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.     

Generation of bright monomeric red fluorescent proteins via computational design of enhanced chromophore packing

Red fluorescent proteins (RFPs) have found widespread application in chemical and biological research due to their longer emission wavelengths. Here, we use computational protein design to increase the quantum yield and thereby brightness of a dim monomeric RFP (mRojoA, quantum yield = 0.02) by optimizing chromophore packing with aliphatic residues, which we hypothesized would reduce torsional motions causing non-radiative decay. Experimental characterization of the top 10 designed sequences yielded mSandy1 (λ_em = 609 nm, quantum yield = 0.26), a variant with equivalent brightness to mCherry, a widely used RFP. We next used directed evolution to further increase brightness, resulting in mSandy2 (λ_em = 606 nm, quantum yield = 0.35), the brightest Discosoma sp. derived monomeric RFP with an emission maximum above 600 nm reported to date. Crystallographic analysis of mSandy2 showed that the chromophore p-hydroxybenzylidene moiety is sandwiched between the side chains of Leu63 and Ile197, a structural motif that has not previously been observed in RFPs, and confirms that aliphatic packing leads to chromophore rigidification. Our results demonstrate that computational protein design can be used to generate bright monomeric RFPs, which can serve as templates for the evolution of novel far-red fluorescent proteins.

We used computational design to increase quantum yield in a fluorescent protein by optimizing chromophore packing to reduce non-radiative decay, resulting in an >10-fold increase in quantum yield that was further improved by directed evolution.     

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.     

Optimized and far-red-emitting variants of fluorescent protein eqFP611

Fluorescent proteins (FPs) emitting in the far-red region of the spectrum are highly advantageous for whole-body imaging applications because scattering and absorption of long-wavelength light is markedly reduced in tissue. We characterized variants of the red fluorescent protein eqFP611 with bright fluorescence emission shifted up to 639 nm. The additional red shift is caused by a trans-cis isomerization of the chromophore. The equilibrium between the trans and cis conformations is strongly influenced by amino acid residues 143 and 158. Pseudo monomeric tags were obtained by further genetic engineering. For the red chromophores of eqFP611 variants, molar extinction coefficients of up to approximately 150,000 were determined by an approach that is not affected by the presence of molecules with nonfunctional red chromophores. The bright fluorescence makes the red-shifted eqFP611 variants promising lead structures for the development of near-infrared fluorescent markers. The red fluorescent proteins performed well in cell biological applications, including two-photon imaging.     

Radiationless decay of red fluorescent protein chromophore models via twisted intramolecular charge-transfer states

We use CASSCF and MRPT2 calculations to characterize the bridge photoisomerization pathways of a model red fluorescent protein (RFP) chromophore model. RFPs are homologues of the green fluorescent protein (GFP). The RFP chromophore differs from the GFP chromophore via the addition of an N-acylimine substitution to a common hydroxybenzylidene-imidazolinone (HBI) motif. We examine the substituent effects on the manifold of twisted intramolecular charge-transfer (TICT) states which mediates radiationless decay via bridge isomerization in fluorescent protein chromophore anions. We find that the substitution destabilizes states associated with isomerization about the imidazolinone-bridge bond and stabilizes states associated with phenoxy-bridge bond isomerization. We discuss the results in the context of chromophore conformation and quantum yield trends in the RFP subfamily, as well as recent studies on synthetic models where the acylimine has been replaced with an olefin.     

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.     

Red Fluorescent Proteins: Advanced Imaging Applications and Future Design

In the past few years a large series of the advanced red‐shifted fluorescent proteins (RFPs) has been developed. These enhanced RFPs provide new possibilities to study biological processes at the levels ranging from single molecules to whole organisms. Herein the relationship between the properties of the RFPs of different phenotypes and their applications to various imaging techniques are described. Existing and emerging imaging approaches are discussed for conventional RFPs, far‐red FPs, RFPs with a large Stokes shift, fluorescent timers, irreversibly photoactivatable and reversibly photoswitchable RFPs. Advantages and limitations of specific RFPs for each technique are presented. Recent progress in understanding the chemical transformations of red chromophores allows the future RFP phenotypes and their respective novel imaging applications to be foreseen.     

