scholarly journals Increasing the brightness of red fluorescent proteins by saturation mutagenesis

2018 ◽  
Vol 1 ◽  
pp. 10-14
Author(s):  
Erin Nguyen ◽  
Antonia Pandelieva ◽  
Roberto A. Chica
Keyword(s):  
Spectral Properties ◽  
Fluorescent Proteins ◽  
Fold Increase ◽  
Saturation Mutagenesis ◽  
Side Chain ◽  
Aromatic Rings ◽  
Screening Assay ◽  
Red Fluorescent Proteins ◽  
Packing Arrangement

Red fluorescent proteins (RFPs) are genetically-encoded fluorophores that are widely used for in vivo imaging. For all applications of RFPs, brighter variants are desired. Previously, we improved the brightness of mRojoA, a red-shifted mutant of the widely-used RFP mCherry, by designing a triple-decker motif of aromatic rings around its chromophore. This yielded the brighter variant mRojo-VHSV, which contains a triple-decker motif consisting of His and Tyr side chains that pack against the chromophore. This improved chromophore packing resulted in an approximately 3-fold brightness increase at physiological pH. However, the His side chain in the triple-decker motif of mRojo-VHSV adopted a perpendicular arrangement to the other two, which may result in a suboptimal packing arrangement. To further improve chromophore packing in mRojo-VHSV, we performed saturation mutagenesis of residues surrounding its triple-decker motif (positions 62, 97, 165, and 199). Using a microplate fluorescence screening assay, a total of 376 colonies were screened for improved brightness. The brightest mutant found, L199M, was expressed and purified, and its spectral properties were characterized in detail. We found that the quantum yield of this variant was improved by two-fold, resulting in a two-fold brightness increase compared to mRojo-VHSV as well as a 5.3-fold increase in brightness compared to mRojoA. The L199M improved variant is the basis for continued engineering with the goal of further improving the spectral properties of this family of RFPs.

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 First Biphotochromic Fluorescent Protein moxSAASoti Stabilized for Oxidizing Environment

2021 ◽  
Author(s):  
Nadya Marynich ◽  
Mariya Khrenova ◽  
Alexandra Gavshina ◽  
Ilya Solovyev ◽  
Alexander Savitsky
Keyword(s):  
Conversion Rate ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Point Mutations ◽  
Md Simulations ◽  
Side Chain ◽  
Dynamic Simulations ◽  
Red Fluorescent Proteins ◽  
Oxidation Resistant ◽  
Red Form

Abstract Biphotochromic proteins simultaneously possesses reversible photoswitching (on-to-off) and irreversible photoconversion (green-to-red). High photochemical reactivity of cysteine residues is one of the reasons for the development of “mox”-monomeric and oxidation resistant proteins. Based on site-saturated simultaneous two points C105 and C117 mutagenesis we have chosen the C21N/C71G/C105G/C117T/C175A as the moxSAASoti variant, since its on-to-off photoswitching rate is higher, off-to-on recovery is more complete and photoconversion rates are higher than for the mSAASoti. We analyzed the conformational behavior of the F177 side chain by classical MD simulations. The conformational flexibility of the F177 side chain is mainly responsible for the off-to-on conversion rate changes and can be further utilized as a measure of the conversion rate. Point mutations in the mSAASoti mainly affect the pKa values of the red form and the off-to-on switching. We demonstrate that the microscopic measure of the observed pKa value is the C – O bond length in the phenyl fragment of the neutral chromophore. According to the molecular dynamic simulations with the QM/MM potentials, larger C – O bond lengths are found for proteins with larger pKa. This feature can be utilized for prediction of the pKa values of red fluorescent proteins.

scholarly journals Orange Fluorescent Proteins: Filling the Spectral Gap

2014 ◽  
Vol 70 (a1) ◽  
pp. C1670-C1670
Author(s):  
Sergei Pletnev ◽  
Daria Shcherbakova ◽  
Oksana Subach ◽  
Vladimir Malashkevich ◽  
Steven Almo ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Spectral Properties ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Stokes Shift ◽  
Emission Wavelength ◽  
Microscopy Imaging ◽  
Large Stokes Shift

