Peptide bond trans-cis isomerization and acylimine formation in chromophore maturation of the red fluorescent proteins

In red fluorescent proteins such as DsRed, an acylimine is formed from the Phe65-Gln66 linkage in GFP-like immature form, while it shows a cis configuration in its mature state. To date, the relationship between acylimine formation and trans-cis isomerization is still unresolved. We have calculated bond rotation profiles for mature and immature chromophores within the protein using our own n-layered integrated molecular orbital and molecular mechanism (ONIOM) approach. The results suggested that the isomerization is barrierless in acylimine formed in the mature state, suggesting that the acylimine formation precedes the trans-cis isomerization in DsRed chromophores. Further decomposition analysis of electrostatic contributions from individual residues has identified several residues and a specific water molecule which could play key roles in controlling the rate of the trans-cis isomerization of peptide bond in immature GFP-like protein. The results also highlight the importance of Gln66-like of tripeptide motif (chromophore) in the maturation of red fluorescent proteins. In view of the considerable interest in developing red fluorescent proteins for numerous biotechnological applications, these results should be useful for design of novel fluorescent proteins.     

Spectral Versatility of Single Reef Coral Fluorescent Proteins Detected by Spectrally‐Resolved Single Molecule Spectroscopy

We use spectrally‐resolved room temperature single molecule spectroscopy to yield insights into the occurrence and dynamics of spectral forms of single tetramers of DsRed and its variants DsRed2, Fluorescent Timer, DsRed_N42H and AG4. The red‐emitting chromophore in DsRed and all studied variants readily converts into a high quantum efficiency super‐red emitting form. We propose the existence of two super‐red forms of different quantum efficiencies. The observed emission from the green‐emitting chromophore is consistent with bulk spectroscopy. We further observe distinct new spectral forms from each variant, which we attribute to a photoinduced chemical reaction leading to a truncated form of the red‐emitting chromophore analogous to the chromophore in the visible fluorescent protein zFP538. Our results have implications for the accurate interpretation of biological and biochemical processes illuminated by fluorescent proteins as well as for choosing appropriate experimental configurations.     

Resonance CARS study of the structure of "green" and "red" chromophores within the red fluorescent protein DsRed

Vibrational spectra of red fluorescent protein DsRed have been studied for the first time by polarization-sensitive multiplex coherent anti-Stokes Raman scattering at two excitation wavelengths, 545 and 583 nm, in resonance with the absorption bands of the immature "green" and mature "red" protein chromophores. Overall vibrational patterns of both DsRed chromophores were found to be similar to each other and to differ from that of S65T-GFP at pH8. The combined analysis of our CARS data and published structural information suggest that both "green" and "red" DsRed species possess an extended chromophore structure. Consequently, our data suggest that pi-bonding system extension during isomerization around the cis peptide bond between Phe 65 and Gln 66 is a necessary but not sufficient step in DsRed chromophore maturation.     

Insight into the common mechanism of the chromophore formation in the red fluorescent proteins: the elusive blue intermediate revealed

Understanding the chromophore maturation process in fluorescent proteins is important for the design of proteins with improved properties. Here, we present the results of electronic structure calculations identifying the nature of a blue intermediate, a key species in the process of the red chromophore formation in DsRed, TagRFP, fluorescent timers, and PAmCherry. The chromophore of the blue intermediate has a structure in which the π-system of the imidazole ring is extended by the acylimine bond, which can be represented by the model N-[(5-hydroxy-1H-imidazole-2yl)methylidene]acetamide (HIMA) compound. Ab initio and QM/MM calculations of the isolated model and protein-bound (mTagBFP) chromophores identify the anionic form of HIMA as the only structure that has absorption that is consistent with the experiment and is stable in the protein binding pocket. The anion and zwitterion are the only protonation forms of HIMA whose absorption (421 and 414 nm, or 2.95 and 3.00 eV) matches the experimental spectrum of the blue form in DsRed (the absorption maximum is 408 nm or 3.04 eV) and mTagBFP (400 nm or 3.10 eV). The QM/MM optimization of the protein-bound anionic form results in a structure that is close to the X-ray one, whereas the zwitterionic chromophore is unstable in the protein binding pocket and undergoes prompt proton transfer. The computed excitation energy of the protein-bound anionic form of the mTagBFP-like chromophore (3.04 eV) agrees with the experimental absorption spectrum of the protein. The DsRed-like chromophore formation in red fluorescent proteins is revisited on the basis of ab initio results and verified by directed mutagenesis revealing a key role of the amino acid residue 70, which is the second after the chromophore tripeptide, in the formation process.     

Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed

Recently, it has been shown that the red fluorescent protein DsRed undergoes photoconversion on intense irradiation, but the mechanism of the conversion has not yet been elucidated. Upon irradiation with a nanosecond-pulsed laser at 532 nm, the chromophore of DsRed absorbing at 559 nm and emitting at 583 nm (R form) converts into a super red (SR) form absorbing at 574 nm and emitting at 595 nm. This conversion leads to a significant change in the fluorescence quantum yield from 0.7 to 0.01. Here we demonstrate that the photoconversion is the result of structural changes of the chromophore and one amino acid. Absorption, fluorescence, and vibrational spectroscopy as well as mass spectrometry suggest that a cis-to-trans isomerization of the chromophore and decarboxylation of a glutamate (E215) take place upon irradiation to form SR. At the same time, another photoproduct (B) with an absorption maximum at 386 nm appears upon irradiation. This species is assigned as a protonated form of the DsRed chromophore. It might be a mixture of several protonated DsRed forms as there is at least two ways of formation. Furthermore, the photoconversion of DsRed is proven to occur through a consecutive two-photon absorption process. Our results demonstrate the importance of the chromophore conformation in the ground state on the brightness of the protein as well as the importance of the photon flux to control/avoid the photoconversion process.     

Photoconversion in the red fluorescent protein from the sea anemone Entacmaea quadricolor: is cis-trans isomerization involved?

Proteins from the family of the green fluorescent protein (GFP) are presently extensively used in molecular and cellular biology. Recent studies suggest that isomerization of the chromophore occurs upon excitation and is involved in nonradiative deactivation. Using Raman spectroscopy, we report on photoinduced cis-trans isomerization in the red fluorescent protein eqFP611 from the sea anemone Entacmaea quadricolor. The crystal structure of eqFP611 shows that the chemical structure of the chromophore, p-hydroxybenzylidene-imidazolinone with an extended -conjugated system, is nearly identical to the chromophore of other red fluorescent proteins such as DsRed and HcRed. However, the chromophore of eqFP611 has a trans configuration whereas the chromophore of DsRed has a cis configuration. Upon irradiation with 532-nm light, the absorption of eqFP611 peaking at 559 nm diminished, and concomitantly a drastic decrease in the quantum yield of fluorescence as well as more complex decay kinetics was observed. Upon irradiation, changes in the Raman spectrum of eqFP611 were observed, and the relative intensities and peak positions of the irradiated eqFP611 showed striking similarity with the peaks in the Raman spectrum of DsRed. These observations are tentatively interpreted as trans-to-cis isomerization of the chromophore taking place upon irradiation together with the opening of new, nonradiative pathways.     

Trans-cis isomerization is responsible for the red-shifted fluorescence in variants of the red fluorescent protein eqFP611

An important class of red fluorescent proteins (RFPs) feature a 2-iminomethyl-5-(4-hydroxybenzylidene)imidazolinone chromophore. Among these proteins, eqFP611 has the chromophore in a coplanar trans orientation, whereas the cis isomer is preferred by other RFPs such as DsRed and its variants. In the photoactivatable protein asFP595, the chromophore can even be switched from the nonfluorescent trans to the fluorescent cis state by light. By using X-ray crystallography, we have determined the structure of dimeric eqFP611 at high resolution (up to 1.1 A). In the far-red emitting eqFP611 variant d2RFP630, which carries an additional Asn143Ser mutation, the chromophore resides predominantly (approximately 80%) in the cis isomeric state, and in RFP639, which has Asn143Ser and Ser158Cys mutations, the chromophore is found completely in the cis form. The pronounced red shift of excitation and emission maxima of RFP639 can thus unambiguously be assigned to trans-cis isomerization of the chromophore. Among RFPs, eqFP611 is thus unique because its chromophore is highly fluorescent in both the cis and trans isomeric forms.     