Electronic excitations of green fluorescent proteins: modeling solvatochromatic shifts of red fluorescent protein chromophore model compound in aqueous solutions

While green fluorescent proteins (GFPs) have been widely used as tools in biochemistry, cell biology, and molecular genetics, novel red fluorescent proteins (RFPs) with red fluorescence emission have also been identified, as complements to the existing GFP technology. The unusual spectrophotometric and fluorescence properties of GFPs and RFPs are controlled by the protonation states and possibly cis/trans isomerization within their chromophores. In this work, we have investigated the electronic structures, liquid structures, and solvent shifts of the possible neutral and anionic protonated states and the cis/trans isomerization of a RFP chromophore model compound HBMPI in aqueous solutions. The calculations reproduced the experimental absorption solvatochromatic shifts of dilute HBMPI in water under neutral and anionic conditions. Unlike the GFP chromophore, the RFP chromophore model compound HBMPI in basic solution can only adopt a conformation where the C=C bond between the bridge group and the imidazolinone ring and the C-C bond between the imidazolinone and ethylene groups exist in cis and trans conformations, respectively. Moreover, the solvent-solute hydrogen-bonding interactions are found to contribute significantly to the total solvent shifts of pi-pi* excitations of aqueous HBMPI solutions, signifying the importance of protein environment in the determination of the conformation of the chromophores in red fluorescent proteins.     

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.     

Single‐Molecule Localization Microscopy using mCherry

We demonstrate the potential of the commonly used red fluorescent protein mCherry for single‐molecule super‐resolution imaging. mCherry can be driven into a light‐induced dark state in the presence of a thiol from which it can recover spontaneously or by irradiation with near UV light. We show imaging of subcellular protein structures such as microtubules and the nuclear pore complex with a resolution below 40 nm. We were able to image the C‐terminus of the nuclear pore protein POM121, which is on the inside of the pore and not readily accessible for external labeling. The photon yield for mCherry is comparable to that of the latest optical highlighter fluorescent proteins. Our findings show that the widely used mCherry red fluorescent protein and the vast number of existing mCherry fusion proteins are readily amenable to super‐resolution imaging. This obviates the need for generating novel protein fusions that may compromise function or the need for external fluorescent labeling.     

Stalling chromophore synthesis of the fluorescent protein Venus reveals the molecular basis of the final oxidation step

Fluorescent proteins (FPs) have revolutionised the life sciences, but the mechanism of chromophore maturation is still not fully understood. Here we show that incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; a crystal structure indicates the presence of O₂ located above a dehydrated enolate form of the imidazolone ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an “open” conformation so forming a channel that allows O₂ access to the immature chromophore. Absorbance spectroscopy supported by QM/MM simulations suggests that the first oxidation step involves formation of a hydroperoxyl intermediate in conjunction with dehydrogenation of the methylene bridge. A fully conjugated mature chromophore is formed through release of H₂O₂, both in vitro and in vivo. The possibility of interrupting and photochemically restarting chromophore maturation and the mechanistic insights open up new approaches for engineering optically controlled fluorescent proteins.

Fluorescent proteins (FPs) have revolutionised the life sciences, but the chromophore maturation mechanism is still not fully understood. Here we photochemically trap maturation at a crucial stage and structurally characterise the intermediate.     

Are the Fluorescent Properties of the Cyan Fluorescent Protein Sensitive to Conditions of Oxidative Stress?

The modifications induced by reactive oxygen species (ROS) on fluorescent proteins (FPs) may have important implications for live cell fluorescence imaging. Using quantitative γ-radiolysis, we have studied the ROS-induced biochemical and photophysical perturbations on recombinant cyan fluorescent protein (CFP). After oxidation by the ˙OH radical, the protein displays a modified RP-HPLC elution profile, while the CFP fluorescence undergoes pronounced decreases in intensity and lifetime, without changes in its excitation and emission spectra. Meanwhile, the Förster resonant energy transfer (FRET) between the single W⁵⁷ and the chromophore remains unperturbed. These results rule out a direct oxidation of the CFP chromophore and of W⁵⁷ as well as major changes in the protein 3D structure, but show that new fluorescent forms associated to a higher level of dynamic quenching have been generated. Thus, strict in situ controls are required when CFP is to be used for FRET studies in situations of oxidative activity, or under strong illumination.     

