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.     

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.     

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.     

利用聚合物自组装实现绿色荧光蛋白生色团的荧光增强

根据绿色荧光蛋白的发光原理,采用聚乙二醇与聚甲基丙烯酸甲酯的两亲性两嵌段聚合物通过自组装包覆生色团的方式,模拟了绿色荧光蛋白发光,考察了组装行为对光学性能的影响,并将其用于细胞成像.通过核磁共振、高分辨质谱、傅里叶变换红外光谱、凝胶渗透色谱、紫外-可见吸收光谱及荧光光谱等表征了生色团分子和聚合物的结构及性能.生色团紫外最大吸收在371 nm,荧光最大发射峰在428 nm.聚合物和生色团进行组装后,其紫外吸收消失,而最大荧光发射峰强度大大增强,且发生了约70 nm的红移,这是因为组装使得生色团的自由旋转受到了限制,且生色团共平面性增加.动态光散射(DLS)和透射电镜(TEM)证明了纳米粒子的结构和尺寸.由于尺寸适合且具有较好的荧光性能,纳米粒子成功应用于细胞成像.这种绿色荧光蛋白生色团的简单自组装方式在生物成像领域具有良好应用前景.     

Mechanistic aspects of proton chain transfer: a computational study for the green fluorescent protein chromophore

We explore several models for the ground-state proton chain transfer pathway between the green fluorescent protein chromophore and its surrounding protein matrix, with a view to elucidating mechanistic aspects of this process. We have computed quantum chemically the minimum energy pathways (MEPs) in the ground electronic state for one-, two-, and three-proton models of the chain transfer. There are no stable intermediates for our models, indicating that the proton chain transfer is likely to be a single, concerted kinetic step. However, despite the concerted nature of the overall energy profile, a more detailed analysis of the MEPs reveals clear evidence of sequential movement of protons in the chain. The ground-state proton chain transfer does not appear to be driven by the movement of the phenolic proton off the chromophore onto the neutral water bridge. Rather, this proton is the last of the three protons in the chain to move. We find that the first proton movement is from the bridging Ser205 moiety to the accepting Glu222 group. This is followed by the second proton moving from the bridging water to the Ser205--for our model this is where the barrier occurs. The phenolic proton on the chromophore is hence the last in the chain to move, transferring to a bridging "water" that already has substantial negative charge.     

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.     

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.     

POLARIZED PHOTO ACOUSTIC SPECTRA OF PHYCOBILISOMES IN POLYVINYL ALCOHOL FILMS

Abstract— Phycobilisomes from Porphyridium cruentum , suspended in polyvinyl alcohol were found to be highly stable, and had normal absorption and fluorescence spectra. Intact phycobilisomes had a major emission peak at 680 nm, whereas upon partial dissociating the major emission was at 580 nm. Incorporation of phycobilisomes in thin polyvinyl alcohol films facilitated examination by photoacoustic spectroscopy. The photoacoustic spectra had a broad absorption maximum at 545–565 nm (phycoerythrin), which resolved as two peaks (545 and 563 nm) in absorption spectra. Stretching of films resulted in apparent chromophore reorientation in partially dissociated, but not in intact phycobilisomes. Only in dissociated phycobilisomes was observed a differential chromophore orientation at 685 nm by polarized fluorescence, which is attributed to a change in orientation of the terminal phycobilisome pigment relative to phycoerythrin.     

Functional characterization of permuted enhanced green fluorescent proteins comprising varying linker peptides

New variants of green fluorescent protein (GFP) can be engineered by circular permutation of their amino acid sequence. We characterized a series of permuted enhanced GFP (PEGFP) with new termini introduced at N144-Y145 and linkers of 1, 3, 5 and 6 residues inserted between G232 and M1, as well as a variant with an extended 7-residues linker between K238 and M1. A minimum linker length of 3 residues was necessary for a functional chromophore to be formed, and linkers exceeding 4 residues yielded almost the same fluorescence quantum yield as enhanced GFP (EGFP). PEGFP exhibited dual-wavelength absorption and fluorescence excitation with peaks at 395 and 490 nm but single-wavelength emission at 512 nm. Fluorescence emission increased with increasing pH for all excitation wavelengths with a pKa of 7.7. Between the pH values of 6 and 8 optical absorption showed an isobestic point at 445 nm. PEGFP rapidly denatured in urea between 50 and 60 degrees C. Renaturation proceeded with a short (approximately 29 s) and a longer (> 150 s) time constant. Transient transfection of HEK293 and HeLa cells revealed the expression dynamics of PEGFP to be similar to that of EGFP. Laser-scanning microscopy of HeLa cells demonstrated that the PEGFP are particularly well suited as fluorescent indicators in two-photon imaging.     