Conversion of red fluorescent protein into a bright blue probe

We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in F?rster resonance energy transfer applications.     

Synthesis and spectroscopic studies of model red fluorescent protein chromophores

[reaction: see text]. Here we describe the synthesis and spectroscopic characterization of two compounds designed to model the chromophore in DsRed, a red fluorescent protein. Comparison with model green fluorescent protein (GFP) chromophores indicates that the additional conjugation in the DsRed models can account, in part, for the red-shifted absorption and emission properties of DsRed compared to those of GFP. In contrast to the GFP models, the DsRed models are fluorescent with quantum yields of 0.002-0.01 in CHCl3.     

Competition between energy and proton transfer in ultrafast excited-state dynamics of an oligomeric fluorescent protein red Kaede

We investigated femtosecond and picosecond time-resolved fluorescence dynamics of a tetrameric fluorescent protein Kaede with a red chromophore (red Kaede) to examine a relationship between the excited-state dynamics and a quaternary structure of the fluorescent protein. Red Kaede was obtained by photoconversion from green Kaede that was cloned from a stony coral Trachyphyllia geoffroyi. In common with other typical fluorescent proteins, a chromophore of red Kaede has two protonation states, the neutral and the anionic forms in equilibrium. Time-resolved fluorescence measurements clarified that excitation of the neutral form gives the anionic excited state with a time constant of 13 ps at pH 7.5. This conversion process was attributed to fluorescence resonance energy transfer (FRET) from the photoexcited neutral form to the ground-state anionic form that is located in an adjacent subunit in the tetramer. The time-resolved fluorescence data measured at different pH revealed that excited-state proton transfer (ESPT) also occurs with a time constant of 300 ps and hence that the FRET and ESPT take place simultaneously in the fluorescent protein as competing processes. The ESPT rate in red Kaede was significantly slower than the rate in Aequorea GFP, which highly likely arises from the different hydrogen bond network around the chromophore.     

Origin of the anomalous two-photon absorption in fluorescent protein DsRed

The red fluorescent protein DsRed displays a two-photon excitation band around 760 nm which is not accompanied by any feature in the corresponding one-photon spectral region (380 nm). By means of time-dependent density functional theory, we are able to explain such an effect, as arising from an electronic excitation of the DsRed chromophore with ability to couple with a charge-transfer state, through an effective two-photon absorption channel.     

A synthetic GFP-like chromophore undergoes base-catalyzed autoxidation into acylimine red form

Fluorescent proteins are widely used in modern experimental biology, but much controversy exists regarding details of maturation of different types of their chromophores. Here we studied possible mechanisms of DsRed-type red chromophore formation using synthetic biomimetic GFP-like chromophores, bearing an acylamino substituent, corresponding to an amino acid residue at position 65. We have shown these model compounds to readily react with molecular oxygen to produce a highly unstable DsRed-like acylimine, isolated in the form of stable derivatives. Under the same aerobic conditions an unusual red-shifted imide chromophore--a product of 4-electron oxidation of Gly65 residue--is formed. Our data showed that GFP chromophore is prone to autoxidation at position 65 Cα by its chemical nature with basic conditions being the only key factor required.     

