scholarly journals Structure-guided point mutations on FusionRed produce a brighter red fluorescent protein

2020 ◽  
Author(s):  
Srijit Mukherjee ◽  
Sheng-Ting Hung ◽  
Nancy Douglas ◽  
Premashis Manna ◽  
Connor Thomas ◽  
...  
Keyword(s):  
Rate Constant ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Point Mutations ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Radiative Rate ◽  
Low Cytotoxicity ◽  
Molecular Brightness

AbstractThe development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and appropriate localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, FusionRed-MQV, which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (~140,000 M−1cm−1), increased radiative rate constant and reduced non-radiative rate constant with respect to its precursor. The properties of FusionRed-MQV derive from three mutations - M42Q, C159V and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the phenol and the acylimine ends of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the phenol end of the chromophore were mutated. The M42Q mutation is located near the acylimine end of the chromophore and was discovered using site-directed mutagenesis guided by x-ray crystal structures. FusionRed-MQV exhibits 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells (based on FACS) compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells.

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 Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

2017 ◽  
Author(s):  
Stephen D. Carter ◽  
Shrawan K. Mageswaran ◽  
Zachary J. Farino ◽  
João I. Mamede ◽  
Catherine M. Oikonomou ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Secretory Granules ◽  
Light And Electron Microscopy ◽  
Low Contrast ◽  
Fluorescent Protein Tags ◽  
Protein Tags

AbstractCryogenic correlated light and electron microscopy (cryo-CLEM) is a valuable tool for studying biological processes in situ. In cryo-CLEM, a target protein of interest is tagged with a fluorophore and the location of the corresponding fluorescent signal is used to identify the structure in low-contrast but feature-rich cryo-EM images. To date, cryo-CLEM studies of mammalian cells have relied on very bright organic dyes or fluorescent protein tags concentrated in virus particles. Here we describe a method to expand the application of cryo-CLEM to cells harboring genetically-encoded fluorescent proteins. We discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80K). Compared to fluorescent protein tags, these sources of autofluorescence exhibit a broader spectrum of fluorescence, which we exploited to develop a simple, robust approach to discriminate between the two. We validate this method in INS-1 E cells using a mitochondrial marker, and apply it to study the ultrastructural variability of secretory granules in a near-native state within intact INS-1E pancreatic cells by high-resolution 3D electron cryotomography.

scholarly journals Quantitative assessment of near-infrared fluorescent proteins

2021 ◽  
Author(s):  
Kiryl Piatkevich ◽  
Hanbin Zhang ◽  
Stavrini Papadaki ◽  
Xiaoting Sun ◽  
Luxia Yao ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Quantitative Assessment ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Oligomeric State ◽  
C Elegans ◽  
The Right ◽  
Infrared Fluorescent Protein

Abstract Recent progress in fluorescent protein development has generated a large diversity of near-infrared fluorescent proteins, which are rapidly becoming popular probes for a variety of imaging applications. To assist end-users with a selection of the right near-infrared fluorescent protein for a given application, we will conduct a quantitative assessment of intracellular brightness, photostability, and oligomeric state of 19 near-infrared fluorescent proteins in cultured mammalian cells. The top-performing proteins will be further validated for in vivo imaging of neurons in C. elegans, zebrafish, and mice. We will also assess the applicability of the selected NIR FPs for expansion microscopy and two-photon imaging.

scholarly journals Identifying Functionally Important Mutations from Phenotypically Diverse Sequence Data

2006 ◽  
Vol 72 (5) ◽  
pp. 3696-3701 ◽  
Author(s):  
Kyle Jensen ◽  
Hal Alper ◽  
Curt Fischer ◽  
Gregory Stephanopoulos
Keyword(s):  
Statistical Method ◽  
Fluorescent Protein ◽  
Sequence Data ◽  
Point Mutations ◽  
Multinomial Distribution ◽  
Site Directed Mutagenesis ◽  
Single Mutation ◽  
The Individual ◽  
Green Fluorescent ◽  
Functional Nucleic Acids

ABSTRACT Here we present a simple statistical method to determine the phenotypic contribution of a single mutation from libraries of mutants with diverse phenotypes in which each mutant contains a multitude of mutations. The central premise of this method is that, given M phenotypic classes, mutations that do not affect the phenotype should partition among the M classes according to a multinomial distribution. Deviations from this distribution are indicative of a link between specific mutations and phenotypes. We suggest that this method will aid the engineering of functional nucleic acids, proteins, and other biomolecules by uncovering target sites for rational mutagenesis. As a proof of the principle, we show how the method can be used to deduce the individual effects of mutations in a set of 69 PL-λ promoter variants. Each of these promoters was generated by error-prone PCR and incorporated numerous mutations. The activity of the promoters was assayed using flow cytometry to measure the fluorescence of a green fluorescent protein reporter gene. Our analysis of the sequences of these mutants revealed seven positions having a statistically significant correlation with promoter activity. Using site-directed mutagenesis, we constructed point mutations for several sites, both statistically significant and insignificant, and combinations of these sites. Our results show that the statistical method correctly elucidated the phenotypic manifestations of these mutations. We suggest that this method may be useful for expediting directed evolution experiments by allowing both desired and undesired mutations to be identified and incorporated between rounds of mutagenesis.

scholarly journals A photoswitchable fluorescent protein for hours-time-lapse and sub-second-resolved super-resolution imaging