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.     

An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging

Multicolor imaging based on genetically encoded fluorescent proteins (FPs) is a powerful approach to study several dynamic processes in a live cell. We report a monomeric orange FP with a large Stokes shift (LSS), called LSSmOrange (excitation/emission at 437/572 nm), which fills up an existing spectral gap between the green-yellow and red LSSFPs. Brightness of LSSmOrange is five-fold larger than that of the brightest red LSSFP and similar to the green-yellow LSSFPs. LSSmOrange allows numerous multicolor applications using a single-excitation wavelength that was not possible before. Using LSSmOrange we developed four-color single-laser fluorescence cross-correlation spectroscopy, solely based on FPs. The quadruple cross-correlation combined with photon counting histogram techniques allowed quantitative single-molecule analysis of particles labeled with four FPs. LSSmOrange was further applied to simultaneously image two F?rster resonance energy transfer pairs, one of which is the commonly used CFP-YFP pair, with a single-excitation laser line. The combination of LSSmOrange-mKate2 and CFP-YFP biosensors enabled imaging of apoptotic activity and calcium fluctuations in real time. The LSSmOrange mutagenesis, low-temperature, and isotope effect studies revealed a proton relay for the excited-state proton transfer responsible for the LSS phenotype.     

Common pathway for the red chromophore formation in fluorescent proteins and chromoproteins

The mechanism of the chromophore maturation in members of the green fluorescent protein (GFP) family such as DsRed and other red fluorescent and chromoproteins was analyzed. The analysis indicates that the red chromophore results from a chemical transformation of the protonated form of the GFP-like chromophore, not from the anionic form, which appears to be a dead-end product. The data suggest a rational strategy to achieve the complete red chromophore maturation utilizing substitutions to favor the formation of the neutral phenol in GFP-like chromophore. Our approach to detect the neutral chromophore form expands the application of fluorescent timer proteins to faster promoter activities and more spectrally distinguishable fluorescent colors. Light sensitivity found in the DsRed neutral form, resulting in its instant transformation to the mature red chromophore, could be exploited to accelerate the fluorescence acquisition.     

Two-photon excitation of a phytofluor protein

Phytofluors are highly fluorescent proteins in which the chromophore in a phytochrome is replaced with phycoerythrobilin (PEB), the pigment precursor of the cyanobacterial light harvesting protein phycoerythrin. We examined the fluorescence spectra of the N-terminal region of the cyanobacterial phytochrome 1 from cyanobacterium Synechocystis sp. Pcc 6803 bound to PEB. This protein, Cph1(N514)-PEB, displayed a good two-photon cross-section of 20–30 GM for excitation at 792 nm. This phytofluor also exhibits a high fundamental anisotropy at most practical two-photon excitation (2PE) wavelengths from 700 to 900 nm. Identical lifetimes and correlation times with one and 2PE indicates that the phytofluor is not adversely affected by the intensities needed for 2PE. The one-photon absorption extends well beyond the absorption spectrum and even beyond the emission spectrum to 700 nm. The phytofluor thus appears to be a suitable probe for 2PE and/or cellular imaging.     

The nature of transient dark states in a photoactivatable fluorescent protein

Fluorescent proteins (FPs) of the green fluorescent protein family blink and bleach like all fluorophores. However, contrary to organic dyes, the mechanisms by which transient losses of fluorescence occur in FPs have received little attention. Here, we focus on the photoactivatable IrisFP, for which a transient non-fluorescent chromophoric state with distorted geometry was recently reported (Adam, V.; et al. J. Am. Chem. Soc. 009, 131, 18063). We investigated the chemical nature of this blinked state by employing quantum chemical/molecular mechanical calculations. Our findings suggest two previously unidentified dark states that display similar distorted chromophores with a transiently ruptured π-electron system. Both are protonated at atom C(α) of the chromophore methylene bridge. Transient protonation may occur via proton transfer from the nearby Arg66 either in the triplet state T(1) after intersystem crossing or in an anionic radical (doublet) ground state. As Arg66 is conserved in green-to-red photoconvertible FPs, these dark states are predicted to be common to all these proteins. We also suggest that C(α) protonated dark states may accelerate photobleaching by favoring decarboxylation of the fully conserved Glu212.     

