Download or read book Development of Fluorescent Protein Tags for Live cell Imaging written by Elizabeth Marie Santos and published by . This book was released on 2017 with total page 400 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Development of Novel Fluorescent Protein Tags for No wash Live cell Imaging with Minimum Fluorescent Background written by Rahele Esmatpour Salmani and published by . This book was released on 2022 with total page 331 pages. Available in PDF, EPUB and Kindle. Book excerpt: Recent fluorescence microscopy technologies have revolutionized many areas of biomedical research. Nonetheless, high brightness, far-red/near infra-red emission, deep tissue penetration, and selective fluorescent imaging with the minimum background are among the most desired novel fluorescent labeling. One of our primary goals is to develop flexible fluorescent protein tags capable of being tailored ad infinitum. We successfully demonstrated the ability to fine-tune the absorption and emission spectra of protein-bound chromophores over an unprecedented wide range(~200 nm). In contrast to intrinsically fluorescent proteins that are always "ON" in our systems, fluorescent is activated upon covalent binding of ligand and the target protein leading to temporal control of fluorescence. However, the fluorescence background from unbound free chromophore and non-specific binding has always been a deep concern in fluorescent labeling. This Ph.D. research aimed to develop novel protein-based fluorescent tags emitting in the far-red/NIR region of the spectrum for no-wash background-free live-cell imaging applications. This was accomplished by coupling novel synthetic fluorogenic chromophores with hCRBPII mutants. Unbound free aldehyde ThioPhenol and CyThioPhenol are non-emissive dyes that become highly fluorescent upon imine formation with an active site lysine residue engineered deep in the hCRBPII cavity. We created a hydrogen-bonding network around the ThioPhenol hydroxyl group through rational protein engineering that facilitates its deprotonation upon photoexcitation. On the other hand, engineering the target protein to maintain a high iminium pKa resulted in Protonated Schiff Base (PSB) formation. The resultant complex experiences a strong intramolecular charge transfer (ICT), leading to fluorescence and a large bathochromic shift in the emission (~700 nm). The designed protein-based photoacid provides an unprecedented spatiotemporal control for no-wash bright NIR imaging. Our most recent report demonstrated that hCRBPII/chromophore complexes could be developed as a photobase where the imine is converted to an iminium upon photoexcitation. In the course of optimizing hCRBPII to promote ESPT of the hydroxyl group, we discovered that ThioPhenol is capable of acting as both a photoacid and a photobase upon a single photoirradiation. When bound as a Schiff base (SB) to protein mutants that maintain a low iminium pKa(~5), engineered to deprotonate the hydroxyl group, a dual ESPT process leads to protonation of the imino to iminium (the photobase) and deprotonation of the hydroxyl to alkoxide (the photoacid). This double ESPT feature is recapitulated in a protein ligand micro-environment, yielding bright protein-dye complexes with unapparelled large pseudo-Stokes shifts (~250 nm). Additionally, the double ESPT ThioPhenol/hCRBPII complexes show fast binding rates (half-life of
Download or read book Fluorescent Proteins II written by Gregor Jung and published by Springer Science & Business Media. This book was released on 2012-01-05 with total page 287 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 II highlights the physicochemical and biophysical aspects of fluorescent protein technology beyond imaging. It is tailored to meet the needs of physicists, chemists and biologists who are interested in the fundamental properties of fluorescent proteins, while also focussing on specific applications. The implementations described are cutting-edge studies and exemplify how the physical and chemical properties of fluorescent proteins can stimulate novel findings in life sciences.
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.