Microscopy ◽  
2021 ◽  
Author(s):  
Tetsuichi Wazawa ◽  
Ryohei Noma ◽  
Shusaku Uto ◽  
Kazunori Sugiura ◽  
Takashi Washio ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Ph Dependence ◽  
Super Resolution ◽  
Time Lapse ◽  
Light Irradiation ◽  
Acquisition Time ◽  
Polarization Angle ◽  
Cellular Dynamics

Abstract Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.

scholarly journals A genetically encoded biosensor roKate for monitoring the redox state of the glutathione pool

2019 ◽  
pp. 86-92 ◽  
Author(s):  
AG Shokhina ◽  
VV Belousov ◽  
DS Bilan
Keyword(s):  
Mammalian Cells ◽  
Redox State ◽  
Fluorescent Protein ◽  
Spectral Characteristics ◽  
Disulfide Bridge ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Living Organisms ◽  
Cysteine Amino Acid

Genetically encoded fluorescent sensors are exploited to study a variety of biological processes in living organisms in real time. In recent years, a whole family of biosensors has been developed, serving to visualize changes in the glutathione redox state. The aim of our experiment was to design a biosensor based on the red fluorescent protein mKate2 for measuring the 2GSH/GSSG ratio. A pair of cysteine amino acid residues were introduced into the structure of the fluorescent protein using site-directed mutagenesis. These residues form a disulfide bridge when the surrounding glutathione pool is oxidized, affecting the spectral characteristics of the protein. Our biosensor, which we called roKate, was tested in vitro on an isolated protein. Specifically, we examined the spectral characteristics, pH and the redox potential of the sensor. Additionally, the performance of roKate was evaluated using the culture of living mammalian cells. The fluorescent signal emitted by the sensor was very bright and remarkably stable under pH conditions varying in the physiological range. Irreversibly oxidized in mammalian cells, roKate stands out from other members of this biosensor family. This biosensor should be preferred in the experiments when the time between the manipulations with the biological object and the subsequent analysis of the induced effect is substantial, as is the case with long sample preparation.

scholarly journals An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching

PLoS ONE ◽  
2011 ◽  
Vol 6 (3) ◽  
pp. e17896 ◽  
Author(s):  
Michele L. Markwardt ◽  
Gert-Jan Kremers ◽  
Catherine A. Kraft ◽  
Krishanu Ray ◽  
Paula J. C. Cranfill ◽  
...  
Keyword(s):  
Quantum Yield ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Site Directed Mutagenesis ◽  
Quantum Yields

Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.

scholarly journals Cloning Vectors and Fluorescent Proteins Can Significantly Inhibit Salmonella enterica Virulence in Both Epithelial Cells and Macrophages: Implications for Bacterial Pathogenesis Studies

2005 ◽  
Vol 73 (10) ◽  
pp. 7027-7031 ◽  
Author(s):  
Leigh A. Knodler ◽  
Aaron Bestor ◽  
Caixia Ma ◽  
Imke Hansen-Wester ◽  
Michael Hensel ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Salmonella Enterica ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Bacterial Pathogenesis ◽  
Cloning Vectors ◽  
Serovar Typhimurium ◽  
Reporter Systems ◽  
Fluorescent Protein Reporter

ABSTRACT Plasmid vectors and fluorescent protein reporter systems are commonly used in the study of bacterial pathogenesis. Here we show that they can impair the ability of Salmonella enterica serovar Typhimurium to productively infect either cultured mammalian cells or mice. This has significant implications for studies that rely on these systems.

scholarly journals Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements

2001 ◽  
Vol 21 (8) ◽  
pp. 2826-2837 ◽  
Author(s):  
Arun Venkatesan ◽  
Asim Dasgupta
Keyword(s):  
Internal Ribosome Entry Site ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Independent Manner ◽  
Internal Ribosome Entry

ABSTRACT We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library of bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (EBFP and EGFP) separated by randomized 50-nucleotide-long sequences was amplified in bacteria and delivered into mammalian cells via protoplast fusion. Cells that received functional IRES elements were isolated using the EBFP and EGFP reporters and fluorescence-activated cell sorting, and several small IRES elements were identified. Two of these elements were subsequently shown to possess IRES activity comparable to that of a variant of the encephalomyocarditis virus IRES element in a context-independent manner both in vitro and in vivo, and these elements functioned in multiple cell types. Although no sequence or structural homology was apparent between the synthetic IRES elements and known viral and cellular IRES elements, the two synthetic IRES elements specifically blocked poliovirus (PV) IRES-mediated translation in vitro. Competitive protein-binding experiments suggested that these IRES elements compete with PV IRES-mediated translation by utilizing some of the same factors as the PV IRES to direct translation. The utility of this fluorescent protein-based screen in identifying IRES elements with improved activity as well as in probing the mechanism of IRES-mediated translation is discussed.

scholarly journals Enhancement of a protocol purifying T1 lipase through molecular approach

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5833
Author(s):  
Che Haznie Ayu Che Hussian ◽  
Raja Noor Zaliha Raja Abd. Rahman ◽  
Adam Leow Thean Chor ◽  
Abu Bakar Salleh ◽  
Mohd Shukuri Mohamad Ali
Keyword(s):  
Rational Design ◽  
Double Point ◽  
Specific Activity ◽  
Point Mutations ◽  
Fold Increase ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Site Directed Mutagenesis ◽  
Glutathione S Transferase ◽  
Direct Separation

T1 Lipase is a thermostable secretary protein of Geobacillus zalihae strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification.

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