Quantification of Photosensitized Singlet Oxygen Production by a Fluorescent Protein

Fluorescent proteins are increasingly becoming actuators in a range of cell biology techniques. One of those techniques is chromophore‐assisted laser inactivation (CALI), which is employed to specifically inactivate the function of target proteins or organelles by producing photochemical damage. CALI is achieved by the irradiation of dyes that are able to produce reactive oxygen species (ROS). The combination of CALI and the labelling specificity that fluorescent proteins provide is useful to avoid uncontrolled photodamage, although the inactivation mechanisms by ROS are dependent on the fluorescent protein and are not fully understood. Herein, we present a quantitative study of the ability of the red fluorescent protein TagRFP to produce ROS, in particular singlet oxygen (¹O₂). TagRFP is able to photosensitize ¹O₂ with an estimated quantum yield of 0.004. This is the first estimation of a quantum yield of ¹O₂ production value for a GFP‐like protein. We also find that TagRFP has a short triplet lifetime compared to EGFP, which reflects relatively high oxygen accessibility to the chromophore. The insight into the structural and photophysical properties of TagRFP has implications in improving fluorescent proteins for fluorescence microscopy and CALI.     

Understanding GFP posttranslational chemistry: structures of designed variants that achieve backbone fragmentation, hydrolysis, and decarboxylation

The green fluorescent protein (GFP) creates a fluorophore out of three sequential amino acids by promoting spontaneous posttranslational modifications. Here, we use high-resolution crystallography to characterize GFP variants that not only undergo peptide backbone cyclization but additional denaturation-induced peptide backbone fragmentation, native peptide hydrolysis, and decarboxylation reactions. Our analyses indicate that architectural features that favor GFP peptide cyclization also drive peptide hydrolysis. These results are relevant for the maturation pathways of GFP homologues, such as the kindling fluorescent protein and the Kaede protein, which use backbone cleavage to red-shift the spectral properties of their chromophores. We further propose a photochemical mechanism for the decarboxylation reaction, supporting a role for the GFP protein environment in facilitating radical formation and one-electron chemistry, which may be important in activating oxygen for the oxidation step of chromophore biosynthesis. Together, our results characterize GFP posttranslational modification chemistry with implications for the energetic landscape of backbone cyclization and subsequent reactions, and for the rational design of predetermined spontaneous backbone cyclization and cleavage reactions.     

几个荧光蛋白发色团双光子吸收性质的理论研究

实验测得的荧光蛋白的单、双光子吸收光谱在低频和高频区域都表现出明显不同的特征。为了揭示这些不同点的起源和研究荧光蛋白的构–效关系,我们详细研究了三种荧光蛋白发色团(一种增强型蓝绿色荧光蛋白的中性发色团和两种红色荧光蛋白的阴离子发色团)的单、双光子吸收特性,分别计算了纯的和振动分辨的电子谱。计算结果表明：光谱线形与计算采用的交换相关密度泛函及谱截面计算所采用的近似关系密切;如果在计算光谱截面时,我们利用长程修正的交换相关泛函CAM-B3LYP来计算几何和电子结构参数,然后把Franck-Condon (FC)效应和包含Herzberg-Teller (HT)效果的电-声耦合效应都考虑进去,理论计算的光谱与实验测定的光谱可以很好地符合;对于两种离子态的发色团,HT电-声耦合效应使得对应于基态到第一激发态跃迁的双光子吸收最强峰相对于单光子吸收的最强峰发生了蓝移,但HT电-声耦合效应对高频的双光子吸收谱没有太大的影响;分子内电荷转移是导致高频区的双光子吸收明显强于单光子吸收的主要原因。