Structural features responsible for GFPuv and S147P-GFP’s improved fluorescence

Green fluorescent protein (GFP) is used as a biological marker. It is a protein in the jellyfish, Aequorea victorea, which is found in the cold Pacific Northwest. Mature GFP, i.e. fully fluorescent GFP, is most efficiently formed at temperatures well below 37 °C. The GFPuv (F99S/M153T/V163A) and S147P-GFP mutants mature more efficiently at room temperature than wild-type GFP, and therefore result in increased fluorescence at room temperature. Computational methods have been used to examine whether the low-energy precyclized forms of these improved GFP-mutants are preorganized so that they can more efficiently form the chromophore than the wild-type and S65T-GFP. All mutations examined (S147P, F99S, M153T, V163A and F99S/M153T/V163A) more efficiently preorganize the immature precyclized forms of GFP for chromophore formation than immature wild-type GFP. It has been proposed that Arg96 is involved in chromophore formation. Our calculations suggest that the M153T and V163A mutations in GFPuv maybe partially responsible for the increased maturation efficiency observed in GFPuv because they improve the Arg96–Tyr66 interaction. The same is true for the S147P mutation in S147P-GFP.     

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.     

Light-driven decarboxylation of wild-type green fluorescent protein

The response of wild-type GFP to UV and visible light was investigated using steady state absorption, fluorescence, and Raman spectroscopies. As reported previously [van Thor, Nat. Struct. Biol. 2002, 9, 37-41], irradiation of GFP results in decarboxylation of E222. Here it is reported that the rate of the light-driven decarboxylation reaction strongly depends on the excitation wavelength, decreasing in the order 254 nm > 280 nm > 476 nm. The relative efficiencies of decarboxylation are explained in terms of the Kolbe-type mechanism in which the excited state of the chromophore acts as an oxidant by accepting an electron from E222. Specifically, it is proposed that 254 nm excitation populates the S2 (or higher) excited state of the chromophore, whereas 404 and 476 nm excitation populate the S1 excited state of neutral and anionic forms, respectively, and that the relative oxidizing power of the three excited states controls the rate of the decarboxylation reaction. In addition, the role of W57 in the photophysics of GFP has been probed by mutating this residue to phenylalanine. These studies reveal that while W57 does not affect decarboxylation, this residue is involved in resonance energy transfer with the chromophore, thereby partially explaining the green fluorescence observed upon UV irradiation of wild-type GFP. Finally, comparison of Raman spectra obtained from nonilluminated and decarboxylated forms of wild-type GFP has provided further vibrational band assignments for neutral and anionic forms of the chromophore within the protein. In addition, these spectra provide valuable insight into the specific interactions between the protein and the chromophore that control the optical properties of wild-type GFP.     

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.     

Photoinduced Energy- and Electron-Transfer Processes in a Side-to-Face RuII-Porphyrin/Perylene-bisimide Array

A side-to-face array DPy-gPBI[Ru(4-tBuTPP)(CO)]₂, based on a “green” perylene bisimide chromophore sandwiched between two Ru^II-porphyrins, has been prepared by self-assembly. Its photophysical properties have been characterized in detail by a combination of steady-state and time-resolved techniques upon selective excitation of the two different components. Different photoinduced processes are observed as a function of the excitation wavelength. Electron transfer quenching is attained upon “red light” excitation of the perylene unit, whilst an energy transfer pathway is followed upon “green light” excitation of the metallo-porphyrin moiety. Regardless of the excitation wavelength efficient population of the triplet excited state of the perylene chromophore is achieved. The photophysical results are discussed within the framework of classical electron transfer theory and compared with those of a previously reported system.     

