Computational prediction of absorbance maxima for a structurally diverse series of engineered green fluorescent protein chromophores

By virtue of its self-sufficiency to form a visible wavelength chromophore within the confines of its tertiary structure, the Aequorea victoria green fluorescent protein (GFP) is single-handedly responsible for the ever-growing popularity of fluorescence imaging of recombinant fusion proteins in biological research. Engineered variants of GFP with altered excitation or emission wavelength maxima have helped to expand the range of applications of GFP. The engineering of the GFP variants is usually done empirically by genetic modifications of the chromophore structure and/or its environment in order to find variants with new photophysical properties. The process of identifying improved variants could be greatly facilitated if augmented or guided by computational studies of the chromophore ground and excited-state properties and dynamics. In pursuit of this goal, we now report a thorough investigation of computational methods for prediction of the absorbance maxima for an experimentally validated series of engineered GFP chromophore analogues. The experimental dataset is composed of absorption maxima for 10 chemically distinct GFP chromophore analogues, including a previously unreported Y66D variant, measured under identical denaturing conditions. For each chromophore analogue, excitation energies and oscillator strengths were calculated using configuration interaction with single excitations (CIS), CIS with perturbative correction for double substitutions [CIS(D)], and time-dependent density functional theory (TD DFT) using several density functionals with solvent effects included using a polarizable continuum model. Comparison of the experimental and computational results show generally poor quantitative agreement with all methods attempted. However, good linear correlations between the calculated and experimental excitation energies (R2>0.9) could be obtained. Oscillator strengths obtained with TD DFT using pure density functionals also correlate well with the experimental values. Interestingly, most of the computational methods used in this work fail in the case of nonaromatic Y66S and Y66L protein chromophores, which may be related to a significant contribution of double excitations to their excited-state wavefunctions. These results provide an important benchmark of the reliability of the computational methods as applied to GFP chromophore analogues and lays a foundation for the computational design of GFP variants with improved properties for use in biological imaging.     

Analog Retinal Redshifts Visible Absorption of QuasAr Transmembrane Voltage Sensors into Near-infrared

Opsin-based transmembrane voltage sensors (OTVSs) are increasingly important tools for neuroscience enabling neural function in complex brain circuits to be explored in live, behaving animals. However, the visible wavelengths required for fluorescence excitation of the current generation of OTVSs limit optogenetic imaging in the brain to depths of only a few mm due to the strong absorption and scattering of visible light by biological tissues. We report that substitution of the native A1 retinal chromophore of the widely used QuasAr1/2 OTVSs with the retinal analog MMAR containing a methylamino-modified dimethylphenyl ring results in over a 100-nm redshift of the maxima of the absorption and fluorescence emission bands to near 700 and 840 nm, respectively. FT-Raman spectroscopy reveals that at pH 7 QuasAr1 with both the A1 and MMAR chromophores possess predominantly an all-trans protonated Schiff base configuration with the MMAR chromophore exhibiting increased torsion of the polyene single-/double-bond system similar to the O-intermediate of the BR photocycle. In contrast, the A1 and the MMAR chromophores of QuasAr2 exist partially in a 13-cis PSB configuration. These results demonstrate that QuasArs containing the MMAR chromophore are attractive candidates for use as NIR-OTVSs, especially for applications such as deep brain imaging.     

Dual Illumination Enhances Transformation of an Engineered Green‐to‐Red Photoconvertible Fluorescent Protein

The molecular mechanisms for the photoconversion of fluorescent proteins remain elusive owing to the challenges of monitoring chromophore structural dynamics during the light‐induced processes. We implemented time‐resolved electronic and stimulated Raman spectroscopies to reveal two hidden species of an engineered ancestral GFP‐like protein LEA, involving semi‐trapped protonated and trapped deprotonated chromophores en route to photoconversion in pH 7.9 buffer. A new dual‐illumination approach was examined, using 400 and 505 nm light simultaneously to achieve faster conversion and higher color contrast. Substitution of UV irradiation with visible light benefits bioimaging, while the spectral benchmark of a trapped chromophore with characteristic ring twisting and bridge‐H bending motions enables rational design of functional proteins. With the improved H‐bonding network and structural motions, the photoexcited chromophore could increase the photoswitching‐aided photoconversion while reducing trapped species.     