Download or read book Make Life Visible written by Yoshiaki Toyama and published by Springer Nature. This book was released on 2019-10-02 with total page 292 pages. Available in PDF, EPUB and Kindle. Book excerpt: This open access book describes marked advances in imaging technology that have enabled the visualization of phenomena in ways formerly believed to be completelyimpossible. These technologies have made major contributions to the elucidation of the pathology of diseases as well as to their diagnosis and therapy. The volume presents various studies from molecular imaging to clinical imaging. It also focuses on innovative, creative, advanced research that gives full play to imaging technology inthe broad sense, while exploring cross-disciplinary areas in which individual research fields interact and pursuing the development of new techniques where they fuse together. The book is separated into three parts, the first of which addresses the topic of visualizing and controlling molecules for life. Th e second part is devoted to imaging of disease mechanisms, while the final part comprises studies on the application of imaging technologies to diagnosis and therapy. Th e book contains the proceedings of the 12th Uehara International Symposium 2017, “Make Life Visible” sponsored by the Uehara Memorial Foundation and held from June 12 to 14, 2017. It is written by leading scientists in the field and is an open access publication under a CC BY 4.0 license.
Download or read book Nanoscale Photonic Imaging written by Tim Salditt and published by Springer Nature. This book was released on 2020-06-09 with total page 634 pages. Available in PDF, EPUB and Kindle. Book excerpt: This open access book, edited and authored by a team of world-leading researchers, provides a broad overview of advanced photonic methods for nanoscale visualization, as well as describing a range of fascinating in-depth studies. Introductory chapters cover the most relevant physics and basic methods that young researchers need to master in order to work effectively in the field of nanoscale photonic imaging, from physical first principles, to instrumentation, to mathematical foundations of imaging and data analysis. Subsequent chapters demonstrate how these cutting edge methods are applied to a variety of systems, including complex fluids and biomolecular systems, for visualizing their structure and dynamics, in space and on timescales extending over many orders of magnitude down to the femtosecond range. Progress in nanoscale photonic imaging in Göttingen has been the sum total of more than a decade of work by a wide range of scientists and mathematicians across disciplines, working together in a vibrant collaboration of a kind rarely matched. This volume presents the highlights of their research achievements and serves as a record of the unique and remarkable constellation of contributors, as well as looking ahead at the future prospects in this field. It will serve not only as a useful reference for experienced researchers but also as a valuable point of entry for newcomers.
Download or read book Fluorescent Proteins written by Kevin F. Sullivan and published by Elsevier. This book was released on 2007-12-14 with total page 613 pages. Available in PDF, EPUB and Kindle. Book excerpt: This new edition of Fluorescent Proteins presents current applications of autofluorescent proteins in cell and molecular biology authored by researchers from many of the key laboratories in the field. Starting from a current review of the broad palette of fluorescent proteins available, several chapters focus on key autofluorescent protein variants, including spectral variants, photodynamic variants as well as chimeric FP approaches. Molecular applications are addressed in chapters that detail work with single molecules, approaches to generating protein fusions and biosensors as well as analysis of protein-protein interactions in vivo by FRET, fluorescence polarization and fluorescence cross correlation techniques. A number of approaches to in vivo dynamics are presented, including FRAP, photoactivation, and 4-dimensional microscopy. Behavior of spindle components, membrane proteins, mRNA trafficking as well as analysis of cell types in tissues and in development are detailed and provide models for a wide variety of experimental approaches. In addition, several chapters deal directly with the computational issues involved in processing multidimensional image data and using fluorescent imaging to probe cellular behavior with quantitative modeling. This volume brings together the latest perspective and techniques on fluorescent proteins and will be an invaluable reference in a wide range of laboratories.
