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 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

scholarly journals Acclimatization of symbiotic corals to mesophotic light environments through wavelength transformation by fluorescent protein pigments

2017 ◽  
Vol 284 (1858) ◽  
pp. 20170320 ◽  
Author(s):  
Edward G. Smith ◽  
Cecilia D'Angelo ◽  
Yoni Sharon ◽  
Dan Tchernov ◽  
Joerg Wiedenmann
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Environmental Gradients ◽  
Red Light ◽  
Spectral Composition ◽  
Light Environment ◽  
Homogeneous Distribution ◽  
Red Fluorescent Proteins ◽  
Ecological Importance ◽  
Colour Morphs

The depth distribution of reef-building corals exposes their photosynthetic symbionts of the genus Symbiodinium to extreme gradients in the intensity and spectral quality of the ambient light environment. Characterizing the mechanisms used by the coral holobiont to respond to the low intensity and reduced spectral composition of the light environment in deeper reefs (greater than 20 m) is fundamental to our understanding of the functioning and structure of reefs across depth gradients. Here, we demonstrate that host pigments, specifically photoconvertible red fluorescent proteins (pcRFPs), can promote coral adaptation/acclimatization to deeper-water light environments by transforming the prevalent blue light into orange-red light, which can penetrate deeper within zooxanthellae-containing tissues; this facilitates a more homogeneous distribution of photons across symbiont communities. The ecological importance of pcRFPs in deeper reefs is supported by the increasing proportion of red fluorescent corals with depth (measured down to 45 m) and increased survival of colour morphs with strong expression of pcRFPs in long-term light manipulation experiments. In addition to screening by host pigments from high light intensities in shallow water, the spectral transformation observed in deeper-water corals highlights the importance of GFP-like protein expression as an ecological mechanism to support the functioning of the coral– Symbiodinium association across steep environmental gradients.

ESTABLISHMENT OF VISIBLE ANIMAL METASTASIS MODELS FOR HUMAN NASOPHARYNGEAL CARCINOMA BASED ON A FAR-RED FLUORESCENT PROTEIN

2012 ◽  
Vol 05 (03) ◽  
pp. 1250019 ◽  
Author(s):  
YING ZHENG ◽  
CHUAN HUANG ◽  
ZHIYONG CHENG ◽  
MIN CHEN
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Animal Models ◽  
Tumor Growth ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Vectors ◽  
Red Fluorescent Protein ◽  
Red Fluorescent Proteins ◽  
Metastasis Model

Background and aims: The spectral properties of enhanced green fluorescent protein (EGFP) used in current visualizable animal models for nasopharyngeal carcinoma (NPC) result in a limited imaging depth. Far-red fluorescent proteins have optimal spectral wavelengths that allow deep tissue penetration, thus are well-suited for the imaging of tumor growth and metastases in live animals. This study aims to establish an imageable animal model of NPC using far-red fluorescent proteins. Methods: Eukaryotic expression vectors of far-red fluorescent proteins, mLumin and Katushka S158A, were separately transfected into 5-8F NPC cells, and cell lines stably expressing the far-red fluorescent proteins were obtained. These cells were intraperitoneally or intravenously injected into mice, and their tumorigenic and metastatic potential were examined through fluorescence imaging. Finally, factors affecting their tumorigenic ability were further assessed through testing side population (SP) cells proportion by flow cytometry. Results: NPC cell line with high tumorigenicity and metastasis (5-8F-mL2) was screened out, which stably expressed far-red fluorescent protein. Intraperitoneal and intravenous injection of 5-8F-mL2 cells resulted in an abdomen metastasis model and a lung metastasis model. In addition, NPC cell line without tumorigenicity (5-8F-Katushka S158A) was screened out. The percentage of SP cells between 5-8F-mL2 and 5-8F-Katushka S158A was found different, suggesting that the SP cell proportion may play a key role in the determination of cell tumorigenic ability. Conclusion: We successfully established animal models for NPC with high tumorigenicity and metastasis using a super-bright far-red fluorescent protein. Owing to the super-brightness and excellent wavelength parameters, these models may be applied as useful tools for intuitive and efficient monitoring of tumor growth and metastasis, as well as assessing the efficacy of nasopharyngeal cancer drugs.

