Browse
Submit Data
Databases
API
Help

Dataset Information

14 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity.

ABSTRACT: MCherry, the Discosoma sp. mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. We have used a combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emission profiles. These efforts ultimately led to a long Stokes shift (LSS)-mCherry variant (?ex = 460 nm and ?em = 610 nm) and a red-shifted (RDS)-mCherry variant (?ex = 600 nm and ?em = 630 nm). These new RFPs provide insight into the influence of the chromophore environment on mCherry's fluorescence properties, and may serve as templates for the future development of fluorescent probes for live cell imaging.

SUBMITTER: Shen Y

PROVIDER: S-EPMC5328254 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity.

Shen Yi Y Chen Yingche Y Wu Jiahui J Shaner Nathan C NC Campbell Robert E RE

PloS one 20170227 2

MCherry, the Discosoma sp. mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. We have used a combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emission profiles. These efforts ultimately led to a long Stokes shift (LSS)-mCherry variant (λex = 460 nm and λem = 610 nm) and a red-shifted (RDS)-mCherry ...[more]

PMID: 28241009

Similar Datasets

Engineering ESPT pathways based on structural analysis of LSSmKate red fluorescent proteins with large Stokes shift.

Project description:LSSmKate1 and LSSmKate2 are monomeric red fluorescent proteins (RFPs) with large Stokes shifts (LSSs), which allows for efficient separation of absorbance and emission maxima, as well as for excitation with conventional two-photon laser sources. These LSSmKates differ by a single amino acid substitution at position 160 and exhibit absorbance maxima around 460 nm, corresponding to a neutral DsRed-like chromophore. However, excitation at 460 nm leads to fluorescence emission above 600 nm. Structures of LSSmKate1 and LSSmKate2, determined at resolutions of 2.0 and 1.5 A, respectively, revealed that the predominant DsRed-chromophore configurations are cis for LSSmKate1 but trans for LSSmKate2. Crystallographic and mutagenesis analyses, as well as isotope and temperature dependences, suggest that an excited-state proton transfer (ESPT) is responsible for the LSSs observed in LSSmKates. Hydrogen bonding between the chromophore hydroxyl and Glu160 in LSSmKate1 and a proton relay involving the chromophore tyrosine hydroxyl, Ser158, and the Asp160 carboxylate in LSSmKate2 represent the putative ESPT pathways. Comparisons with mKeima LSS RFP suggest that similar proton relays could be engineered in other FPs. Accordingly, we mutated positions 158 and 160 in several conventional red-shifted FPs, including mNeptune, mCherry, mStrawberry, mOrange, and mKO, and the resulting FP variants exhibited LSS fluorescence emission in a wide range of wavelengths from 560 to 640 nm. These data suggest that different chromophores formed by distinct tripeptides in different environments can be rationally modified to yield RFPs with novel photochemical properties.

| S-EPMC2919691 | biostudies-literature

Photoswitchable red fluorescent protein with a large Stokes shift.

Project description:A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622 nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621 nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.

| S-EPMC4224599 | biostudies-literature

Monomeric red fluorescent proteins with a large Stokes shift.

Project description:Two-photon microscopy has advanced fluorescence imaging of cellular processes in living animals. Fluorescent proteins in the blue-green wavelength range are widely used in two-photon microscopy; however, the use of red fluorescent proteins is limited by the low power output of Ti-Sapphire lasers above 1,000 nm. To overcome this limitation we have developed two red fluorescent proteins, LSS-mKate1 and LSS-mKate2, which possess large Stokes shifts with excitation/emission maxima at 463/624 and 460/605 nm, respectively. These LSS-mKates are characterized by high pH stability, photostability, rapid chromophore maturation, and monomeric behavior. They lack absorbance in the green region, providing an additional red color to the commonly used red fluorescent proteins. Substantial overlap between the two-photon excitation spectra of the LSS-mKates and blue-green fluorophores enables multicolor imaging using a single laser. We applied this approach to a mouse xenograft model of breast cancer to intravitally study the motility and Golgi-nucleus alignment of tumor cells as a function of their distance from blood vessels. Our data indicate that within 40 mum the breast cancer cells show significant polarization towards vessels in living mice.

| S-EPMC2851791 | biostudies-literature

Dynamic Stokes shift in green fluorescent protein variants.

Project description:Solvent reorganization around the excited state of a chromophore leads to an emission shift to longer wavelengths during the excited-state lifetime. This solvation response is absent in wild-type green fluorescent protein, and this has been attributed to rigidity in the chromophore's environment necessary to exclude nonradiative transitions to the ground state. The fluorescent protein mPlum was developed via directed evolution by selection for red emission, and we use time-resolved fluorescence to study the dynamic Stokes shift through its evolutionary history. The far-red emission of mPlum is attributed to a picosecond solvation response that is observed at all temperatures above the glass transition. This time-dependent shift in emission is not observed in its evolutionary ancestors, suggesting that selective pressure has produced a chromophore environment that allows solvent reorganization. The evolutionary pathway and structures of related fluorescent proteins suggest the role of a single residue in close proximity to the chromophore as the primary cause of the solvation response.

| S-EPMC2154406 | biostudies-literature

mBeRFP, an improved large stokes shift red fluorescent protein.