Download or read book Development of Novel Far red near infrared Dye hcrbpii Based Imaging Tags for Background free Live Cell Imaging written by Wei Sheng and published by . This book was released on 2019 with total page 325 pages. Available in PDF, EPUB and Kindle. Book excerpt: Modern fluorescence imaging technologies, including deep-tissue imaging and super-resolution microscopies, require novel fluorescent labeling tags possessing non-conventional optical features, among which most desired ones are high brightness in the far-red/near-infrared (NIR) region and turn-on/off control in a spatiotemporal manner. Previously, we demonstrated the ability of fine tuning the absorption spectra of a protein-bound natural chromophore over an unprecedented range (474 ~ 664 nm). The goal of this PhD research is to exploit protein-ligand interactions for the development of protein-based pigments as NIR fluorescent tags for background-free live cell imaging. In the past half century, tremendous efforts have been invested in the optimization and derivatization of GFP-like fluorescent proteins (FPs). More recently, growing attention on phytochrome-based FPs has even upsized the repertoire of available FPs with many enhanced optical features. Giving this advancement, certain pitfalls are still limiting their uses in modern fluorescence imaging. In this context, synthetic dyes provide a broader chemical space for tailoring desired optic features including spectral wavelengths, brightness, stability, and many more photophysical and/or photochemical functionalities. To achieve high contrast imaging with minimal background interference, three different strategies have been applied here. 1) NIR emission is approached by utilizing a dye capable of specific complexation with a target protein via imine bond formation. Upon protonation of the imine, the complex experiences a large bathochromic shift as a result of a strong intramolecular charge transfer (ICT) process. A light-triggered imine isomerization is further incorporated to furnish a photoswitchable tag and negate the routine wash steps in live cell experiments. Rational protein engineering affords a faster variant that allows unprecedented spatiotemporal control of this no-wash bright NIR imaging. (2) A rare organic super photobase is identified, exhibiting a 14-unit change in pKa upon light excitation. Steady-state and ultrafast spectroscopic measurements ascribe this event to an excited-state proton transfer (ESPT) process. This ESPT feature is recapitulated in a protein-ligand micro-environment, yielding protein-dye complexes with extremely high fluorescence quantum yields (up to 92%) and large pseudo-Stokes shifts (> 200 nm). Our optimal mutant bound to the dye boasts millisecond binding rate and enables live cell imaging with negligible background. (3) A general approach to fluorogenicity, i.e., the ability to turn on fluorescence, is designed by coupling a quenching moiety capable of photoinduced electron transfer (PeT) to our dyes. The fluorescence is negligible before the Michael addition of engineered cysteine residue (the trigger) with the quencher moiety. A 30-fold fluorescence enhancement is achieved in vitro with an electronically tuned quencher group. Currently, further modifications are in progress to optimize the quenched system for in vivo applications.
Download or read book Noncanonical Amino Acids written by Edward A. Lemke and published by . This book was released on 2018 with total page 411 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Fluorescent Protein Revolution written by Richard N. Day and published by . This book was released on 2014 with total page 340 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."
Download or read book Protein Engineering written by Huimin Zhao and published by John Wiley & Sons. This book was released on 2021-08-23 with total page 41 pages. Available in PDF, EPUB and Kindle. Book excerpt: A one-stop reference that reviews protein design strategies to applications in industrial and medical biotechnology Protein Engineering: Tools and Applications is a comprehensive resource that offers a systematic and comprehensive review of the most recent advances in the field, and contains detailed information on the methodologies and strategies behind these approaches. The authors—noted experts on the topic—explore the distinctive advantages and disadvantages of the presented methodologies and strategies in a targeted and focused manner that allows for the adaptation and implementation of the strategies for new applications. The book contains information on the directed evolution, rational design, and semi-rational design of proteins and offers a review of the most recent applications in industrial and medical biotechnology. This important book: Covers technologies and methodologies used in protein engineering Includes the strategies behind the approaches, designed to help with the adaptation and implementation of these strategies for new applications Offers a comprehensive and thorough treatment of protein engineering from primary strategies to applications in industrial and medical biotechnology Presents cutting edge advances in the continuously evolving field of protein engineering Written for students and professionals of bioengineering, biotechnology, biochemistry, Protein Engineering: Tools and Applications offers an essential resource to the design strategies in protein engineering and reviews recent applications.