Color morphs of the coral, Acropora tenuis, show different responses to environmental stress and different expression profiles of fluorescent-protein genes

2020 ◽  
Author(s):  
Noriyuki Satoh ◽  
Koji Kinjo ◽  
Kohei Shintaku ◽  
Daisuke Kezuka ◽  
Hiroo Ishimori ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Profiles ◽  
Biological Significance ◽  
Gene Expression Profiles ◽  
Gfp Expression ◽  
Red Fluorescent Proteins ◽  
Color Morphs ◽  
Acropora Tenuis ◽  
Green Fluorescent

ABSTRACTCorals of the family Acroporidae are key structural components of reefs that support the most diverse marine ecosystems. Due to increasing anthropogenic stresses, coral reefs are in decline. Along the coast of Okinawa, Japan, three different color morphs of Acropora tenuis have been recognized for decades. These include brown (N morph), yellow-green (G) and purple (P) forms. The tips of axial coral polyps exhibit specific fluorescence spectra. This attribute is inherited asexually, and color morphs do not change seasonally. In Okinawa Prefecture, during the summer of 2017, the N and P morphs experienced bleaching, in which some N morphs died while P morphs recovered. In contrast, G morphs successfully withstood the stress. Symbiotic dinoflagellates are essential symbiotic partners of scleractinian corals. Photosynthetic activity of symbionts was reduced in July in N and P morphs; however, the three color-morphs host similar sets of Clade-C zoothanthellae, suggesting that beaching of N and P morphs cannot be attributed to differences in symbiont clades. The decoded Acropora tenuis genome includes five genes for green fluorescent proteins (GFP), two for cyan fluorescent proteins (CFP), three for red fluorescent proteins (RFP), and seven genes for chromoprotein (ChrP). A summer survey of gene expression profiles demonstrated that (a) expression of CFP and REP was quite low in all three morphs, (b) P morphs expressed higher levels of ChrP, (c) both N and G morphs expressed GFP highly, and (d) GFP expression was reduced in N morphs, compared to G morphs, which maintained higher levels of GFP expression throughout the summer. Although further studies are required to understand the biological significance of these color morphs of Acropora tenuis, our results suggest that thermal stress resistance is modified by genetic mechanisms that coincidentally lead to diversification of color morphs.

scholarly journals Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching

2015 ◽  
Vol 7 (2) ◽  
pp. 263-273 ◽  
Author(s):  
Kevin M. Dean ◽  
Jennifer L. Lubbeck ◽  
Lloyd M. Davis ◽  
Chola K. Regmi ◽  
Prem P. Chapagain ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Microfluidic Platform ◽  
Red Fluorescent Proteins ◽  
New Variant ◽  
Selection Of

We use a high-throughput microfluidic platform that sorts cells on the basis of fluorescent protein photostability to identify a new variant with improved photon output.

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 Chromatic bacteria – A broad host-range plasmid and chromosomal insertion toolbox for fluorescent protein expression in bacteria

2018 ◽  
Author(s):  
Rudolf O. Schlechter ◽  
Hyunwoo Jun ◽  
Michał Bernach ◽  
Simisola Oso ◽  
Erica Boyd ◽  
...  
Keyword(s):  
Host Range ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Broad Host Range ◽  
Complex Environments ◽  
Chloramphenicol Resistance ◽  
Red Fluorescent Proteins ◽  
Broad Host Range Plasmid ◽  
Environmental Interactions ◽  
Yellow Orange