Fluorescent protein barrel fluctuations and oxygen diffusion pathways in mCherry

Fluorescent proteins (FPs) are valuable tools as biochemical markers for studying cellular processes. Red fluorescent proteins (RFPs) are highly desirable for in vivo applications because they absorb and emit light in the red region of the spectrum where cellular autofluorescence is low. The naturally occurring fluorescent proteins with emission peaks in this region of the spectrum occur in dimeric or tetrameric forms. The development of mutant monomeric variants of RFPs has resulted in several novel FPs known as mFruits. Though oxygen is required for maturation of the chromophore, it is known that photobleaching of FPs is oxygen sensitive, and oxygen-free conditions result in improved photostabilities. Therefore, understanding oxygen diffusion pathways in FPs is important for both photostabilites and maturation of the chromophores. In this paper, we use molecular dynamics calculations to investigate the protein barrel fluctuations in mCherry, which is one of the most useful monomeric mFruit variant. We employ implicit ligand sampling to determine oxygen pathways from the bulk solvent into the mCherry chromophore in the interior of the protein. We also show that these pathways can be blocked or altered and barrel fluctuations can be reduced by strategic amino acid substitutions.     

Dipolar relaxation within the protein matrix of the green fluorescent protein: a red edge excitation shift study

The fluorophore in green fluorescent protein (GFP) is localized in a highly constrained environment, protected from the bulk solvent by the barrel-shaped protein matrix. We have used the wavelength-selective fluorescence approach (red edge excitation shift, REES) to monitor solvent (environment) dynamics around the fluorophore in enhanced green fluorescent protein (EGFP) under various conditions. Our results show that EGFP displays REES in buffer and glycerol, i.e., the fluorescence emission maxima exhibit a progressive shift toward the red edge, as the excitation wavelength is shifted toward the red edge of the absorption spectrum. Interestingly, EGFP displays REES when incorporated in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT), independent of the hydration state. We interpret the observed REES to the constrained environment experienced by the EGFP fluorophore in the rigid protein matrix, rather than to the dynamics of the bulk solvent. These results are supported by the temperature dependence of REES and characteristic wavelength-dependent changes in fluorescence anisotropy.     

Dipolar stabilization of emissive singlet charge transfer excited states in polyfluorene copolymers

The singlet excited-state dynamics in poly[(9,9-dioctylfluorene)-(dibenzothiophene-S,S-dioxide)] (PFSx ) random copolymers with different contents of dibenzothiophene-S,S-dioxide (S) units have been studied by steady-state and time resolved fluorescence spectroscopies. Emission from PFSx copolymers shows a pronounced solvatochromism in polar chloroform, relative to the less polar toluene. An excited intramolecular charge transfer state (ICT) is stabilized by dipole-dipole interactions with the polar solvent cage, and possibly accompanied by conformational rearrangement of the molecular structure, in complete analogy with their small oligomer counterparts. The spectral dynamics clearly show that the ICT stabilization is strongly affected by the surrounding medium. In the solid state, emission from PFSx copolymers depends on the content of S units, showing an increase of inhomogeneous broadening and a red shift of the optical transitions. This observation is consistent with stabilization of the emissive ICT state, by the local reorientation of the surrounding molecules at the location of the excited chromophore, which results in favorable dipole-dipole interactions driven by the increase in the dielectric constant of the bulk polymer matrix with increasing S content, in analogy to what happens in polar solvent studies. Furthermore, in clear agreement with the interpretation described above, a strong increase in the emission quantum efficiency is observed in the solid state by decreasing the temperature and freezing out the molecular torsions and dipole-dipole interactions necessary to stabilize the ICT state.     

Resolving the ultrafast dynamics of the anionic green fluorescent protein chromophore in water

The chromophore of the green fluorescent protein (GFP) is critical for probing environmental influences on fluorescent protein behavior. Using the aqueous system as a bridge between the unconfined vacuum system and a constricting protein scaffold, we investigate the steric and electronic effects of the environment on the photodynamical behavior of the chromophore. Specifically, we apply ab initio multiple spawning to simulate five picoseconds of nonadiabatic dynamics after photoexcitation, resolving the excited-state pathways responsible for internal conversion in the aqueous chromophore. We identify an ultrafast pathway that proceeds through a short-lived (sub-picosecond) imidazolinone-twisted (I-twisted) species and a slower (several picoseconds) channel that proceeds through a long-lived phenolate-twisted (P-twisted) intermediate. The molecule navigates the non-equilibrium energy landscape via an aborted hula-twist-like motion toward the one-bond-flip dominated conical intersection seams, as opposed to following the pure one-bond-flip paths proposed by the excited-state equilibrium picture. We interpret our simulations in the context of time-resolved fluorescence experiments, which use short- and long-time components to describe the fluorescence decay of the aqueous GFP chromophore. Our results suggest that the longer time component is caused by an energetically uphill approach to the P-twisted intersection seam rather than an excited-state barrier to reach the twisted intramolecular charge-transfer species. Irrespective of the location of the nonadiabatic population events, the twisted intersection seams are inefficient at facilitating isomerization in aqueous solution. The disordered and homogeneous nature of the aqueous solvent environment facilitates non-selective stabilization with respect to I- and P-twisted species, offering an important foundation for understanding the consequences of selective stabilization in heterogeneous and rigid protein environments.

