Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light

Green fluorescent protein (GFP) and GFP-like proteins represent invaluable genetically encoded fluorescent probes. In the last few years a new class of photoactivatable fluorescent proteins (PAFPs) capable of pronounced light-induced spectral changes have been developed. Except for tetrameric KFP1 (ref. 4), all known PAFPs, including PA-GFP, Kaede, EosFP, PS-CFP, Dronpa, PA-mRFP1 and KikGR require light in the UV-violet spectral region for activation through one-photon excitation--such light can be phototoxic to some biological systems. Here, we report a monomeric PAFP, Dendra, derived from octocoral Dendronephthya sp. and capable of 1,000- to 4,500-fold photoconversion from green to red fluorescent states in response to either visible blue or UV-violet light. Dendra represents the first PAFP, which is simultaneously monomeric, efficiently matures at 37 degrees C, demonstrates high photostability of the activated state, and can be photoactivated by a common, marginally phototoxic, 488-nm laser line. We demonstrate the suitability of Dendra for protein labeling and tracking to quantitatively study dynamics of fibrillarin and vimentin in mammalian cells.     

Structural characterization of a thiazoline-containing chromophore in an orange fluorescent protein, monomeric Kusabira Orange

Monomeric Kusabira Orange (mKO) is a green fluorescent protein (GFP)-like protein that emits orange light at a peak of 559 nm. We analyzed its X-ray structure at 1.65 A and found a novel three-ring chromophore that developed autocatalytically from a Cys65-Tyr66-Glu67 tripeptide in which the side chain of Cys65 formed the third 2-hydroxy-3-thiazoline ring. As a result, the chromophore contained the CNCOH group at the 2-position of the imidazolinone moiety such that the conjugated pi-electron system of the chromophore was more extended than that of GFP but less extended than that of the Discosoma sp. red fluorescent protein (DsRed). Since a sulfur atom has potent nucleophilic character, the third 3-thiazoline ring is rapidly and completely cyclized. Furthermore, our structure reveals the presence of a pi-pi stacking interaction between His197 and the chromophore as well as a pi-cation interaction between Arg69 and the chromophore. These structural findings are sufficient to account for the orange emission, pH tolerance, and photostability of mKO.     

Lyophilization and enhanced stability of fluorescent protein nanoparticles

Protein nanoparticles (PNPs) that are nanostructured biomaterials with intrinsic biological function have been widely employed as three-dimensional nanobiomaterials for sensitive bioassays, MRI contrast, semiconductor devices, template for hybrid materials, etc., and stable and long-term maintenance of PNPs seems to be of crucial importance. We evaluated the stability of PNPs and the efficacy of lyophilization for the long-term stability of PNPs, especially using green fluorescent protein nanoparticles (gFPNPs) as a model PNP. Fluorescence intensities and TEM images of gFPNPs were analyzed to monitor their functional and structural stabilities. Unlike the green fluorescent protein monomers (eGFP) that were gradually inactivated in aqueous solution, gFPNP in the same aqueous solution retained the initial fluorescence activity and spherical nanoparticle structure even for 2 weeks at 4 °C. To ensure stable and long-term maintenance of gFPNPs, gFPNPs in aqueous solution were converted to the dried solid forms through lyophilization. It is notable that fluorescence activity and nanoparticle structure of gFPNPs that were lyophilized with both Tween 80 and sucrose were very stably maintained even for 10 weeks at various storage temperatures (−20 °C, 4 °C, 25 °C, and 37 °C). During the period of 10 weeks, the fluorescence of gFPNP was always more than 80% level of initial fluorescence at a wide range of temperature. Although this stability study was focused on gFPNPs, the developed optimal lyophilization conditions for gFPNPs can be applied in general to stable and long-term maintenance of many other PNP-derived biomaterials.     

Mutants of Discosoma red fluorescent protein with a GFP-like chromophore

The green fluorescent protein (GFP)-homologous red fluorescent protein (RFP) from Discosoma (drFP583) which emits bright red fluorescence peaking at 583 nm is an interesting novel genetic marker. We show here that RFP maturation involves a GFP-like fluorophore which can be stabilized by point mutations selected from a randomly mutated expression library. By homology modeling, these point mutations cluster near the imidazolidinone ring of the chromophore. Exciting the GFP-like absorption band in the mutant proteins produces both green and red fluorescence. Upon unfolding and heating, the absorption spectrum of the RFP chromophore slowly becomes similar to that of the GFP chromophore. This can be interpreted as a covalent modification of the GFP chromophore in RFP that appears to occur in the final maturation step.     