Fluorescent proteins (FPs) have become valuable tools for molecular biology, biochemistry, medicine, and cancer research. Starting from parent green fluorescent protein (GFP), most challenging task of the FPs studies was the development of FPs with longer excitation/emission wavelength. This pursuit was motivated by advantages of so-called red-shifted FPs, namely, lower background of cellular autofluorescence in microscopy, lower light scattering and reduced tissue absorbance of longer wavelengths for in vivo imaging. In addition to FPs with regular spectral properties, there are proteins of other types available, including FPs with a large Stokes shift and photoconvertible FPs. These special kinds of FPs have become useful in super-resolution microscopy, imaging of enzyme activities, protein-protein interactions, photolabeling, and in vivo imaging. According to their emission wavelength, red-shifted FPs could be divided in the following groups: 520-540 nm yellow FPs (YFPs), 540-570 nm orange FPs (OFPs), 570-620 nm red FPs (RFPs), and > 620 nm far-RFPs. Red shift of the excitation/emission bands of these FPs is predominantly achieved by extension of the conjugated system of the chromophore and its protonation/deprotonation. The variety of spectral properties of FPs (excitation and emission wavelength, quantum yield, brightness, photo- and pH- stability, photoconversion, large Stokes shift, etc) results from the different chromophore structures and its interactions with surrounding amino acid residues. In this work we focus on structural studies and molecular mechanisms of FPs with orange emission.

Fluorescent Nanodiamond – A Novel Nanomaterial for In Vivo Applications

2011 ◽  
Vol 1362 ◽  
Author(s):  
Nitin Mohan ◽  
Bailin Zhang ◽  
Cheng-Chun Chang ◽  
Liling Yang ◽  
Chao-Sheng Chen ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Ion Irradiation ◽  
Spectroscopic Characterization ◽  
Correlation Spectroscopy ◽  
Model Organisms ◽  
C Elegans ◽  
Red Fluorescent Proteins ◽  
Fluorescent Nanodiamonds ◽  
High Fluorescence

ABSTRACTFluorescent nanodiamonds (FNDs) with a size in the range of 10 – 100 nm have been produced by ion irradiation and annealing, and isolated by differential centrifugation. Single particle spectroscopic characterization with confocal fluorescence microscopy and fluorescence correlation spectroscopy indicates that they are photostable and useful as an alternative to far-red fluorescent proteins for bioimaging applications. We demonstrate the application by performing in vivo imaging of bare and bioconjugated FND particles (100 nm in diameter) in C. elegans and zebrafishes and exploring the interactions between this novel nanomaterial and the model organisms. Our results indicate that FNDs can be delivered to the embryos of both organisms by microinjection and eventually into the hatched larvae in the next generation. No deleterious effects have been observed for the carbon-based nanoparticles in vivo. The high fluorescence brightness, excellent photostability, and nontoxic nature of the nanomaterial have allowed long-term imaging and tracking of embryogenesis in the organisms.

scholarly journals A Comparison of Red Fluorescent Proteins to Model DNA Vaccine Expression by Whole Animal In Vivo Imaging

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0130375 ◽  
Author(s):  
Ekaterina Kinnear ◽  
Lisa J. Caproni ◽  
John S. Tregoning
Keyword(s):  
Dna Vaccine ◽  
In Vivo Imaging ◽  
Fluorescent Proteins ◽  
Red Fluorescent Proteins

scholarly journals Click Chemistry-Facilitated Structural Diversification of Nitrothiazoles, Nitrofurans, and Nitropyrroles Enhances Antimicrobial Activity against Giardia lamblia

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Wan Jung Kim ◽  
Keith A. Korthals ◽  
Suhua Li ◽  
Christine Le ◽  
Jarosław Kalisiak ◽  
...  
Keyword(s):  
Click Chemistry ◽  
Giardia Lamblia ◽  
Diarrheal Disease ◽  
Wide Spectrum ◽  
Fold Increase ◽  
Side Chain ◽  
Full Potential ◽  
Content Type

