scholarly journals Circularly Permuted Far-Red Fluorescent Proteins

Biosensors ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 438
Author(s):  
Tianchen Wu ◽  
Yu Pang ◽  
Hui-wang Ai
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Circular Permutation ◽  
Tissue Imaging ◽  
Tissue Penetration ◽  
Optical Window ◽  
Color Palette ◽  
Red Fluorescent Proteins

The color palette of genetically encoded fluorescent protein indicators (GEFPIs) has expanded rapidly in recent years. GEFPIs with excitation and emission within the “optical window” above 600 nm are expected to be superior in many aspects, such as enhanced tissue penetration, reduced autofluorescence and scattering, and lower phototoxicity. Circular permutation of fluorescent proteins (FPs) is often the first step in the process of developing single-FP-based GEFPIs. This study explored the tolerance of two far-red FPs, mMaroon1 and mCarmine, towards circular permutation. Several initial constructs were built according to previously reported circularly permuted topologies for other FP analogs. Mutagenesis was then performed on these constructs and screened for fluorescent variants. As a result, five circularly permuted far-red FPs (cpFrFPs) with excitation and emission maxima longer than 600 nm were identified. Some displayed appreciable brightness and efficient chromophore maturation. These cpFrFPs variants could be intriguing starting points to further engineer far-red GEFPIs for in vivo tissue imaging.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

scholarly journals Supramolecular Encapsulation of Small-Ultra Red Fluorescent Proteins in Virus-Like Nanoparticles for Non-Invasive In Vivo Imaging Agents

2020 ◽  
Author(s):  
Fabian C. Herbert ◽  
Olivia Brohlin ◽  
Tyler Galbraith ◽  
Candace Benjamin ◽  
Cesar A. Reyes ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Ex Vivo ◽  
Imaging Agents ◽  
Non Invasive ◽  
Red Fluorescent Proteins ◽  
Ex Vivo Imaging

<div> <div> <div> <p>Icosahedral virus-like particles (VLPs) derived from bacteriophages Qβ and PP7 encapsulating small-ultra red fluorescent protein (smURFP) were produced using a versatile supramolecualr capsid dissassemble-reassemble approach. The generated fluorescent VLPs display identical structural properties to their non-fluorescent analogs. Encapsulated smURFP shows indistinguishable photochemical properties to its unencapsulated counterpart, exhibits outstanding stability towards pH, and produces bright in vitro images following phagocytosis by macrophages. In vivo imaging allows biodistribution to be imaged at different time points. Ex vivo imaging of intravenously administered encapsulated smURFP reveleas localization in the liver and </p> </div> </div> <div> <div> <p>kidneys after 2 h blood circulation and substantial elimination constructs as non-invasive in vivo imaging agents. </p> </div> </div> </div>

scholarly journals Structural Insights into the Binding of Nanobodies Lam2 and Lam4 to the Red Fluorescent Protein mCherry

2021 ◽  
Author(s):  
Ziying Wang ◽  
Long Li ◽  
Rongting Hu ◽  
Peiyu Zhong ◽  
Yiran Zhang ◽  
...  
Keyword(s):  
Binding Sites ◽  
Fluorescent Protein ◽  
Structural Information ◽  
Fluorescent Proteins ◽  
Binding Interface ◽  
Red Fluorescent Proteins ◽  
Multiple Operation ◽  
Structural Insights ◽  
Biology Research

