book was released on 2010 with total page 55 pages. Available in PDF, EPUB and Kindle. Book excerpt: The goal of this project is to develop methods for the site-specific insertion in vivo of one or more amino acid analogues into proteins in eubacteria and in eukaryotes. The amino acids to be used include those that are photoactivatable, those that are fluorescent, those that carry reactive side chains such as keto groups, heavy atoms such as iodine, those that act as spectroscopic probes, and those that mimic phosphoserine, phosphothreonine, or phosphotyrosine. Besides providing a method for the production of new biomateirals with novel chemical and biological properties, proteins carrying such amino acid analogues will have wide applications in biology. These include studies on the folding, structure, stability and function of proteins, protein-protein interactions, protein localization and protein dynamics in vivo and signal transduction.
Book Synopsis Studies Directed Towards the Development of an in Vitro Method for the Site-specific Incorporation of Unnatural Amino Acids Into Proteins Using Unassigned Codons in Micrococcus Luteus by : Anne Kathryn Kowal
Download or read book Studies Directed Towards the Development of an in Vitro Method for the Site-specific Incorporation of Unnatural Amino Acids Into Proteins Using Unassigned Codons in Micrococcus Luteus written by Anne Kathryn Kowal and published by . This book was released on 1996 with total page 254 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis In Vivo Incorporation of Unnatural Amino Acids by :
Download or read book In Vivo Incorporation of Unnatural Amino Acids written by and published by . This book was released on 2008 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The invention provides methods and compositions for in vivo incorporation of unnatural amino acids. Also provided are compositions including proteins with unnatural amino acids.
Book Synopsis Fluorescent Proteins I by : Gregor Jung
Download or read book Fluorescent Proteins I written by Gregor Jung and published by Springer Science & Business Media. This book was released on 2011-10-25 with total page 275 pages. Available in PDF, EPUB and Kindle. Book excerpt: Fluorescent proteins are intimately connected to research in the life sciences. Tagging of gene products with fluorescent proteins has revolutionized all areas of biosciences, ranging from fundamental biochemistry to clinical oncology, to environmental research. The discovery of the Green Fluorescent Protein, its first, seminal application and the ingenious development of a broad palette of fluorescence proteins of other colours, was consequently recognised with the Nobel Prize for Chemistry in 2008. Fluorescent Proteins I is devoted to the basic photophysical and photochemical aspects of fluorescent protein technology. Experienced experts highlight colour tuning, the exploration of switching phenomena and respective methods for their investigation. The book provides a thorough understanding of primary molecular processes allowing the design of fluorescent proteins for specific applications.
Book Synopsis Unnatural Amino Acid Incorporation for Genetic Code Expansion in Mammalian Cells by : Jeffrey Kunio Takimoto
Download or read book Unnatural Amino Acid Incorporation for Genetic Code Expansion in Mammalian Cells written by Jeffrey Kunio Takimoto and published by . This book was released on 2011 with total page 150 pages. Available in PDF, EPUB and Kindle. Book excerpt: The genetic code of most organisms was evolved to encode 20 amino acids. Although the ability to encode 20 amino acids provides the basis to translate proteins necessary for life, researchers are also limited to these 20 amino acids for conventional site-directed mutagenesis. The ability to encode unnatural amino acids provides researchers the ability to circumvent the limitation imposed by the genetic code. Genetically encoding unnatural amino acids provides researchers the means to not only mimic naturally occurring posttranslational modifications but also the ability to encode amino acids with new physical or chemical properties to study biological processes. The incorporation of unnatural amino acids into proteins had been developed in Escherichia coli and also in yeast. We have developed a methodology to genetically incorporate unnatural amino acids in mammalian cells in response to an amber codon (UAG). The incorporation of unnatural amino acids is high in E. coli and yeast, but the incorporation in mammalian cells is relatively low. In addition to developing the system to incorporate unnatural amino acids in mammalian cells, we have also improved suppression efficiencies by modifying the synthetase and unnatural amino acid. To incorporate unnatural amino acids in response to an amber codon, the tRNA anticodon is mutated from a GUA to a CUA. We were able to show that engineering the anticodon-binding domain of the synthetase could enhance the recognition of the tRNA and thus increased suppression efficiencies. Furthermore, by masking the carboxyl group of the amino acid by an ester group, we were able to increase the bioavailability of an unnatural amino acid to further increase suppression efficiencies. Most evolved synthetases aminoacylate unnatural amino acids that are structurally similar to the native substrate of the wild-type synthetase. We were able to adapt Methanosarcina mazei pyrrolysine synthetase (PylRS) to charge a considerable disparate amino acid from its native substrate, O-methyl-L-tyrosine. In addition, the X-ray crystal structure was solved for the evolved PylRS complexed with O-methyl-L-tyrosine at 1.75Å. This multifaceted approach provides the basis to engineer the PylRS to incorporate a significantly diverse selection of unnatural amino acids than previously anticipated.