ABSTRACT Giardia lamblia is an important and ubiquitous cause of diarrheal disease. The primary agents in the treatment of giardiasis are nitroheterocyclic drugs, particularly the imidazoles metronidazole and tinidazole and the thiazole nitazoxanide. Although these drugs are generally effective, treatment failures occur in up to 20% of cases, and resistance has been demonstrated in vivo and in vitro. Prior work had suggested that side chain modifications of the imidazole core can lead to new effective 5-nitroimidazole drugs that can combat nitro drug resistance, but the full potential of nitroheterocycles other than imidazole to yield effective new antigiardial agents has not been explored. Here, we generated derivatives of two clinically utilized nitroheterocycles, nitrothiazole and nitrofuran, as well as a third heterocycle, nitropyrrole, which is related to nitroimidazole but has not been systematically investigated as an antimicrobial drug scaffold. Click chemistry was employed to synthesize 442 novel nitroheterocyclic compounds with extensive side chain modifications. Screening of this library against representative G. lamblia strains showed a wide spectrum of in vitro activities, with many of the compounds exhibiting superior activity relative to reference drugs and several showing >100-fold increase in potency and the ability to overcome existing forms of metronidazole resistance. The majority of new compounds displayed no cytotoxicity against human cells, and several compounds were orally active against murine giardiasis in vivo. These findings provide additional impetus for the systematic development of nitroheterocyclic compounds with nonimidazole cores as alternative and improved agents for the treatment of giardiasis and potentially other infectious agents.

scholarly journals Structural heterogeneity in biliverdin modulates spectral properties of Sandercyanin fluorescent protein

2021 ◽  
Author(s):  
Swagatha Ghosh ◽  
Sayan Mondal ◽  
Keerti Yadav ◽  
Shantanu Aggarwal ◽  
Wayne F. Schaefer ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Spectral Properties ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Binding Pocket ◽  
Structural Heterogeneity ◽  
Wild Type ◽  
Absorbing States

Sandercyanin, a blue homo-tetrameric lipocalin protein purified from Canadian walleye (Stizostedion vitreus), is the first far-red fluorescent protein reported in vertebrates. Sandercyanin binds non-covalently to biliverdin IXα (BLA) and fluoresces at 675nm on excitation at 375nm and 635nm. Sandercyanin fluorescence can be harnessed for many in vivo applications when engineered into a stable monomeric form. Here, we report the spectral properties and crystal structures of engineered monomeric Sandercyanin-BLA complexes. Compared to wild-type protein, monomeric Sandercyanin (~18kDa) binds BLA with similar affinities and show a broad red- shifted absorbance spectra but possess reduced quantum efficiency. Crystal structures reveal D-ring pyrrole of BLA rotated around the C14-C15 bond, which is stabilized by neighboring aromatic residues and increased water-mediated polar contacts in the BLA-binding pocket. A tetrameric Sandercyanin variant (Tyr-142-Ala) co-displaying red- and far-red absorbing states, and reduced fluorescence shows similar conformational changes in BLA binding pocket. Our results suggest that D-ring flexibility of BLA and its rearrangement reduces the fluorescence quantum-yield of monomeric Sandercyanin. Structures of monomeric Sandercyanin could be utilized as prototypes to generate bright BLA-inducible fluorescent proteins. Further, our study postulates a mechanism for modulating photo-states in BLA- bound lipocalins, known only in phytochromes till date.

Three-Dimensional In Vivo Imaging of Tumors Expressing Red Fluorescent Proteins

2012 ◽  
pp. 97-114 ◽  
Author(s):  
Alexander P. Savitsky ◽  
Irina G. Meerovich ◽  
Victoria V. Zherdeva ◽  
Lyaysan R. Arslanbaeva ◽  
Olga S. Burova ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Fluorescent Proteins ◽  
Three Dimensional ◽  
Red Fluorescent Proteins

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.

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