Rational control of structural off-state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, a key parameter that largely dictates the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

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tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

10.1101/2021.04.27.441613 ◽

2021 ◽

Author(s):

Y. Bousmah ◽

H. Valenta ◽

G. Bertolin ◽

U. Singh ◽

V. Nicolas ◽

...

Keyword(s):

Single Molecule ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy ◽

Live Cell ◽

Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

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Properties of Fluorescent Far-Red Anti-TNF Nanobodies

Antibodies ◽

10.3390/antib7040043 ◽

2018 ◽

Vol 7 (4) ◽

pp. 43 ◽

Cited By ~ 2

Author(s):

Ekaterina Gorshkova ◽

Grigory Efimov ◽

Ksenia Ermakova ◽

Ekaterina Vasilenko ◽

Diana Yuzhakova ◽

...

Keyword(s):

Autoimmune Diseases ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Humanized Mice ◽

Whole Body ◽

Whole Body Imaging ◽

Theranostic Agent ◽

Variable Heavy

Upregulation of the expression of tumor necrosis factor (TNF-α, TNF) has a significant role in the development of autoimmune diseases. The fluorescent antibodies binding TNF may be used for personalized therapy of TNF-dependent diseases as a tool to predict the response to anti-TNF treatment. We generated recombinant fluorescent proteins consisting of the anti-TNF module based on the variable heavy chain (VHH) of camelid antibodies fused with the far-red fluorescent protein Katushka (Kat). Two types of anti-TNF VHH were developed: one (BTN-Kat) that was bound both human or mouse TNF, but did not neutralize their activity, and a second (ITN-Kat) that was binding and neutralizing human TNF. BTN-Kat does not interfere with TNF biological functions and can be used for whole-body imaging. ITN-Kat can be evaluated in humanized mice or in cells isolated from humanized mice. It is able to block human TNF (hTNF) activities both in vitro and in vivo and may be considered as a prototype of a theranostic agent for autoimmune diseases.

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C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽

10.1098/rsob.200010 ◽

2020 ◽

Vol 10 (5) ◽

pp. 200010

Author(s):

Navaneethan Palanisamy ◽

Mehmet Ali Öztürk ◽

Emir Bora Akmeriç ◽

Barbara Di Ventura

Keyword(s):

Escherichia Coli ◽

In Silico ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Time Lapse ◽

Enhanced Yellow Fluorescent Protein ◽

Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

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Cellular Uptake of Carbon Nanotubes Conjugated to Semiconductor Quantum Dots for Breast Cancer Imaging and Treatment Applications

ASME 2010 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2010-19504 ◽

2010 ◽

Author(s):

Kristen A. Zimmermann ◽

Jianfei Zhang ◽

Harry Dorn ◽

Christopher Rylander ◽

Marissa Nichole Rylander

Keyword(s):

Carbon Nanotubes ◽

Quantum Dots ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Fluorescent Properties ◽

Breast Cancer Imaging ◽

Fluorescent Markers ◽

Green Fluorescent

Carbon nanotubes (CNTs) are attractive materials for early detection, treatment, and imaging of cancer malignancies; however, they are limited by their inability to be monitored in vitro and in vivo [1]. Unlabeled CNTs are difficult to distinguish using elemental analysis because they are composed entirely of carbon, which is also characteristic of cellular membranes. Although some single walled nanotubes (SWNT) have been found to exhibit fluorescent properties, not all particles in a single batch fluoresce [2]. Additionally, these emissions may be too weak to be detected using conventional imaging modalities [3]. Incorporating fluorescent markers, such as fluorescent proteins or quantum dots, allows the non-fluorescent particles to be visualized. Previously, fluorophores, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), have been used to visualize and track cells or other particles in biological environments, but their low quantum yield and tendency to photobleach generate limitations for their use in such applications.

