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Unnatural Amino Acids As Novel Probes For Ultrafast 2d Ir Spectroscopy Of Proteins
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Book Synopsis Unnatural Amino Acids as Novel Probes for Ultrafast 2D-IR Spectroscopy of Proteins by : Henrike Müller-Werkmeister
Download or read book Unnatural Amino Acids as Novel Probes for Ultrafast 2D-IR Spectroscopy of Proteins written by Henrike Müller-Werkmeister and published by . This book was released on 2014 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Unnatural Amino Acids as Vibrational Spectroscopy Probes for Protein Structure and Dynamics by : Anne Creon
Download or read book Unnatural Amino Acids as Vibrational Spectroscopy Probes for Protein Structure and Dynamics written by Anne Creon and published by . This book was released on 2020 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis The Development of Unnatural Amino Acid-based Probes and Methods for Biological Studies by : Ismail A. Ahmed
Download or read book The Development of Unnatural Amino Acid-based Probes and Methods for Biological Studies written by Ismail A. Ahmed and published by . This book was released on 2019 with total page 418 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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
Book Synopsis Unnatural Amino Acids by : Loredano Pollegioni
Download or read book Unnatural Amino Acids written by Loredano Pollegioni and published by Humana Press. This book was released on 2011-10-05 with total page 409 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
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.
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 Unnatural Amino Acids in Proteins for Development of Novel Biochemical Tools by : Jordan Villa
Download or read book Unnatural Amino Acids in Proteins for Development of Novel Biochemical Tools written by Jordan Villa and published by . This book was released on 2015 with total page 150 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Book Synopsis Development of Novel Chemical Techniques to Address Biological Questions by : Johnathan C. Maza
Download or read book Development of Novel Chemical Techniques to Address Biological Questions written by Johnathan C. Maza and published by . This book was released on 2015 with total page 182 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Book Synopsis Synthesis of a Novel Unnatural Amino Acid for Protein Incorporation and Click Mediated Conjugation by : Christopher A. Farley
Download or read book Synthesis of a Novel Unnatural Amino Acid for Protein Incorporation and Click Mediated Conjugation written by Christopher A. Farley and published by . This book was released on 2014 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
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.
Book Synopsis Genetic Incorporation of Unnatural Amino Acids Into Proteins by : Isaac Nnamdi Ugwumba
Download or read book Genetic Incorporation of Unnatural Amino Acids Into Proteins written by Isaac Nnamdi Ugwumba and published by . This book was released on 2010 with total page 308 pages. Available in PDF, EPUB and Kindle. Book excerpt: The introduction of unnatural amino acids with novel properties into proteins is potentially a useful tool for understanding and manipulating their functions. The development of an in vitro method for the incorporation of a fluorescent amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco), into proteins is described here.
Book Synopsis Ultraviolet Spectroscopy of Proteins by : Aleksandr Petrovich Demchenko
Download or read book Ultraviolet Spectroscopy of Proteins written by Aleksandr Petrovich Demchenko and published by Springer. This book was released on 1986 with total page 332 pages. Available in PDF, EPUB and Kindle. Book excerpt: The aim of this book is to give a comprehensive description of the basic methods used in the ultraviolet spectroscopy of proteins, to discuss new trends and development of these methods, and to analyze their different applications in the study of various aspects of protein structure and dynamics. Ultraviolet spectroscopy is one of the oldest and most popular methods in the field of biochemistry and molecular biophysics. At present, it is difficult to imagine the biochemical laboratory without a recording spectrophotometer or spectrofluorimeter. There are several hundreds of publications directly devoted to protein ultraviolet spectroscopy and in a great number of studies UV spectroscopic methods are used for the structural analysis of different proteins. Meanwhile a unified description of the theoretical basis of the methods, experimental techniques, data analysis, and generalization of results obtained in solving the specific problems of protein structure are lacking. There are three reasons for which a monograph on ultraviolet spectroscopy is needed today. Firstly, there has been significant growth in facilities of experimental technique, its precision, and versatility associated with computer data analysts. This new technique is available to a wide circle of scientists engaged in the field of protein research. Most of them are not spectroscopists and, thus, there is a need for a conceivable and precise source of information on how to use this method and what kind of data it should provide.
