Browse
Submit Data
Databases
API
Help

Dataset Information

29 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching.

ABSTRACT: 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.

SUBMITTER: Markwardt ML

PROVIDER: S-EPMC3066204 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa.

Project description:Reversible photoswitching of individual molecules has been demonstrated for a number of mutants of the green fluorescent protein (GFP). To date, however, a limited number of switching events with slow response to light have been achieved at the single-molecule level. Here, we report reversible photoswitching characteristics observed in individual molecules of Dronpa, a mutant of a GFP-like fluorescent protein that was cloned from a coral Pectiniidae. Ensemble spectroscopy shows that intense irradiation at 488 nm changes Dronpa to a dim protonated form, but even weak irradiation at 405 nm restores it to the bright deprotonated form. Although Dronpa exists in an acid-base equilibrium, only the photoinduced protonated form shows the switching behavior. At the single-molecule level, 488- and 405-nm lights can be used to drive the molecule back and forth between the bright and dim states. Such reversible photoswitching could be repeated >100 times. The response speed to irradiation depends almost linearly on the irradiation power, with the response time being in the order of milliseconds. The perfect reversibility of the Dronpa photoswitching allows us to propose a detailed model, which quantitatively describes interconversion among the various states. The fast response of Dronpa to light holds great promise for following fast diffusion or transport of signaling molecules in live cells.

| S-EPMC1157093 | biostudies-literature

A structural basis for reversible photoswitching of absorbance spectra in red fluorescent protein rsTagRFP.

Project description:rsTagRFP is the first monomeric red fluorescent protein (FP) with reversibly photoswitchable absorbance spectra. The switching is realized by irradiation of rsTagRFP with blue (440 nm) and yellow (567 nm) light, turning the protein fluorescence ON and OFF, respectively. It is perhaps the most useful probe in this color class that has yet been reported. Because of the photoswitchable absorbance, rsTagRFP can be used as an acceptor in photochromic Förster resonance energy transfer. Yellow FPs, YPet and mVenus, are demonstrated to be excellent photochromic Förster resonance energy transfer donors for the rsTagRFP acceptor in its fusion constructs. Analysis of X-ray structures has shown that photoswitching of rsTagRFP is accompanied by cis-trans isomerization and protonation/deprotonation of the chromophore, with the deprotonated cis- and protonated trans-isomers corresponding to its ON and OFF states, respectively. Unlike in other photoswitchable FPs, both conformers of rsTagRFP chromophore are essentially coplanar. Two other peculiarities of the rsTagRFP chromophore are an essentially hydrophobic environment of its p-hydroxyphenyl site and the absence of direct hydrogen bonding between this moiety and the protein scaffold. The influence of the immediate environment on rsTagRFP chromophore was probed by site-directed mutagenesis. Residues Glu145 and His197 were found to participate in protonation/deprotonation of the chromophore accompanying the photoswitching of rsTagRFP fluorescence, whereas residues Met160 and Leu174 were shown to spatially restrict chromophore isomerization, favoring its radiative decay.

| S-EPMC3294044 | biostudies-literature

Crystal structure of the cyan fluorescent protein Cerulean-S175G.

Project description:Enhanced cyan fluorescent protein (ECFP) was derived from Aequorea victoria green fluorescent protein (avGFP), notably with S65T/Y66W mutations. Its chromophore consists of a tripeptide comprised of Thr65, Trp66 and Gly67 (TWG) residues, while that of avGFP consists of a Ser65, Tyr66 and Gly67 (SYG) tripeptide. Cerulean and SCFP3A were derived from ECFP-S72A/H148D (a double mutation) with additional Y145A and S175G mutations, respectively, while Cerulean-S175G has both mutations (Y145A and S175G). The crystal structures of these ECFP variants at neutral pH were reported to adopt two distinct major conformations called ECFP and Cerulean. In this study, Cerulean-S175G was revealed to adopt only the Cerulean conformation, while Cerulean has been reported to adopt both the ECFP and the Cerulean conformations in its crystal structures. Sharing the same S175G mutation with SCFP3A, Cerulean-S175G showed a slightly increased quantum yield, like SCFP3A, but did not adopt the ECFP conformation adopted by SCFP3A. Detailed comparison of Cerulean-S175G and other ECFP variants revealed that the notable conformational changes in ECFP variants can be understood mainly in terms of the interaction between the Trp66 residue of the chromophore and residues 145-148 of β-strand 7.

| S-EPMC4933001 | biostudies-literature

Microfluidic System for In-Flow Reversible Photoswitching of Near-Infrared Fluorescent Proteins.