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Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

Journal of Bacteriology ◽

10.1128/jb.00864-15 ◽

2016 ◽

Vol 198 (7) ◽

pp. 1035-1043 ◽

Cited By ~ 35

Author(s):

Na Ke ◽

Dirk Landgraf ◽

Johan Paulsson ◽

Mehmet Berkmen

Keyword(s):

Escherichia Coli ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Living Cells ◽

Functional Protein ◽

Content Type ◽

Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

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

10.26434/chemrxiv.12067851.v1 ◽

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>

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First Biphotochromic Fluorescent Protein moxSAASoti Stabilized for Oxidizing Environment

10.21203/rs.3.rs-1131671/v1 ◽

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.

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Stalling chromophore maturation of the fluorescent protein Venus reveals the molecular basis of the final oxidation step

10.1101/2020.10.13.337386 ◽

2020 ◽

Author(s):

Husam Sabah Auhim ◽

Bella L. Grigorenko ◽

Tessa Harris ◽

Igor V. Polyakov ◽

Colin Berry ◽

...

Keyword(s):

Molecular Basis ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Life Sciences ◽

Intermediate Stage ◽

Open Conformation ◽

Oxidation Step ◽

Canonical Amino Acid

AbstractFluorescent proteins (FPs) have revolutionised the life sciences but the mechanism of chromophore maturation is still not fully understood. Incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; the crystal structure reveals the presence of O2 located above a dehydrated enolate imidazolone (I) ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an “open” conformation, potentially allowing O2 access to the chromophore. Absorption spectroscopy supported by QM/MM simulations suggest that the first oxidation step involves formation of a hydroperoxyl intermediate in conjunction with dehydrogenation of the methylene bridge. A fully conjugated mature chromophore is formed through release of H2O2 upon irradiation of this intermediate, both in vitro and in vivo. The possibility of interrupting and photochemically restarting chromophore maturation, and the mechanistic insights opens up new approaches for engineering optically controlled fluorescent proteins.

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275.Calmodulin-dependent nuclear import pathway of the testis-determining factor SRY

Reproduction Fertility and Development ◽

10.1071/srb04abs275 ◽

2004 ◽

Vol 16 (9) ◽

pp. 275

Author(s):

G. Kaur ◽

A. Delluc-Clavieres ◽

I. Poon ◽

D. A. Jans

Keyword(s):

Nuclear Import ◽

Protein Import ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Gonadal Development ◽

Nuclear Accumulation ◽

Related Factor ◽

Testis Development ◽

New Findings

Modulation of the nuclear entry of transcription factors (TFs) and chromatin components is a means by which eukaryotic cells can regulate gene expression in response to extracellular signals and the cell cycle during differentiation and development. TFs and chromatin components access the nucleus through nuclear localisation sequences (NLSs), which mediate interaction with components of the cellular nuclear import machinery, such as members of the importin superfamily. The Ca2+-binding protein calmodulin (CaM ) has previously been shown to bind at or near NLSs in several nuclear-localising proteins that have important roles in testis development including the Y chromosome-encoded HMG-domain-carrying chromatin remodelling factor SRY, and related factor SOX9, both of which are key regulators of gonadal development. SRY function in the nucleus of somatic cells of the fetal gonad, in particular, is essential for development of a testis in males. Here we present new findings implicating a role for CaM in modulating SRY nuclear accumulation, whereby treatment of transfected cells with CaM antagonists significantly reduces nuclear accumulation of green fluorescent protein (GFP)-fusion proteins encoding either full length SRY or the SRY HMG domain alone. An in vitro nuclear transport assay using bacterially expressed fluorescent proteins showed similar results, with native gel electrophoresis/fluorimaging and fluorescence polarisation assays, indicating direct binding of CaM to the SRY HMG domain in Ca2+-dependent fashion. Since clinical mutations resulting in sex reversal occur within SRY's CaM-binding NLS, these results may shed new insight into CaM-dependent pathways of nuclear protein import, and how this may relate to testis development.

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

10.1101/2021.02.05.429969 ◽

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.

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