Effect of microhydration on the electronic structure of the chromophores of the photoactive yellow and green fluorescent proteins

Electronic structure calculations of microhydrated model chromophores (in their deprotonated anionic forms) of the photoactive yellow and green fluorescent proteins (PYP and GFP) are reported. Electron-detachment and excitation energies as well as binding energies of mono- and dihydrated isomers are computed and analyzed. Microhydration has different effects on the excited and ionized states. In lower-energy planar isomers, the interaction with one water molecule blueshifts the excitation energies by 0.1-0.2 eV, whereas the detachment energies increase by 0.4-0.8 eV. The important consequence is that microhydration by just one water molecule converts the resonance (autoionizing) excited states of the bare chromophores into bound states. In the lower-energy microhydrated clusters, interactions with water have negligible effect on the chromophore geometry; however, we also identified higher-energy dihydrated clusters of PYP in which two water molecules form hydrogen-bonding network connecting the carboxylate and phenolate moieties and the chromophore is strongly distorted resulting in a significant shift of excitation energies (up to 0.6 eV).     

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.     

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.     

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.     

An alternate proton acceptor for excited-state proton transfer in green fluorescent protein: rewiring GFP

The neutral form of the chromophore in wild-type green fluorescent protein (wtGFP) undergoes excited-state proton transfer (ESPT) upon excitation, resulting in characteristic green (508 nm) fluorescence. This ESPT reaction involves a proton relay from the phenol hydroxyl of the chromophore to the ionized side chain of E222, and results in formation of the anionic chromophore in a protein environment optimized for the neutral species (the I* state). Reorientation or replacement of E222, as occurs in the S65T and E222Q GFP mutants, disables the ESPT reaction and results in loss of green emission following excitation of the neutral chromophore. Previously, it has been shown that the introduction of a second mutation (H148D) into S65T GFP allows the recovery of green emission, implying that ESPT is again possible. A similar recovery of green fluorescence is also observed for the E222Q/H148D mutant, suggesting that D148 is the proton acceptor for the ESPT reaction in both double mutants. The mechanism of fluorescence emission following excitation of the neutral chromophore in S65T/H148D and E222Q/H148D has been explored through the use of steady state and ultrafast time-resolved fluorescence and vibrational spectroscopy. The data are contrasted with those of the single mutant S65T GFP. Time-resolved fluorescence studies indicate very rapid (< 1 ps) formation of I* in the double mutants, followed by vibrational cooling on the picosecond time scale. The time-resolved IR difference spectra are markedly different to those of wtGFP or its anionic mutants. In particular, no spectral signatures are apparent in the picosecond IR difference spectra that would correspond to alteration in the ionization state of D148, leading to the proposal that a low-barrier hydrogen bond (LBHB) is present between the phenol hydroxyl of the chromophore and the side chain of D148, with different potential energy surfaces for the ground and excited states. This model is consistent with recent high-resolution structural data in which the distance between the donor and acceptor oxygen atoms is < or = 2.4 A. Importantly, these studies indicate that the hydrogen-bond network in wtGFP can be replaced by a single residue, an observation which, when fully explored, will add to our understanding of the various requirements for proton-transfer reactions within proteins.     

Intramolecular energy hopping and energy trapping in polyphenylene dendrimers with multiple peryleneimide donor chromophores and a terryleneimide acceptor trap chromophore

Intramolecular F?rster-type excitation energy transfer (FRET) processes in a series of first-generation polyphenylene dendrimers substituted with spatially well-separated peryleneimide chromophores and a terryleneimide energy-trapping chromophore at the rim were investigated by steady-state and time-resolved fluorescence spectroscopy. Energy-hopping processes among the peryleneimide chromophores are revealed by anisotropy decay times of 50--80 ps consistent with a FRET rate constant of k(hopp) = 4.6 ns(-1). If a terryleneimide chromophore is present at the rim of the dendrimer together with three peryleneimide chromophores, more than 95% of the energy harvested by the peryleneimide chromophores is transferred and trapped in the terryleneimide. The two decay times (tau(1) = 52 ps and tau(2) = 175 ps) found for the peryleneimide emission band are recovered as rise times at the terryleneimide emission band proving that the energy trapping of peryleneimide excitation energy by the terryleneimide acceptor occurs via two different, efficient pathways. Molecular- modeling-based structures tentatively indicate that the rotation of the terryleneimide acceptor group can lead to a much smaller distance to a single donor chromophore, which could explain the occurrence of two energy-trapping rate constants. All energy-transfer processes are quantitatively describable with F?rster energy transfer theory, and the influence of the dipole orientation factor in the F?rster equation is discussed.