Modeling photoabsorption of the asFP595 chromophore

The fluorescent protein asFP595 is a promising photoswitchable biomarker for studying processes in living cells. We present the results of a high level theoretical study of photoabsorption properties of the model asFP595 chromophore molecule in biologically relevant protonation states: anionic, zwitterionic, and neutral. Ground state equilibrium geometry parameters are optimized in the PBE0/(aug)-cc-pVDZ density functional theory approximation. An augmented version of multiconfigurational quasidegenerate perturbation theory (aug-MCQDPT2) following the state-averaged CASSCF/(aug)-cc-pVDZ calculations is used to estimate the vertical S0-S1 excitation energies for all chromophore species. An accuracy of this approach is validated by comparing the computed estimates of the S0-S1 absorption maximum of the closely related chromophore from the DsRed protein to the known experimental value in the gas phase. An influence of the CASSCF active space on the aug-MCQDPT2 excitation energies is analyzed. The zwitterionic form of the asFP595 chromophore is found to be the most sensitive to a particular choice and amount of active orbitals. This observation is explained by the charge-transfer type of the S0-S1 transition involving the entire conjugated pi-electron system for the zwitterionic protonation state. According to the calculation results, the anionic form in the trans conformation is found to possess the most red-shifted absorption band with the maximum located at 543 nm. The bands of the zwitterionic and neutral forms are considerably blue-shifted compared to those of the anionic form. These conclusions are at variance with the results obtained in the TDDFT approximation for the asFP595 chromophore. The absorption wavelengths computed in the aug-MCQDPT2/CASSCF theory are as follows: 543 (535), 470 (476), and 415 (417) nm for the anionic, zwitterionic, and neutral forms of the trans and cis (in parentheses) isomers of the asFP595 chromophore. These data can be used as a reference for further theoretical studies of the asFP595 chromophore in different media and for modeling photoabsorption properties of the asFP595 fluorescent protein.     

Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes

Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. GFP-like proteins constitute a fast growing family as several naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by conformational stability, quantum yield, spectroscopic properties (positions of absorption and fluorescence spectra) and by photochemical properties. GFP-like proteins are very diverse, as they can be not only green, but also blue, orange-red, far-red, cyan, and yellow. They also can have dual-color fluorescence (e.g., green and red) or be non-fluorescent. Some of them possess kindling property, some are photoactivatable, and some are photoswitchable. This review is an attempt to characterize the main color groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation, systemize data on their conformational stability and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology.     

The role of the protein matrix in green fluorescent protein fluorescence

In the ground state of the highly conjugated green fluorescent protein (GFP), the chromophore should be planar. However, numerous crystal structures of GFP and GFP-like proteins have been reported with slightly twisted chromophores. We have previously shown that the protein cavity surrounding the chromophore in wild-type GFP is not complementary with a planar chromophore. This study shows that the crystal structure of wild-type GFP is not an anomaly: most of the GFP and GFP-like proteins in the protein databank have a protein matrix that is not complementary with a planar chromophore. When the pi-conjugation across the ethylenic bridge of the chromophore is removed the protein matrix will significantly twist the freely rotating chromophore from the relatively planar structures found in the crystal structures. The possible consequences of this nonplanar deformation on the photophysics of GFP are discussed. A volume analysis of the cis-trans-isomerization of HBDI, a GFP chromophore model compound, reveals that its hula-twist motion is volume conserving. This means that, if the GFP chromophore or GFP chromophore model compounds undergo a cis-trans-isomerization in a volume-constricting medium, such as a protein matrix or viscous liquid, it will probably isomerize by means of a HT-type motion.     

Anthozoa red fluorescent protein in biosensing

The identification and cloning of a red fluorescent protein (DsRed) obtained from Anthozoa corals has provided an alternative to commonly used green fluorescent proteins (GFPs) in bioanalytical and biomedical research. DsRed in tandem with GFPs has enhanced the feasibility of multicolor labeling studies. Properties of DsRed, for example high photostability, red-shifted fluorescence emission, and stability to pH changes have proven valuable in its use as a fluorescent tag in cell-biology applications. DsRed has some limitations, however. Its slow folding and tendency to form tetramers have been a hurdle. Several different mutational studies have been performed on DsRed to overcome these problems. In this paper, applications of DsRed in biosensing, specifically in FRET/BRET assays, whole-cell assays, and in biosensors, is discussed. In the future, construction of DsRed mutants with unique characteristics will further expand its applications in bioanalysis.     

