Unnatural Amino Acids as Novel Probes for Ultrafast 2D-IR Spectroscopy of Proteins PDF Download
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Author: Henrike Müller-Werkmeister
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Author: Henrike Müller-Werkmeister
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ISBN:
Category :
Languages : en
Pages :
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Author: Anne Creon
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Author: Ismail A. Ahmed
Publisher:
ISBN:
Category :
Languages : en
Pages : 418
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Proteins form a diverse ensemble of dynamic structures to carry out all life-sustaining functions. Therefore, many efforts have gone into studying the structure-dynamics-function relationship of proteins using a wide range of techniques, including fluorescence and infrared (IR) spectroscopies. While very useful, intrinsic fluorescence and IR signals arising from the natural amino acid side chains within the protein are often insufficient or unable to provide the information needed to understand the biological question of interest. To this end, various extrinsic spectroscopic probes, such as fluorescent dyes, have been used to increase the information content in specific measurements and applications. However, incorporation of a foreign moiety into any protein unavoidably affects its native structure and dynamics; hence effort must be made to reduce such perturbation. In this regard, the overarching aim of this thesis is to develop novel spectroscopic probes based on scaffolds of natural amino acids (NAAs). Because of their small size and similarity to NAAs, such unnatural amino acid-based (UAA-based) probes are expected to be minimally perturbing. Specifically, we show that (1) 4-cyanotryptophan (4CN-Trp) is a blue fluorescent amino acid useful for fluorescence microscopy applications; (2) 4CN-Trp and DiO (a common dye used to stain membranes) are a useful FRET pair to study peptide-membrane interactions; (3) 4CN-Trp, and tryptophan constitutes a dual FRET-PET pair which was used to study peptide end-to-end termini interactions and protein ligand-binding; and (4) the functional group of 4CN-Trp, 4-cyanoindole can be used in the form of a nucleoside as a dual fluorescence-IR reporter for DNA-protein studies. Furthermore, we extended applications of previously known UAAs and showed (5) p-cyanophenylalanine is useful as a fluorescence-based pH sensor which we used to determine peptide pKa's and peptide membrane penetration kinetics and (6) we use a simple synthetic method for post-translationally installing an ester moiety on to proteins via cysteine alkylation as an UAA-based vibrational probe in proteins to study fibril formation and protein-ligand interactions
Author: Loredano Pollegioni
Publisher: Humana Press
ISBN: 9781617793301
Category : Science
Languages : en
Pages : 409
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Even though they are present in nature, non-proteinogenic amino acids are usually defined as unnatural or non-natural. Beside their structural diversity, interest in these compounds is due to their occurrence in nature, their biological properties, the analytical aspects, their use as probes, and their incorporation into peptides and proteins, among other reasons. Divided into five convenient sections, Unnatural Amino Acids: Methods and Protocols deals with enzymatic methods used to produce non-natural amino acids, aspects concerning the presence of unnatural amino acids in peptides with antimicrobial properties, genetic incorporation of unnatural amino acids into proteins (yeast and mammalian cells), and detection and quantification of D-amino acids and related enzymes. Written in the highly successful Methods in Molecular BiologyTM series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and accessible, Unnatural Amino Acids: Methods and Protocols serves as an ideal guide for scientists and contributes to directing the attention of researchers to the many fields of growing scientific interest in non-natural amino acids.
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Publisher: Academic Press
ISBN: 0080921639
Category : Science
Languages : en
Pages : 334
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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
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Publisher: GRIN Verlag
ISBN: 3668663467
Category : Science
Languages : en
Pages : 53
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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.
Author: Jordan Villa
Publisher:
ISBN:
Category : Amino acids
Languages : en
Pages : 150
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Unnatural amino acids (UAAs) permit the incorporation of novel biochemical functionalities into proteins. This expansion of the genetic code has enabled enhanced spatial and temporal control of protein activity and conferred novel protein reactivity. This study examines the incorporation of three UAAs: fluoro-tyrosine, ortho-nitrobenzyl-tyrosine, and propargyloxy-phenylalanine towards various applications. Each UAA was successfully incorporated into a protein of interest (GFP or PRMT1) to facilitate the desired manipulation of protein function. The resulting alterations to GFP fluorescence, PRMT1 activity, or immobilization using Glaser-Hay bioconjugation demonstrate the success and practicality of the utilization of UAAs in the development of novel biochemical tools.
Author: Johnathan C. Maza
Publisher:
ISBN:
Category : Amino acids
Languages : en
Pages : 182
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The current biological toolkit has been vital in advancing our understanding of the world. That being said, the toolkit has limitations. As such, chemical biologists have been developing novel means to probe biological systems using chemical techniques. Bioorthogonal chemistry represents a new avenue to address biological questions that cannot be answered using current techniques. Herein, we describe a novel technique to probe proteins-of-interest using unnatural amino acid (UAA) mutagenesis. We have found that our UAAs allow us to access bioorthogonal chemistries for the conjugation of fluorophores to UAA-containing proteins. Additionally, we have extended these findings towards the application of protein immobilization. Finally, we used microwave technology to investigate novel means to transform bacterial cells with exogenous DNA.
Author: Christopher A. Farley
Publisher:
ISBN:
Category : Amino acids
Languages : en
Pages :
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Unnatural amino acids (UAAs) contain side chains, or R groups, that are not found in the 20 canonical amino acids. These noncanonical groups afford the capability to incorporate powerful chemical capabilities in proteins that are ordinarily unavailable with the naturally-occurring amino acids. Among the most useful moieties to incorporate into proteins are functional groups that can undergo Huisgen [3+2] cycloadditions, or “click,” reactions. This reaction occurs between azides and alkynes, and its mild conditions and high regioselectivity and reactivity make it an ideal process for bioconjugation. Photoreactivity is another useful characteristic that can be conferred to UAAs. Photolabile caging groups can inhibit the function of a protein until brief irradiation with UV light induces an intramolecular rearrangement and its displacement, reestablishing normal function. In this thesis, we propose a synthesis to incorporate both of these moieties into a single UAA.
Author: Itthipol Sungwienwong
Publisher:
ISBN:
Category :
Languages : en
Pages : 306
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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.