AbstractDifferential fluorescent labelling of bacteria has become instrumental for many aspects of microbiological research, such as the study of biofilm formation, bacterial individuality, evolution, and bacterial behaviour in complex environments. We designed a variety of plasmids, each bearing one of eight unique, constitutively expressed fluorescent protein genes in conjunction with one of four different antibiotic resistance combinations. The fluorophores mTagBFP2, mTurquoise2, sGFP2, mClover3, sYFP2, mOrange2, mScarlet-I, and mCardinal, encoding for blue, cyan, green, green-yellow, yellow, orange, red, and far-red fluorescent proteins, respectively, were combined with selectable markers conferring tetracycline, gentamicin, kanamycin, and/or chloramphenicol resistance. These constructs were cloned into three different plasmid backbones: a broad host-range plasmid, a Tn5transposon delivery plasmid, and a Tn7transposon delivery plasmid. The utility of the plasmids and transposons was tested in bacteria from the phyla Actinobacteria, Proteobacteria, and Bacteroidetes. We were able to tag representatives from the phylum Proteobacteria at least via our Tn5transposon delivery system. The here constructed plasmids are available to the community and provide a valuable tool to investigate bacteria-bacteria, bacteria-host, and bacteria-environmental interactions.

scholarly journals Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

2015 ◽  
Author(s):  
Anton Khmelinskii ◽  
Matthias Meurer ◽  
Chi-Ting Ho ◽  
Birgit Besenbeck ◽  
Julia Fueller ◽  
...  
Keyword(s):  
Protein Turnover ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Proteasomal Degradation ◽  
Time Range ◽  
Green Fluorescent Proteins ◽  
Red Fluorescent Proteins ◽  
Dependent Change ◽  
The Stability ◽  
Green Fluorescent

Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far tFTs were constructed by combining different slower-maturing red fluorescent proteins (redFPs) with the same faster-maturing superfolder green fluorescent protein (sfGFP). Towards a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing greenFPs, while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT towards slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for design of new tFTs.

scholarly journals Insights into the mechanisms of LOV domain color tuning from a set of high-resolution X-ray structures

2021 ◽  
Author(s):  
Alina Remeeva ◽  
Vera V. Nazarenko ◽  
Kirill Kovalev ◽  
Ivan Goncharov ◽  
Anna Yudenko ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Resolution ◽  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Side Chain ◽  
Lov Domain ◽  
Charged Amino Acids ◽  
C Terminus ◽  
Maximal Shift

AbstractLight-oxygen-voltage (LOV) domains are widespread photosensory modules that can be used in fluorescence microscopy, optogenetics and controlled production of reactive oxygen species. All of the currently known LOV domains have absorption maxima in the range of ∼440 to ∼450 nm, and it is not clear whether they can be shifted significantly using mutations. Here, we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine amino acid of a thermostable flavin based fluorescent protein CagFbFP (Gln148) to asparagine, aspartate, glutamate, histidine, lysine and arginine. Absorption spectra of all of the mutants are blue-shifted, with the maximal shift of 8 nm observed for the Q148H variant. While CagFbFP and its Q148N/D/E variants are not sensitive to pH, Q148H/K/R reveal a moderate red shift induced by acidic pH. To gain further insight, we determined high resolution crystal structures of all of the mutants studied at the resolutions from 1.07 Å for Q148D to 1.63 Å for Q148R. Whereas in some of the variants, the amino acid 148 remains in the vicinity of the flavin, in Q148K, Q148R and partially Q148D, the C-terminus of the protein unlatches and the side chain of the residue 148 is reoriented away from the chromophore. Our results explain the absence of color shifts from replacing Gln148 with charged amino acids and pave the way for rational design of color-shifted flavin based fluorescent proteins.

scholarly journals mCherry contains a fluorescent protein isoform that interferes with its reporter function

2021 ◽  
Author(s):  
Maxime Fages-Lartaud ◽  
Lisa Tietze ◽  
Florence Elie ◽  
Rahmi Lale ◽  
Martin Frank Hohmann-Marriott
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Red Fluorescent Protein ◽  
Functional Protein ◽  
Red Fluorescent Proteins ◽  
Expression Studies ◽  
Protein Isoform

AbstractFluorescent proteins are essential reporters in cell biology and molecular biology. Here, we reveal that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform. The short isoform creates significant background fluorescence that biases the outcome of expression studies. Our investigation identifies the short protein isoform, traces its origin, and determines the extent of the issue within the family of red fluorescent protein. Our analysis shows that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. Finally, we provide a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

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