Single-molecule fluorescence lifetime and anisotropy measurements of the red fluorescent protein,DsRed, in solution

Fluorescence lifetime and anisotropy measurements were made on the red fluorescent protein (DsRed) from tropical coral of the Discosoma genus, both at single-molecule and bulk concentrations. As expected from previous work, the fluorescence lifetime of DsRed in solution is dependent on laser power, decreasing from an average fluorescence lifetime in the beam of about 3.3 ns at low power (3.5 ns if one extrapolates to zero power) to about 2.1 ns at 28 kW/cm2. At the single-molecule level, exciting with 532 nm, 10 ps laser pulses at 80 MHz repetition rate, DsRed particles entering the laser beam initially have a lifetime of about 3.6 ns and convert to a form having a lifetime of about 3.0 ns with a quantum yield of photoconversion on the order of 10(-3) (calculated in terms of photons per DsRed tetramer). The particles then undergo additional photoconversion with a quantum yield of roughly 10(-5), generating a form with an average lifetime of 1.6 ns. These results may be explained by rapid photoconversion of one DsRed monomer in a tetramer, which acts as an energy transfer sink, resulting in a lower quantum yield for photoconversion of subsequent monomers. Multiparameter correlation and selective averaging can be used to identify DsRed in a mixture of fluorophores, in part exploiting the fact that fluorescent lifetime of DsRed changes as a function of excitation intensity.     

Identification of sites within a monomeric red fluorescent protein that tolerate peptide insertion and testing of corresponding circular permutations

In recent years the class of known fluorescent proteins (FPs) has dramatically expanded as an ever-increasing numbers of variants and homologs of the green fluorescent protein (GFP) from Aequorea jellyfish have been either engineered in the lab or discovered in other marine organisms. The red fluorescent protein (RFP) from Discosoma coral (also known as dsFP583 and DsRed) has proven to be a particularly fruitful progenitor of variants with biochemical and spectroscopic properties conducive to applications in live cell imaging. We have investigated the tolerance of an engineered monomeric descendent of Discosoma RFP, known as mCherry, towards peptide insertion and circular permutation. Starting from a random library of insertion variants, we identified six genetically distinct sites localized in three different loops where a sequence of five residues could be inserted without abolishing the ability of the protein to form its intrinsic red fluorescent chromophore. For each of these insertion variants, a corresponding circular permutation variant was created in which the original N- and C-termini were connected by a six-residue linker and new termini were introduced at the site of the insertion. All six circular permutation variants had significantly diminished brightness relative to the analogous insertion variants. The most promising circular permutation variant has termini at the position corresponding to residue 184 of mCherry and retains 37% of the intrinsic fluorescent brightness of mCherry. These circularly permuted variants may serve as the foundation for construction of genetically encoded Ca2+ sensors analogous to the previously reported camgaroo, pericam and G-CaMP sensors based on variants of Aequorea GFP.     

Metal Affinity-Based Purification of a Red Fluorescent Protein

A red fluorescent protein, DsRed, which emits fluorescence in the red region of the spectrum has become a popular alternative to green fluorescent protein as a label in biochemical and bioanalytical applications. In this study, we have developed a simple purification method for DsRed variants utilizing their inherent copper binding property. A purification procedure was developed and optimized using immobilized copper ions yielding a single strong band corresponding to purified DsRed proteins on the SDS-PAGE gel. A purification efficiency of higher than 95% was achieved. A spectral analysis and copper binding study was performed to verify activity of the purified proteins. The development of this method allows DsRed to play a dual role as a fluorescent reporter protein and as a purification affinity tag for a target protein. This simpler approach of purification should expand the utility of DsRed.