Cysteineless non-glycosylated monomeric blue fluorescent protein,secBFP2, for studies in the eukaryotic secretory pathway

Fluorescent protein (FP) technologies suitable for use within the eukaryotic secretory pathway are essential for live cell and protein dynamic studies. Localization of FPs within the endoplasmic reticulum (ER) lumen has potentially significant consequences for FP function. All FPs are resident cytoplasmic proteins and have rarely been evolved for the chemically distinct environment of the ER lumen. In contrast to the cytoplasm, the ER lumen is oxidizing and the site where secretory proteins are post-translationally modified by disulfide bond formation and N-glycosylation on select asparagine residues. Cysteine residues and N-linked glycosylation consensus sequences were identified within many commonly utilized FPs. Here, we report mTagBFP is post-translationally modified when localized to the ER lumen. Our findings suggest these modifications can grossly affect the sensitivity and reliability of FP tools within the secretory pathway. To optimize tools for studying events in this important intracellular environment, we modified mTagBFP by mutating its cysteines and consensus N-glycosylation sites. We report successful creation of a secretory pathway-optimized blue FP, secBFP2.     

Green fluorescent protein with tryptophan-based chromophore stable at low pH

Using directed (substitution T203Y) and subsequent random mutagenesis of the monomeric cyan fluorescent protein mTurquoise2, we obtained a protein with a tryptophan-based chromophore that fluoresces in the green region of the spectrum (excitation maximum 482 nm, emission maximum 519 nm). Fluorescence of the new protein is highly stable in a wide range of pH (pK _a 4.9), more stable than all monomeric green fluorescent proteins with a tyrosine-based chromophore.     

A photostable monomeric superfolder green fluorescent protein

The green fluorescent protein (GFP) from Aequorea victoria has been engineered extensively in the past to generate variants suitable for protein tagging. Early efforts produced the enhanced variant EGFP and its monomeric derivative mEGFP, which have useful photophysical properties, as well as superfolder GFP, which folds efficiently under adverse conditions. We previously generated msGFP, a monomeric superfolder derivative of EGFP. Unfortunately, compared to EGFP, msGFP and other superfolder GFP variants show faster photobleaching. We now describe msGFP2, which retains monomeric superfolder properties while being as photostable as EGFP. msGFP2 contains modified N‐ and C‐terminal peptides that are expected to reduce nonspecific interactions. Compared to EGFP and mEGFP, msGFP2 is less prone to disturbing the functions of certain partner proteins. For general‐purpose protein tagging, msGFP2 may be the best available derivative of A. victoria GFP.     

The fluorescent monomeric protein Kusabira Orange. Pressure effect on its structure and stability

The structure and stability of the fluorescent protein monomeric Kusabira Orange (mKO), a GFP-like protein, was studied under different pressure levels and in different chemical environments. At different pH values (between pH 7.4 and pH 4.0) and under a pressure up to 600 MPa (at 25 °C), mKO did not show significant fluorescence spectral changes, indicating a structural stability of the protein. In more extreme chemical conditions (at pH 4.0 in the presence of 0.8 M guanidine hydrochloride), a marked reduction of mKO fluorescence intensity emission was observed at pressures above 300 MPa. This fluorescence emission quenching may be due to the loss of the intermolecular bonds and, consequently, to the destructuration of the mKO chromophore structure. Since the electrostatic and hydrophobic interactions as well as the salt bridges present in proteins are usually perturbed under high pressure, the reduction of mKO fluorescence intensity emission is associated to the perturbation of the protein salt bridges network.     

Bright monomeric red fluorescent protein with an extended fluorescence lifetime

Fluorescent proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of proteins in living cells. Here we report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.     

Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins

The variant of Aequorea green fluorescent protein (GFP) known as blue fluorescent protein (BFP) was originally engineered by substituting histidine for tyrosine in the chromophore precursor sequence. Herein we report improved versions of BFP along with a variety of engineered fluorescent protein variants with novel and distinct chromophore structures that all share the property of a blue fluorescent hue. The two most intriguing of the new variants are a version of GFP in which the chromophore does not undergo excited-state proton transfer and a version of mCherry with a phenylalanine-derived chromophore. All of the new blue fluorescing proteins have been critically assessed for their utility in live cell fluorescent imaging. These new variants should greatly facilitate multicolor fluorescent imaging by legitimizing blue fluorescing proteins as practical and robust members of the fluorescent protein "toolkit".     

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

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.     

Vibrational analysis of a solvated green fluorescent protein chromophore

Resonance Raman (RR) spectra of green fluorescent protein (GFP) model chromophores in solution have been simulated with the CASSCF/MM methodology. Although several reports on vibrational analysis of GFP model chromophores have been recently published, the RR spectra were simulated for the first time in explicit solution with the inclusion of the counterion, as these effects are crucial for unambiguously reproducing the vibrational band assignment in the anionic form of the GFP chromophore. This strategy allows for a one-to-one correspondence of the calculated vibrational modes to the observed RR bands, concerning both the location and intensity pattern. In addition, these simulations were complemented with total energy distribution calculations to aid in the unambiguous assignment of the measured spectra. The current study helps to clarify some of the previous RR bands assignments as well as producing some new assignment for the anionic form of GFP chromophore. The explicit solvent simulations and PCM-based calculations are compared to the measured spectra, and these results demonstrate that explicit solvent simulations provide better agreement with experiment, both in terms of vibrational frequencies and intensity distribution. Figure a Correlation of explicit hydration calculations (CASSCF/6-31G*/MM) for the HBI model chromophore and experimental RR data [21]; slope = 0.982, intercept = 27.210 and regression coefficient = 0.997. b Correlation of implicit PCM calculations (CASSCF/6-31G*) for the HBI model chromophore and experimental RR data [21], slope = 1.017, intercept = −48.838 and regression coefficient = 0.984     

Optical properties of the monomeric red fluorescent protein mRFP1

For the expression in E. coli, the PCR-amplified fragment encoding mRFP1 from vector pMT-mRFP1 (Fungal Genetic Stock Center) was placed in the pQE-60 vector. Chemically competent E. coli ER1821 were transformed and grown overnight at 37°C. The protein was purified by Ni-NTA chromatography and dialyzed against 67mM Na₂HPO₄, 67mM KH₂PO₄, pH 7.5. There are two peaks (at 503 and 584 nm) in the mRFP1 absorption spectrum. The green component (503 nm) may correspond to a green fraction of the protein (a fraction that never matures beyond the green intermediate or a fraction which is trapped as a dead-end product such as the nonproductive trans conformation for the F65-Q66 peptide bond). The mRFP1’s extinction coefficient is equal to 42 mM^?1 cm^?1 at 584 nm; the emission maximum is at 607 nm; the excitation maximum is at 584–586 nm; the Stokes shift is equal to 23 nm; the fluorescence lifetime is about 1.8 ns; the quantum yield is 0.27; pKa is 4.0. Analysis of the mRFP1 absorption spectrum by high-order derivative spectroscopy shows that electron transition systems of both the fully matured form (absorption maximum at 584 nm) and the green fraction of the protein (absorption maximum at 503 nm) are practically identical.     

Control of the blue fluorescent protein with advanced evolutionary pulse shaping

We demonstrate optical coherent control of the two-photon fluorescence of the blue fluorescent protein (BFP), which is of interest in investigations of protein-protein interactions. In addition to biological relevance, BFP represents an interesting target for coherent control from a chemical perspective due to its many components of highly nonexponential fluorescence decay and low quantum yield resulting from excited state isomerization. Using a genetic algorithm with a multiplicative (rather than ratiometric) fitness parameter, we are able to control the ratio of BFP fluorescence to second-harmonic generation without a considerable drop in the maximized signal. The importance of linear chirp and power-scaling on the discrimination process is investigated in detail.     

On photoabsorption of the neutral form of the green fluorescent protein chromophore

We present results of theoretical studies of the photoabsorption band corresponding to the vertical electronic transition S₀–S₁ between first two singlet states of the model chromophore from the green fluorescent protein (GFP) in its neutral form. Predictions of quantum chemical approaches including ab initio and semi-empirical approximations are compared for the model systems which mimic the GFP chromophore in different environments. We provide evidences that the protein matrix in GFP accounts for a fairly large shift of about 40 nm in the S₀–S₁ absorption band as compared to the gas phase.     