Project description:We have developed a microfluidic flow cytometry system to screen reversibly photoswitchable fluorescent proteins for contrast and stability of reversible photoconversion between high- and low-fluorescent states. A two-color array of 20 excitation and deactivation beams generated with diffractive optics was combined with a serpentine microfluidic channel geometry designed to provide five cycles of photoswitching with real-time calculation of photoconversion fluorescence contrast. The characteristics of photoswitching in-flow as a function of excitation and deactivation beam fluence, flow speed, and protein concentration were studied with droplets of the bacterial phytochrome from Deinococcus radiodurans (DrBphP), which is weakly fluorescent in the near-infrared (NIR) spectral range. In agreement with measurements on stationary droplets and HeLa S3 mammalian cells expressing DrBphP, optimized operation of the flow system provided up to 50% photoconversion contrast in-flow at a droplet rate of few hertz and a coefficient of variation (CV) of up to 2% over 10?000 events. The methods for calibrating the brightness and photoswitching measurements in microfluidic flow established here provide a basis for screening of cell-based libraries of reversibly switchable NIR fluorescent proteins.

| S-EPMC6554737 | biostudies-literature

The 1.6 A resolution structure of a FRET-optimized Cerulean fluorescent protein.

Project description:Genetically encoded cyan fluorescent proteins (CFPs) bearing a tryptophan-derived chromophore are commonly used as energy-donor probes in Förster resonance energy transfer (FRET) experiments useful in live cell-imaging applications. In recent years, significant effort has been expended on eliminating the structural and excited-state heterogeneity of these proteins, which has been linked to undesirable photophysical properties. Recently, mCerulean3, a descendant of enhanced CFP, was introduced as an optimized FRET donor protein with a superior quantum yield of 0.87. Here, the 1.6 Å resolution X-ray structure of mCerulean3 is reported. The chromophore is shown to adopt a planar trans configuration at low pH values, indicating that the acid-induced isomerization of Cerulean has been eliminated. β-Strand 7 appears to be well ordered in a single conformation, indicating a loss of conformational heterogeneity in the vicinity of the chromophore. Although the side chains of Ile146 and Leu167 appear to exist in two rotamer states, they are found to be well packed against the indole group of the chromophore. The Ser65 reversion mutation allows improved side-chain packing of Leu220. A structural comparison with mTurquoise2 is presented and additional engineering strategies are discussed.

| S-EPMC3640468 | biostudies-literature

Reversible Photoswitching of Carbon Dots.

Project description:We present a method of reversible photoswitching in carbon nanodots with red emission. A mechanism of electron transfer is proposed. The cationic dark state, formed by the exposure of red light, is revived back to the bright state with the very short exposure of blue light. Additionally, the natural on-off state of carbon dot fluorescence was tuned using an electron acceptor molecule. Our observation can make the carbon dots as an excellent candidate for the super-resolution imaging of nanoscale biomolecules within the cell.

| S-EPMC4468586 | biostudies-literature

Reversible fluorescence photoswitching in DNA.