Download or read book Green Fluorescent Proteins written by American Society for Cell Biology and published by Gulf Professional Publishing. This book was released on 1999 with total page 430 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume is an authoritative and comprehensive treatment of the approaches and techniques used for Green Fluorescent Proteins (GFP). The primary focus of this work is on research using biological systems. The volume covers all aspects of GFP, from its expression in different organisms to specific microscopic and data analysis methods. Key Features * Only volume on Green Fluorescent Protein research * Covers all aspects of GFP * Provides specific microscopic and data analysis methods * Discusses the design and construction of GFP fusion proteins * Covers GFP expression in animals, insects, plants, and microbes * Details procedures for time lapse imaging of living cells * Explains how to implement single molecule fluorescence detection with GFP * Discusses dual label GFP strategies for multicolor fluorescence * Presents fluorescence resonance energy transfer methods with GFPs * Details quantitative fluorescence imaging techniques * Extensively illustrated with color photographs
Download or read book Single Molecule Enzymology Fluorescence Based and High Throughput Methods written by and published by Academic Press. This book was released on 2016-10-28 with total page 618 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-Molecule Enzymology, Part A, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in single-molecule enzymology, and includes sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, nanopores, and tethered particle motion. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers research methods in single-molecule enzymology Contains sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, nanopores, and tethered particle motion
Download or read book Development of Dye hcrbpii Based Novel Photoswitchable Fluorescent Proteins written by Soham Maity and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Modern fluorescence imaging technologies such as super-resolution microscopies require novel fluorescent labeling tags possessing nonconventional optical features, including light-controlled turn-on/off of the fluorescence. Our previous reports have demonstrated the ability to engineer hCRBPII to bind a myriad of fluorescent dyes and tune their optical properties. Based on these earlier reports, the goal of this Ph.D. research was to find novel photo-controlled pathways for fluorescence activation of hCRBPII bound fluorophore. In the past two decades, tremendous effort has been invested in the optimization and derivatization of GFP-like fluorescent proteins (FPs). This includes the discovery of photoactivable fluorescent protein (PAFP) variants that becomes fluorescent or change color when they are triggered with light. In contrast to the conventional fluorescent protein, which is permanently fluorescent, photoactive proteins become fluorescent only at the site of interest. In this context, fusion protein which uses synthetic dyes for its optical phenomena provides a broader chemical space for tailoring desired optic features including spectral wavelengths, brightness, stability, and many more photophysical and/or photochemical functionalities. To achieve light-controlled fluorescence activation, two different strategies have been applied here-(1) a cysteine residue containing sulfur was engineered inside hCRBPII, which can participate in a reversible addition with the fluorophore. Utilizing spectroscopic analyses along with X-ray crystallographic studies, we demonstrated that conjugation via Michael addition of cysteine with a coumarin analog that creates a non-fluorescent complex. UV illumination reverses the conjugation, yielding a fluorescent species, presumably through a retro-Michael process. This series of events can be repeated between a bound and non-bound form of the cysteine reversibly, resulting in the ON-OFF control of fluorescence. The details of the mechanism of photoswitching were illuminated by recapitulation of the process in light-irradiated single crystals, confirming the mechanism at atomic resolution. (2) a light induced double proton transfer that results in switching between two spectrally different states of the hCRBPII bound fluorophore. Through spectroscopic and high-resolution structural data, we showed that the protein can be engineered to support selective protonation of the chromophore's aryl amine instead of its imine even at low pH. However, the UV absorbing ammonium ion can be reversibly deprotonated, yielding a highly red-shifted fluorophore upon exposure to UV light. Structural data before and after UV irradiation shows that the light-triggered event alters the protein's interaction with the fluorophore, correlating with the spectral change. The last major endeavor was to develop fluorene based fluorescent dyes with improved optical properties. We have previously reported two fluorene-based dyes, FR0 and FR1V, for fluorescence imaging of the live cells. In this study, effort was made to engineer the dye skeleton to minimize different non-radiating pathways based on literature studies. Spectral data of the new derivatives were collected in different solvents and compared with the previous dyes. We have also been able to demonstrate members of the dyes with red-shifted absorption and emission, high fluorescence QY, and improved water solubility.