Abstract Background Red fluorescent proteins (RFPs) are widely used in molecular biology research, especially in deep tissues and animal models, because of their superior autofluorescence, light scattering, and phototoxicity to GFP. Although RFP can be easily monitored in vivo, improved manipulation of RFP is still desired. Using suitable nanobodies (Nbs) to bind to different epitopes of RFP is the most promising approach; thus, it is crucial to obtain structural information on how the different Nbs interact with RFP. Results We determined the crystal structures of the LaM2-mCherry and LaM4-mCherry complexes at 1.4 Å and 1.9 Å resolution. Our results showed that LaM2 binds to the side of the mCherry β-barrel, while Lam4 binds to the bottom of the β-barrel and does not interfere with the homo-oligomerization interface. The distinct binding sites of LaM2 and LaM4 were further verified by ITC, F-SEC and DLS assays. Our results also showed that LaM2 and LaM4 can bind simultaneously to mCherry, which is crucial for recruiting multiple operation elements to the RFP. The binding of LaM2 or LaM4 did not significantly change the chromophore environment of mCherry, which is important for fluorescence quantification assays, while several GFP Nbs significantly altered the fluorescence. Mutation of the residues of the LaM2 or LaM4 binding interface to mCherry significantly decreased the binding affinity of the Nb to mCherry. Conclusions Our results provided atomic resolution interaction information on the binding of Nbs LaM2 and LaM4 binding with mCherry, which is important for developing detection and manipulation methods for RFP-based biotechnology.

scholarly journals Supramolecular Encapsulation of Small-Ultra Red Fluorescent Proteins in Virus-Like Nanoparticles for Non-Invasive In Vivo Imaging Agents

2020 ◽  
Author(s):  
Fabian C. Herbert ◽  
Olivia Brohlin ◽  
Tyler Galbraith ◽  
Candace Benjamin ◽  
Cesar A. Reyes ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Ex Vivo ◽  
Imaging Agents ◽  
Non Invasive ◽  
Red Fluorescent Proteins ◽  
Ex Vivo Imaging

<div> <div> <div> <p>Icosahedral virus-like particles (VLPs) derived from bacteriophages Qβ and PP7 encapsulating small-ultra red fluorescent protein (smURFP) were produced using a versatile supramolecualr capsid dissassemble-reassemble approach. The generated fluorescent VLPs display identical structural properties to their non-fluorescent analogs. Encapsulated smURFP shows indistinguishable photochemical properties to its unencapsulated counterpart, exhibits outstanding stability towards pH, and produces bright in vitro images following phagocytosis by macrophages. In vivo imaging allows biodistribution to be imaged at different time points. Ex vivo imaging of intravenously administered encapsulated smURFP reveleas localization in the liver and </p> </div> </div> <div> <div> <p>kidneys after 2 h blood circulation and substantial elimination constructs as non-invasive in vivo imaging agents. </p> </div> </div> </div>

scholarly journals mBeRFP, a versatile fluorescent tool to enhance multichannel live imaging and its applications

2022 ◽  
Author(s):  
Emmanuel Martin ◽  
Magali Suzanne
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Stokes Shift ◽  
Live Imaging ◽  
Red Fluorescent Protein ◽  
Red Fluorescent Proteins ◽  
Living Organisms ◽  
Illumination Source

Cell and developmental biology increasingly require live imaging of protein dynamics in cells, tissues or living organisms. Thanks to the discovery and the development of a panel of fluorescent proteins over the last decades, live imaging has become a powerful and commonly used approach. However, multicolor live imaging remains challenging. The generation of long Stokes shift red fluorescent proteins, such as mBeRFP, offers interesting new perspectives to bypass this limitation. Here, we constructed a set of mBeRFP-expressing vectors and provided a detailed characterization of this fluorescent protein for in vivo live imaging and its applications in Drosophila. Briefly, we showed that a single illumination source is sufficient to simultaneously stimulate mBeRFP and GFP. We demonstrated that mBeRFP can be easily combined with classical green and red fluorescent protein without any crosstalk. We also showed that the low photobleaching of mBeRFP is suitable for live imaging, and that this protein can be used for quantitative applications such as FRAP or laser ablation. Finally, we believe that this fluorescent protein, with the set of new possibilities it offers, constitutes an important tool for cell, developmental and mechano biologists in their current research.

scholarly journals Comparative assessment of fluorescent proteins forin vivoimaging in an animal model system

2016 ◽  
Author(s):  
Jennifer K Heppert ◽  
Daniel J Dickinson ◽  
Ariel M Pani ◽  
Christopher D Higgins ◽  
Annette Steward ◽  
...  
Keyword(s):  
Animal Model ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Model System ◽  
C Elegans ◽  
Red Fluorescent Proteins ◽  
Fluorescent Protein Tags ◽  
Protein Tags