Book Synopsis Unnatural Amino Acids as Metal-mediated Probes of Biological Function by : Bhaskar Bhushan
Download or read book Unnatural Amino Acids as Metal-mediated Probes of Biological Function written by Bhaskar Bhushan and published by . This book was released on 2014 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Temperature-jump 2D IR Spectroscopy to Study Protein Conformational Dynamics by : Kevin Chapman Jones
Download or read book Temperature-jump 2D IR Spectroscopy to Study Protein Conformational Dynamics written by Kevin Chapman Jones and published by . This book was released on 2012 with total page 324 pages. Available in PDF, EPUB and Kindle. Book excerpt: Temperature-jump (T-jump) two-dimensional infrared spectroscopy (2D IR) is developed, characterized, and applied to the study of protein folding and association. In solution, protein conformational changes span a wide range of timescale from nanoseconds to minutes. Ultrafast 2D IR spectroscopy measures time-dependent structural changes within the protein ensemble by probing the frequency changes associated with amide I backbone vibrations. Combining 2D IR with a perturbing laser-induced T-jump enables the study of conformational dynamics from 5 ns to 50 ms. To access a finer time-sampling of the conformational evolution, a one-dimensional variant of 2D IR, heterodyne-detected dispersed vibrational echo spectroscopy (HDVE), is implemented. The framework for interpreting transient HDVE and 2D IR spectra is developed, and we propose a method to remove the linear absorption distortions along both frequency axes. We first present the T-jump 2D IR spectra of a dipeptide to reveal the general amide I baseline response expected in the absence of conformational change. To facilitate the analysis of T-jump data, singular value decomposition (SVD) is employed for reducing noise, identifying the number of distinguishable states, and separating spectral changes based on shared timescales. Finally, T-jump 2D IR spectroscopy is applied to study the unfolding of ubiquitin, disordering of the 12-residue p-hairpin peptide trpzip2 (TZ2), and the dissociation of insulin dimers to monomers. Experimental results for ubiquitin highlight the importance of linear absorption corrections for interpretation of the data. In response to the T-jump, 2D IR results indicate p-sheet structure melts in ubiquitin with a small amplitude (~10 gs) and large amplitude (17 ms) response. Isotope-labeling T-jump experiments on TZ2 allow for the proposal of a free energy surface in which transitions from a native and misfolded state proceed through a disordered hub-like state with a 1-2 gs timescale. Multiple timescales are observed in the T-jump induced dissociation of insulin. Based on their spectral features and concentration dependence, the insulin timescales can be assigned to dissociation, disordering, and oligomerization processes. With these applications, we demonstrate the capability of T-jump 2D IR spectroscopy to reveal detailed molecular dynamics.
Book Synopsis 2D-IR Spectroscopy of Peptide-protein Binding by : Klemens L. Koziol
Download or read book 2D-IR Spectroscopy of Peptide-protein Binding written by Klemens L. Koziol and published by . This book was released on 2017 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Characterizing Membrane Proteins with 2D IR Spectroscopy by : Prabuddha Mukherjee
Download or read book Characterizing Membrane Proteins with 2D IR Spectroscopy written by Prabuddha Mukherjee and published by . This book was released on 2008 with total page 213 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Two-dimensional Infrared Spectroscopy as a General Approach for the Study of Protein Dynamics by : Sashary Ramos
Download or read book Two-dimensional Infrared Spectroscopy as a General Approach for the Study of Protein Dynamics written by Sashary Ramos and published by . This book was released on 2020 with total page 250 pages. Available in PDF, EPUB and Kindle. Book excerpt: Complete understanding of protein function requires knowledge of protein conformational dynamics, or the structural fluctuations of a protein. However, characterization of protein dynamics is challenged by protein complexity, as they are large, heterogeneous molecules with potentially important motions on very fast timescales. This complexity demands the use of a technique with high spatial and temporal resolution. Two-dimensional infrared (2D IR) spectroscopy has emerged as a powerful tool for the characterization and direct measurement of molecular heterogeneity and dynamics due to its excellent spatial and temporal resolution. However, application to proteins is hindered by their severely congested spectra due to the large number of similar bonds. To overcome this issue, proteins can be site-specifically labeled with spectrally resolved IR probes that are active in the transparent frequency region (~1800 - 2500 cm-1) and are sensitive to their environment. The studies presented here take advantage of the combination of site-specific labeling and IR spectroscopy to study the environments and dynamics at specific locations in three distinct protein systems. Herein, I describe our investigations of dynamic complexes of proteins that have challenged experimental characterization with conventional methods: plastocyanin (Pc) and its binding partner cytochrome f (cyt f); cytochrome P450cam (P450cam) and substrates or its redox partner, putidaredoxin; and the SH3Sho1 domain and the proline-rich (PR) recognition motif of its binding partner Pbs2. In addition, we describe my attempts at improving the experimental technique of site-specific IR spectroscopy as a general biophysical approach for protein characterization. Overall, I present evidence for the importance of fast dynamics in protein function and illustrate the rich information provided by 2D IR spectroscopy to complement existing biophysical methods.