Download or read book Rational Design and Directed Evolution of Probe Ligases for Site specific Protein Labeling and Live cell Imaging written by Katharine Alice White and published by . This book was released on 2012 with total page 241 pages. Available in PDF, EPUB and Kindle. Book excerpt: Chemical fluorophores have superior photophysical properties to fluorescent proteins and are much smaller. However, in order to use these probes for live-cell protein imaging, highly specific labeling methods are required. Here, we will describe three efforts to re-engineer the E. coli enzyme, lipoic acid ligase (LplA), to catalyze the ligation of small-molecule probes onto recombinant proteins. We call this collection of methods the PRIME (PRobe Incorporation Mediated by Enzymes) methodologies. First, we describe the structure-guided mutagenesis of LplA and the identification of an LplA variant that can ligate a blue coumarin fluorophore onto a 13-amino acid LplA acceptor peptide (LAP2). This "coumarin ligase" can be used to image cellular proteins with high specificity, sensitivity, and minimal perturbation of the biology of the protein of interest. We also demonstrate how subpopulations of a protein of interest can be labeled using genetically targeted coumarin ligase. Second, we describe our attempts to use yeast display evolution and fluorescence activated cell sorting (FACS) to evolve a truncated LplA enzyme. The original truncated enzyme had severely decreased activity for LplA's natural substrate, lipoic acid. We created a 107 library of LplA mutants and, after four rounds of selection, produced a truncated LplA mutant with lipoylation activity equivalent to full-length LplA. We next sought to evolve activity for an unnatural small molecule probe, but found that this strategy was limited by both increased hydrophobic probe sticking when using the truncated enzyme and some enzyme-dependent nonspecificity. Finally, from a library of 107 LplA mutants, we evolved a full-length LplA capable of ligating an unnatural picolyl azide (pAz) substrate. We demonstrated improved activity of the "pAz ligase" in the secretory pathway and cell surface, two regions where coumarin ligase is inactive. This enzyme can also be used to image cell surface protein-protein interactions as well as label proteins as they are trafficked through the endoplasmic reticulum. These probe ligases will be useful tools for cell biologists interested in studying protein function or protein-protein interactions in the context of living cells.
Download or read book Green Fluorescent Protein written by Martin Chalfie and published by Wiley-Liss. This book was released on 2006 with total page 496 pages. Available in PDF, EPUB and Kindle. Book excerpt: Since the discovery of the gene for green fluorescent protein (GFP), derived from jellyfish, this protein that emits a green glow has initiated a revolution in molecular biosciences. With this tool, it is now possible to visualize nearly any protein of interest in any cell or tissue of any species. Since the publication of the first edition, there have been tremendously significant technological advances, including development of new mutant variants. Proteins are now available in yellow and blue, and Novel Fluorescent Proteins (NFPs) have expanded their utility in developing biosensors, biological markers, and other biological applications. This updated, expanded new edition places emphasis on the rise of NFPs, including new chapters on NFP properties with detailed protocols, applications of GFPs and NFPs in industry research, and biosensors. This book provides a solid theoretical framework, along with detailed, practical guidance on use of GFPs and NFPs with discussion of potential pitfalls. The expert contributors provide real examples in showing how to tailor GFP/NFP to specific systems, maximize expression, and enhance detection.
Download or read book In Vivo Cellular Imaging Using Fluorescent Proteins written by Robert Hoffman and published by Humana Press. This book was released on 2012-06-15 with total page 269 pages. Available in PDF, EPUB and Kindle. Book excerpt: The discovery and genetic engineering of fluorescent proteins has revolutionized cell biology. What was previously invisible in the cell often can be made visible with the use of fluorescent proteins. In Vivo Cellular Imaging Using Fluorescent Proteins: Methods and Protocols presents state-of-the-art research that has contributed to the fluorescent protein revolution to visualize biological processes in the live animal. This volume covers an array of topics from the employment of the chick CAM model using fluorescent proteins and other fluorescent probes, to intravital fluorescent imaging, as well as 3-dimensional imaging, and design instructions on how to create new and improved far-red and infrared fluorescent proteins, to name a few. Written in the successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, In Vivo Cellular Imaging Using Fluorescent Proteins: Methods and Protocols is the first volume in the new field of in vivo cell biology and it serves both professionals and novices with its well-honed methodologies.