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamicsin vivo. Recent advances in genome editing have enabled precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential ofin vivoimaging experiments, and they facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been madein vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap we quantitatively assessed fluorescent protein propertiesin vivoin an animal model system. We generated transgenicC. elegansstrains expressing green, yellow, or red fluorescent proteins in embryos, and we imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not brightin vivoas predicted based onin vitrodata, but that mNeonGreen is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imagingin vivoinC. elegansembryos, and they suggest good candidate fluorescent proteins to test in other animal model systems.

scholarly journals Structural Insights Into The Binding of Nanobodies LaM2 and LaM4 to the Red Fluorescent Protein mCherry

2021 ◽  
Author(s):  
Ziying Wang ◽  
Long Li ◽  
Rongting Hu ◽  
Peiyu Zhong ◽  
Yiran Zhang ◽  
...  
Keyword(s):  
Binding Sites ◽  
Fluorescent Protein ◽  
Structural Information ◽  
Fluorescent Proteins ◽  
Binding Interface ◽  
Red Fluorescent Proteins ◽  
Multiple Operation ◽  
Structural Insights ◽  
Biology Research

Abstract Red fluorescent proteins (RFPs) are widely used in molecular biology research, especially in deep tissues and animal models, because of their superior autofluorescence, light scattering, and phototoxicity to GFP. Although RFP can be easily monitored in vivo, improved manipulation of RFP is still desired. Using suitable nanobodies (Nbs) to bind to different epitopes of RFP is the most promising approach; thus, it is crucial to obtain structural information on how the different Nbs interact with RFP. We determined the crystal structures of the LaM2-mCherry and LaM4-mCherry complexes at 1.4 Å and 1.9 Å resolution. Our results showed that LaM2 binds to the side of the mCherry β-barrel, while Lam4 binds to the bottom of the β-barrel and does not interfere with the homo-oligomerization interface. The distinct binding sites of LaM2 and LaM4 were further verified by ITC, F-SEC and DLS assays. Our results also showed that LaM2 and LaM4 can bind simultaneously to mCherry, which is crucial for recruiting multiple operation elements to the RFP. The binding of LaM2 or LaM4 did not significantly change the chromophore environment of mCherry, which is important for fluorescence quantification assays, while several GFP Nbs significantly altered the fluorescence. Mutation of the residues of the LaM2 or LaM4 binding interface to mCherry significantly decreased the binding affinity of the Nb to mCherry. Our results provided atomic resolution interaction information on the binding of Nbs LaM2 and LaM4 binding with mCherry, which is important for developing detection and manipulation methods for RFP-based biotechnology.

scholarly journals Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system

2016 ◽  
Vol 27 (22) ◽  
pp. 3385-3394 ◽  
Author(s):  
Jennifer K. Heppert ◽  
Daniel J. Dickinson ◽  
Ariel M. Pani ◽  
Christopher D. Higgins ◽  
Annette Steward ◽  
...  
Keyword(s):  
Animal Model ◽  
In Vivo Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Model System ◽  
Red Fluorescent Proteins ◽  
Fluorescent Protein Tags ◽  
Protein Tags

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments.

scholarly journals Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yusaku Hontani ◽  
Mikhail Baloban ◽  
Francisco Velazquez Escobar ◽  
Swetta A. Jansen ◽  
Daria M. Shcherbakova ◽  
...  
Keyword(s):  
Real Time ◽  
Rational Design ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Covalent Bond ◽  
Binding Pocket ◽  
Tissue Imaging ◽  
Covalent Attachment ◽  
Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

scholarly journals Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent l abeling of endogenous proteins in Caenorhabditis elegans

Genetics ◽  
2021 ◽  
Author(s):  
Jérôme Goudeau ◽  
Catherine S Sharp ◽  
Jonathan Paw ◽  
Laura Savy ◽  
Manuel D Leonetti ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Large Fragment ◽  
C Elegans ◽  
High Affinity ◽  
Red Fluorescent Proteins ◽  
Invaluable Tool ◽  
Endogenous Proteins ◽  
Fluorescent Tags

Abstract We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

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