Project description:Herein, we describe the generation of a monomeric large Stokes shift (LSS) red fluorescent protein, mBeRFP, with excitation and emission peaks at 446 and 615 nm, respectively. Compared with two previously reported LSS-RFPs (mKeima and LSS-mKate2), mBeRFP is approximately three times brighter. In addition, mBeRFP is characterized by improved photostability, rapid maturation, an extended lifetime, and a monomeric nature. Additionally, mBeRFP can be paired with the Alexa 647 dye as a FRET donor to detect caspase 3 activity. This FRET pair has an extremely dynamic range and a large Förster radius (approximately 6.5 nm). To demonstrate the applicability of mBeRFP for imaging in living cells, we performed dual-color imaging of mBeRFP and CFP simultaneously excited by a single excitation source, and we demonstrated that these fluorescent proteins allow the clear visualization of the dynamics of Bax during cancer cell apoptosis. Thus, mBeRFP appears to be particularly useful for cellular imaging applications.

| S-EPMC3688735 | biostudies-other

mRhubarb: Engineering of monomeric, red-shifted, and brighter variants of iRFP using structure-guided multi-site mutagenesis.

Project description:Far-red and near-infrared fluorescent proteins (FPs) enable in vivo tissue imaging with greater depth and clarity compared to FPs in the visible spectrum due to reduced light absorbance and scatter by tissues. However current tools are limited by low brightness, limited red-shifting, and a non-ideal dimeric oligomerization state. In this study we developed a monomeric variant of iRFP, termed mRhubarb713, and subsequently used a targeted and expansive multi-site mutagenesis approach to screen for variants with red-shifted spectral activity. Two monomeric variants were discovered, deemed mRhubarb719 and mRhubarb720, with red-shifted spectra and increased quantum yield compared to iRFP. These tools build on previously developed near-IR FPs and should enable improved in vivo imaging studies with a genetically encoded reporter.

| S-EPMC6821797 | biostudies-literature

Hydrogen bond flexibility correlates with Stokes shift in mPlum variants.

Project description:Fluorescent proteins have revolutionized molecular biology research and provide a means of tracking subcellular processes with extraordinary spatial and temporal precision. Species with emission beyond 650 nm offer the potential for deeper tissue penetration and lengthened imaging times; however, the origin of their extended Stokes shift is not fully understood. We employed spectrally resolved transient grating spectroscopy and molecular dynamics simulations to investigate the relationship between the flexibility of the chromophore environment and Stokes shift in mPlum. We examined excited state solvation dynamics in a panel of strategic point mutants of residues E16 and I65 proposed to participate in a hydrogen-bonding interaction thought responsible for its red-shifted emission. We observed two characteristic relaxation constants of a few picoseconds and tens of picoseconds that were assigned to survival times of direct and water-mediated hydrogen bonds at the 16-65 position. Moreover, variants of the largest Stokes shift (mPlum, I65V) exhibited significant decay on both time scales, indicating the bathochromic shift correlates with a facile switching between a direct and water-mediated hydrogen bond. This dynamic model underscores the role of environmental flexibility in the mechanism of excited state solvation and provides a template for engineering next-generation red fluorescent proteins.

| S-EPMC4084698 | biostudies-literature

Structure-fluorescence activation relationships of a large Stokes shift fluorogenic RNA aptamer.

Project description:The Chili RNA aptamer is a 52 nt long fluorogen-activating RNA aptamer (FLAP) that confers fluorescence to structurally diverse derivatives of fluorescent protein chromophores. A key feature of Chili is the formation of highly stable complexes with different ligands, which exhibit bright, highly Stokes-shifted fluorescence emission. In this work, we have analyzed the interactions between the Chili RNA and a family of conditionally fluorescent ligands using a variety of spectroscopic, calorimetric and biochemical techniques to reveal key structure-fluorescence activation relationships (SFARs). The ligands under investigation form two categories with emission maxima of ?540 or ?590 nm, respectively, and bind with affinities in the nanomolar to low-micromolar range. Isothermal titration calorimetry was used to elucidate the enthalpic and entropic contributions to binding affinity for a cationic ligand that is unique to the Chili aptamer. In addition to fluorescence activation, ligand binding was also observed by NMR spectroscopy, revealing characteristic signals for the formation of a G-quadruplex only upon ligand binding. These data shed light on the molecular features required and responsible for the large Stokes shift and the strong fluorescence enhancement of red and green emitting RNA-chromophore complexes.

| S-EPMC7145527 | biostudies-literature

Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins.

Project description:We describe a red-shifted fluorescence resonance energy transfer (FRET) pair optimized for dual-color fluorescence lifetime imaging (FLIM). This pair utilizes a newly developed FRET donor, monomeric cyan-excitable red fluorescent protein (mCyRFP1), which has a large Stokes shift and a monoexponential fluorescence lifetime decay. When used together with EGFP-based biosensors, the new pair enables simultaneous imaging of the activities of two signaling molecules in single dendritic spines undergoing structural plasticity.

| S-EPMC5322478 | biostudies-literature

Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein.

Project description:We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (?180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (?120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein-protein interactions. We also use MPE-FCCS to detect drug-protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.

| S-EPMC4472016 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data