Visible‐Light‐Induced Photodimerization of a Photoactive Yellow Protein (PYP) Chromophore Model in a Single Crystal

The chromophore of the photoactive yellow protein (PYP), the photoreceptor in the photomotility of the bacterium Halorhodospira halophila, is a deprotonated para‐coumaric thioester linked to the side residue of a cysteine residue. The photophysics of the PYP chromophore is conveniently modeled with para‐hydroxycinnamic thiophenyl esters. Herein, we report the first direct evidence, obtained with X‐ray diffraction, of photodimerization of a para‐hydroxycinnamic thiophenyl ester in single crystalline state. This result represents the first direct observation of [2+2] dimerization of a model PYP chromophore, and demonstrates that even very weak light in the visible region is capable of inducing parallel radical reactions in PYP from the excited state of the chromophore, in addition to the main reaction pathway (trans–cis isomerization). This PYP model system adds an interesting example to the known solid‐state photodimerizations, because unlike the anhydrous crystal (which is not capable of sustaining the stress and disintegrates in the course of photodimerization), a single water molecule “dilutes” the structure to the extent sufficient for single‐crystal‐to‐single‐crystal reaction.     

A general efficient implementation of the BSSE-free SCF and MP2 methods based on the chemical Hamiltonian approach

We describe some details related to a new, general, and efficient implementation of the BSSE‐free SCF and second‐order Møller–Plesset perturbation theories of intermolecular interactions, based on the “Chemical Hamiltonian Approach” (CHA). The program is applicable for both open‐shell and closed‐shell systems and for an arbitrary number of interacting subsystems. With the new program the CHA method is faster than the usual “counterpoise correction” scheme for single point calculations, especially for clusters consisting of several molecules. The numerical results provided by these conceptually different schemes, however, have again found to be very close to each other. The CHA scheme is particularly good for providing truly BSSE‐free MP2 data for intermolecular potentials. © 2006 Wiley Periodicals, Inc. J Comput Chem 27: 1505–1516, 2006     

Light-activated reassembly of split green fluorescent protein

Truncated green fluorescent protein (GFP) with the 11th β-strand removed is potentially interesting for bioconjugation, imaging, and the preparation of semisynthetic proteins with novel spectroscopic or functional properties. Surprisingly, the truncated GFP generated by removing the 11th strand, once refolded, does not reassemble with a synthetic peptide corresponding to strand 11 but does reassemble following light activation. The mechanism of this process has been studied in detail by absorption, fluorescence, and Raman spectroscopy. The chromophore in this refolded truncated GFP is found to be in the trans configuration. Upon exposure to light a photostationary state is formed between the trans and cis conformations of the chromophore, and only truncated GFP with the cis configuration of the chromophore binds the peptide. A kinetic model describing the light-activated reassembly of this split GFP is discussed. This unique light-driven reassembly is potentially useful for controlling protein-protein interactions.     

Manipulation of Single‐Stranded DNA by Using an Artificial Site‐Selective DNA Cutter Composed of Cerium(IV)/EDTA and Phosphonate–Oligonucleotide Conjugates

An artificial site‐selective DNA cutter to hydrolyze single‐stranded DNA at a desired site was prepared from Ce^IV/ethylenediamintetraacetic acid (EDTA) and two ethylenediamine‐N,N,N′,N′‐tetrakis(methylenephosphonic acid)–oligonucleotide conjugates. By using this cutter, the sense strand of a blue fluorescent protein (BFP) gene was selectively cut at a predetermined site in the chromophore‐coding region. The upstream fragment obtained by the site‐selective scission was ligated with the downstream fragment of the closely related green fluorescent protein (GFP) gene so that the 5′‐ and 3′‐end portions of the chromophore came from the BFP fragment and the GFP fragment, respectively. The recombinant gene was successfully expressed in E. coli and the chimeric chromophore emitted green fluorescence as expected.     

Effects of water re-location and cavity trimming on the CASPT2//CASSCF/AMBER excitation energy of Rhodopsin

We have used quantum-mechanics/molecular-mechanics computations based on ab initio multiconfigurational perturbation theory to determine and rationalize the effect of the re-location of one crystallographic water molecule on the vertical excitation energy of the visual pigment rhodopsin. It is found that the re-location of one water molecule to the opposite side of the 11-cis retinal chromophore leads to a large 0.7–0.8 Å contraction in the chromophore—counterion salt-bridge distance. In spite of this structural effect, the change in excitation energy is found to be limited (< 1.5 kcal mol^?1). Through an analysis of different rhodopsin models in terms of “components” (isolated chromophore, isolated chromophore—counterion ion-pair and models deprived of the counterion charges) we show that the limited change of the excitation energy can be related to a displacement of the retinal chromophore to a different spot of the protein cavity.     

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.     