Book Synopsis Protein Engineering Using Unnatural Amino Acids by : Yi Tang
Download or read book Protein Engineering Using Unnatural Amino Acids written by Yi Tang and published by . This book was released on 2002 with total page 354 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Ex Vivo Incorporation of Unnatural Amino Acids Into Recombinant Proteins by : Sviatlana Smolskaya
Download or read book Ex Vivo Incorporation of Unnatural Amino Acids Into Recombinant Proteins written by Sviatlana Smolskaya and published by . This book was released on 2015 with total page 83 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis The Fluorescent Protein Revolution by : Richard N. Day
Download or read book The Fluorescent Protein Revolution written by Richard N. Day and published by CRC Press. This book was released on 2014-04-28 with total page 350 pages. Available in PDF, EPUB and Kindle. Book excerpt: Advances in fluorescent proteins, live-cell imaging, and superresolution instrumentation have ushered in a new era of investigations in cell biology, medicine, and physiology. From the identification of the green fluorescent protein in the jellyfish Aequorea victoria to the engineering of novel fluorescent proteins, The Fluorescent Protein Revolution explores the history, properties, and applications of these important probes. The book first traces the history of fluorescent proteins and the revolution they enabled in cellular imaging. It then discusses fluorescent proteins with novel photophysical properties. The book also covers several cutting-edge imaging applications. These include superresolution microscopy of cellular fine structures, FRET microscopy to visualize protein interactions and cell-signaling activities inside living cells, photobleaching and photoactivation techniques to visualize protein behaviors, techniques that exploit plant and algal photoreceptors to enable light-regulated control of enzymatic activities, and the noninvasive imaging of tumor–host interactions in living animals. In color throughout, this book presents the fundamental principles and latest advances in the field, including the associated development of imaging techniques that exploit fluorescent proteins. It is accessible to a broad audience, from optical imaging experts to novices needing an introduction to the field.
Book Synopsis Enzyme Engineering by : James Samuelson
Download or read book Enzyme Engineering written by James Samuelson and published by Humana Press. This book was released on 2016-05-01 with total page 266 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides guidance to those wishing to create enzyme variants. It covers such topics as a simple method for generating site-specific mutations within bacterial chromosomes and the engineering of two difference types of rare-cutting endonucleases.
Book Synopsis Engineering the Genetic Code by : Nediljko Budisa
Download or read book Engineering the Genetic Code written by Nediljko Budisa and published by John Wiley & Sons. This book was released on 2006-05-12 with total page 312 pages. Available in PDF, EPUB and Kindle. Book excerpt: The ability to introduce non-canonical amino acids in vivo has greatly expanded the repertoire of accessible proteins for basic research and biotechnological application. Here, the different methods and strategies to incorporate new or modified amino acids are explained in detail, including a lot of practical advice for first-time users of this powerful technique. Novel applications in protein biochemistry, genomics, biotechnology and biomedicine made possible by the expansion of the genetic code are discussed and numerous examples are given. Essential reading for all molecular life scientists who want to stay ahead in their research.