Project description:We describe the engineering of reversible fluorescence photoswitching in DNA with high-density substitution, and its applications in advanced fluorescence microscopy methods. High-density labeling of DNA with cyanine dyes can be achieved by polymerase chain reaction using a modified DNA polymerase that has been evolved to efficiently incorporate Cy3- and Cy5-labeled cytosine base analogues into double-stranded DNA. The resulting biopolymer, "CyDNA", displays hundreds of fluorophores per DNA strand and is strongly colored and highly fluorescent, although previous observations suggest that fluorescence quenching at such high density might be a concern, especially for Cy5. Herein, we first investigate the mechanisms of fluorescence quenching in CyDNA and we suggest that two different mechanisms, aggregate formation and resonance energy transfer, are responsible for fluorescence quenching at high labeling densities. Moreover, we have been able to re-engineer CyDNA into a reversible fluorescence photoswitchable biopolymer by using the properties of the Cy3-Cy5 pair. This novel biopolymer constitutes a new class of photoactive DNA-based nanomaterial and is of great interest for advanced microscopy applications. We show that reversible fluorescence photoswitching in CyDNA can be exploited in optical lock-in detection imaging. It also lays the foundations for improved and sequence-specific super-resolution fluorescence microscopy of DNA.

| S-EPMC3439957 | biostudies-literature

Expression, purification, crystallization and preliminary X-ray analysis of eCGP123, an extremely stable monomeric green fluorescent protein with reversible photoswitching properties.

Project description:Enhanced consensus green protein variant 123 (eCGP123) is an extremely thermostable green fluorescent protein (GFP) that exhibits useful negative reversible photoswitching properties. eCGP123 was derived by the application of both a consensus engineering approach and a recursive evolutionary process. Diffraction-quality crystals of recombinant eCGP123 were obtained by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The eCGP123 crystal diffracted X-rays to 2.10 Å resolution. The data were indexed in space group P1, with unit-cell parameters a = 74.63, b = 75.38, c = 84.51 Å, ? = 90.96, ? = 89.92, ? = 104.03°. The Matthews coefficient (V(M) = 2.26 Å(3) Da(-1)) and a solvent content of 46% indicated that the asymmetric unit contained eight eCGP123 molecules.

| S-EPMC3212379 | biostudies-literature

Reversible photoswitching of encapsulated azobenzenes in water.

Project description:Efficient molecular switching in confined spaces is critical for the successful development of artificial molecular machines. However, molecular switching events often entail large structural changes and therefore require conformational freedom, which is typically limited under confinement conditions. Here, we investigated the behavior of azobenzene-the key building block of light-controlled molecular machines-in a confined environment that is flexible and can adapt its shape to that of the bound guest. To this end, we encapsulated several structurally diverse azobenzenes within the cavity of a flexible, water-soluble coordination cage, and investigated their light-responsive behavior. Using UV/Vis absorption spectroscopy and a combination of NMR methods, we showed that each of the encapsulated azobenzenes exhibited distinct switching properties. An azobenzene forming a 1:1 host-guest inclusion complex could be efficiently photoisomerized in a reversible fashion. In contrast, successful switching in inclusion complexes incorporating two azobenzene guests was dependent on the availability of free cages in the system, and it involved reversible trafficking of azobenzene between the cages. In the absence of extra cages, photoswitching was either suppressed or it involved expulsion of azobenzene from the cage and consequently its precipitation from the solution. This finding was utilized to develop an information storage medium in which messages could be written and erased in a reversible fashion using light.

| S-EPMC6156622 | biostudies-literature

Structural basis for reversible photoswitching in Dronpa.

Project description:Dronpa is a novel GFP-like fluorescent protein with exceptional light-controlled switching properties. It may be reversibly switched between a fluorescent on-state and a nonfluorescent off-state by irradiation with light. To elucidate the molecular basis of the switching mechanism, we generated reversibly switchable Dronpa protein crystals. Using these crystals we determined the elusive dark-state structure of Dronpa at 1.95-A resolution. We found that the photoswitching results in a cis-trans isomerization of the chromophore accompanied by complex structural rearrangements of four nearby amino acid residues. Because of this cascade of intramolecular events, the chromophore is exposed to distinct electrostatic surface potentials, which are likely to influence the protonation equilibria at the chromophore. We suggest a comprehensive model for the light-induced switching mechanism, connecting a cascade of structural rearrangements with different protonation states of the chromophore.

| S-EPMC1941826 | 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