Simulations on the aqueous green fluorescent protein (GFP) chromophore (in the equilibrium and non-equilibrium regimes) reveal that observed biexponential fluorescence originates from two competing torsional deactivation pathways.     

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.     

Structural chemistry of a green fluorescent protein Zn biosensor

We designed a green fluorescent protein mutant (BFPms1) that preferentially binds Zn(II) (enhancing fluorescence intensity) and Cu(II) (quenching fluorescence) directly to a chromophore ligand that resembles a dipyrrole unit of a porphyrin. Crystallographic structure determination of apo, Zn(II)-bound, and Cu(II)-bound BFPms1 to better than 1.5 A resolution allowed us to refine metal centers without geometric restraints, to calculate experimental standard uncertainty errors for bond lengths and angles, and to model thermal displacement parameters anisotropically. The BFPms1 Zn(II) site (KD = 50 muM) displays distorted trigonal bipyrimidal geometry, with Zn(II) binding to Glu222, to a water molecule, and tridentate to the chromophore ligand. In contrast, the BFPms1 Cu(II) site (KD = 24 muM) exhibits square planar geometry similar to metalated porphyrins, with Cu(II) binding to the chromophore chelate and Glu222. The apo structure reveals a large electropositive region near the designed metal insertion channel, suggesting a basis for the measured metal cation binding kinetics. The preorganized tridentate ligand is accommodated in both coordination geometries by a 0.4 A difference between the Zn and Cu positions and by distinct rearrangements of Glu222. The highly accurate metal ligand bond lengths reveal different protonation states for the same oxygen bound to Zn vs Cu, with implications for the observed metal ion specificity. Crystallographic anisotropic thermal factor analysis validates metal ion rigidification of the chromophore in enhancement of fluorescence intensity upon Zn(II) binding. Thus, our high-resolution structures reveal how structure-based design has effectively linked selective metal binding to changes in fluorescent properties. Furthermore, this protein Zn(II) biosensor provides a prototype suitable for further optimization by directed evolution to generate metalloprotein variants with desirable physical or biochemical properties.     

Native Biomolecules in the Gas Phase? The Case of Green Fluorescent Protein

Green fluorescent protein (GFP) was ionized by native electrospray ionization and trapped for many seconds in high vacuum, allowing fluorescence emission to be measured as a probe of its biological function, to answer the question whether GFP exists in the native form in the gas phase or not. Although a narrow charge‐state distribution, a collision cross‐section very close to that expected for correctly folded GFP, and a large stability against dissociation all support a near‐native gas‐phase structure, no fluorescence emission was observed. The loss of the native form is attributed to the absence of residual water in the gas phase, which normally stabilizes the para‐hydroxybenzylidene imidazolone chromophore of GFP.     

Pockels-effect relaxation in poled side chain polymers: Decoupling of chromophor reorientation from α-relaxation

 The relaxation of the polar order of poled side chain polymers carrying NLO-active chromophores was monitored by Pockels-effect relaxation studies. Dielectric relaxation investigations were performed in order to analyze the coupling or decoupling of the chromophore reorientation to the relaxation modes of the side chain polymers. It was found that the chromophores perform their own reorientation relaxation mode both in the molten and the glassy state, which is not coupled to backbone relaxations. The chromophore reorientation process is characterized by a narrow distribution of the relaxation times and high activation energies. Studies on physical aging reveal that the chromophore reorientation is controlled by the free volume. The chromophore reorientation process can be influenced by the chemical linkage of the chromophore to the polymer backbone and by the nature of the backbone. Received: 1 August 1996 Accepted: 22 November 1996     

Self-restricted oxazolone GFP chromophore for construction of reaction-based fluorescent probe toward dopamine

The β-barrel provides a confined environment for chromophores of the green fluorescent protein (GFP) family, defining their emission profiles by the chromophore/β-barrel interactions. Here, we describe the generation of self-restricted oxazolone GFP chromophore (GFPc) for construction of reaction-based fluorescent probe toward dopamine by mimicking the confinement effect of the β-barrel. Through standard synthetic method, the first self-restricted GFPc oxazolone analogue (MBDO) and the conventional pyrenyl-based chromophore (PDO) were prepared respectively. Under the same condition, MBDO shows much better emission response with fluorescent quantum yield (QY) over one order of magnitude higher than that of PDO due to the generation of the self-restricted effect. And, the fluorescent QY of MBDO reaches above 30% in dimethyl sulfoxide, which is the largest ever recorded for unlocked GFPc analogues in highly polar solvents. Moreover, theoretical calculations further reveal that the enhanced emission of MBDO is due to the inhibition of conformational motions around the exocyclic CC bonds. Combination the enhanced emission and the reactivity of the lactone, MBDO is applied to construct reaction-based fluorescent probe toward dopamine via a ring-opening reaction of the lactone. Prospectively, the destruction of the oxazolone would break the effective conjugated structure of the chromophore, which can decrease the corresponding fluorescence. This work puts forward a novel approach to generate highly emissive GFPc oxazolone analogue, which can be used to fabricate reaction-based fluorescent probe toward dopamine, potentially promoting the biochemical applications using synthetic GFP chromophore analogues.