Use of enhanced green fluorescent protein to determine pepsin at high sensitivity

A fluorometric assay for pepsin and pepsinogen was developed using enhanced green fluorescent protein (EGFP) as a substrate. Acid denaturation of EGFP resulted in a complete loss of fluorescence that was completely reversible on neutralization. In the proteolytic assay procedure, acid-denatured EGFP was digested by pepsin or activated pepsinogen. After neutralization, the remaining amount of undigested EGFP refolded and was determined by fluorescence. Under standard digestion conditions, 4.8-24.0 ng pepsin or pepsinogen was used. Using porcine pepsin as a standard, 38+/-6.7 ng EGFP was digested per min-1 ng pepsin-1. Activated porcine pepsinogen revealed a similar digestion rate (37.2+/-5.2 ng EGFP min-1 ng activated pepsinogen-1). The sensitivity of the proteolysis assay depended on the time of digestion and the temperature. Increasing temperature and incubation time allowed quantification of pepsin or pepsinogen in a sample even in the picogram range. The pepsin-catalyzed EGFP digestion showed typical Michaelis-Menten kinetics. Km and Vmax values were determined for the pepsin and activated pepsinogen. Digestion of EGFP by pepsin revealed distinct cleavage sites, as analyzed by SDS-PAGE.     

Ground state isomerization of a model green fluorescent protein chromophore

The relationship between ground state cis-trans isomerization and protonation state is explored for a model green fluorescent protein chromophore, 4-hydroxybenzylidene-1,2-dimethylimidazolinone (HBDI). We find that the protonation state has only a modest effect on the free energy differences between cis and trans isomers and on the activation energies for isomerization. Specifically, the experimental free energy differences are 3.3, 8.8, and 9.6 kJ/mol for cationic, neutral, and anionic forms of HBDI, respectively, and the activation energies are 48.9, 54.8, and 54.8 kJ/mol for cationic, neutral, and anionic forms, respectively. Furthermore, these activation energies are much smaller than might be expected based on comparison with similar systems. These results suggest that there may be a sub-population of the chromophore, which is nearly equally accessible to all three protonation states, through which thermal isomerization may proceed.     

A conserved interaction with the chromophore of fluorescent proteins

The chromophore of fluorescent proteins, including the green fluorescent protein (GFP), contains a highly conjugated imidazolidinone ring. In many fluorescent proteins, the carbonyl group of the imidazolidinone ring engages in a hydrogen bond with the side chain of an arginine residue. Prior studies have indicated that such an electrophilic carbonyl group in a protein often accepts electron density from a main-chain oxygen. A survey of high-resolution structures of fluorescent proteins indicates that electron lone pairs of a main-chain oxygen-Thr62 in GFP-donate electron density into an antibonding orbital of the imidazolidinone carbonyl group. This n→π* electron delocalization prevents structural distortion during chromophore excitation that could otherwise lead to fluorescence quenching. In addition, this interaction is present in on-pathway intermediates leading to the chromophore, and thus could direct its biogenesis. Accordingly, this n→π* interaction merits inclusion in computational and photophysical analyses of the chromophore, and in speculations about the molecular evolution of fluorescent proteins.     

Photoabsorption of green and red fluorescent protein chromophore anions in vacuo

Photoabsorption properties of green and red fluorescent protein chromophore anions in vacuo were investigated theoretically, based on the experimental results in gas phase [Phys. Rev. Lett. 2001, 87, 228102; Phys. Rev. Lett. 2003, 90, 118103]. Their calculated transition energies in absorption with TD-DFT and ZINDO methods are directly compared to the experimental reports in gas phase, and the calculations with ZINDO method can correctly reproduce the absorption spectra. The orientation and strength of their transition dipole moments were revealed with transition density. We also showed the orientation and result of their intramolecular charge transfer with transition difference density. The calculated results show that with the increase of the extended conjugated system, the orientation of transition dipole moments and the orientation of charge transfer can be reversed. They are the linear responds with the external electric fields. These theoretical results reveal the insight understanding of the photoinduced dynamics of green and red fluorescent protein chromophore anions and cations in vacuo.