“Atomic Bremsstrahlung”: Retrospectives,current status and perspectives

We describe here the “Atomic bremsstrahlung” (AB)—emission of continuous spectrum electromagnetic radiation, which is generated in collisions of particles that have internal deformable structure that includes positively and negatively charged constituents. The deformation of one or both colliding partners induces multiple, mainly dipole, time-dependent electrical moments that become a source of radiation. The history of AB invention is presented and its unusual in comparison to ordinary bremsstrahlung (OB) properties, are discussed. As examples, fast electron atom, non-relativistic and relativistic collisions are considered. Attention is given to ion–atom and atom–atom collisions. Specifics of “elastic” and “inelastic” (i.e. radiation accompanied by destruction of collision partners) AB will be mentioned. Attention will be given to possible manifestation of AB in nature and in some exotic systems, for instance scattering of electrons upon muonic hydrogen. Some cooperative effects connected to AB will be considered. New classical schemes similar to AB will be presented.     

Primary photoprocesses in oxyblepharismin interacting with its native protein partner

The primary photoprocesses in the photoreceptor for the step-up photophobic response of the light-adapted cells of Blepharisma japonicum (OBIP, OxyBlepharismin bInding Protein) have been studied by ultrafast UV–vis transient spectroscopy. The results are rationalized in terms of heterogeneity of the OBIP sample. Two independent classes of chromoprotein are proposed: a “reactive” species, which presents a specific 680-nm band decaying in 4 and 56 ps and a “non-reactive” one, which behaves like the free chromophore (OxyBP) in solution. A bimolecular photooxidation of OxyBP in the presence of 1,4-benzoquinone was performed to record the absorption spectrum of the OxyBP radical cation. Comparison with reactive OBIP suggests that an electron transfer could be involved in the primary photoprocesses of OBIP and possibly trigger the sensory transduction chain of B. japonicum. In addition, the specificity of the chromophore–protein interaction was investigated by studying the artificial complex that OxyBP forms with human serum albumin (HSA). OxyBP–HSA happens to be spectroscopically much closer to free OxyBP than to OBIP. This highlights the specific nature of the interaction between OxyBP and its native protein partner and further supports the proposal that OBIP is the actual photoreceptor for the photophobic response of B. japonicum.     

Controlling Radical Formation in the Photoactive Yellow Protein Chromophore

To understand how photoactive proteins function, it is necessary to understand the photoresponse of the chromophore. Photoactive yellow protein (PYP) is a prototypical signaling protein. Blue light triggers trans–cis isomerization of the chromophore covalently bound within PYP as the first step in a photocycle that results in the host bacterium moving away from potentially harmful light. At higher energies, photoabsorption has the potential to create radicals and free electrons; however, this process is largely unexplored. Here, we use photoelectron spectroscopy and quantum chemistry calculations to show that the molecular structure and conformation of the isolated PYP chromophore can be exploited to control the competition between trans–cis isomerization and radical formation. We also find evidence to suggest that one of the roles of the protein is to impede radical formation in PYP by preventing torsional motion in the electronic ground state of the chromophore.     

Benzylidene-oxazolones as molecular photoswitches

The synthesis and photochemical study of a family of molecular switches inspired by the green fluorescent protein (GFP) chromophore is presented. These compounds can be easily synthesized, and their photophysical properties may be tuned. Due to their efficient photoisomerization and high stability, these compounds can be switched on/off by using light and heat or light with different wavelengths.     

Enabling visible-light water photooxidation by coordinative incorporation of Co(II/III) cocatalytic sites into organic-inorganic hybrids: quantum chemical modeling and photoelectrochemical performance

Coordinative incorporation of Co(II/III) cocatalytic sites into organic–inorganic hybrids of TiO₂ and “polyheptazine” (PH, poly(aminoimino)heptazine, melon, or “graphitic carbon nitride”) has been investigated both by quantum chemical calculations and experimental techniques. Specifically, density-functional theory (DFT) calculations (PBE/def2-TZVPP) suggest that Co(II/III) and Zn(II) ions adsorb in nanocavities at the surface of the hybrid PH–TiO₂ cluster, a prediction which can be further confirmed experimentally by ¹⁵N nuclear magnetic resonance in the case of the Zn complex. The absorption spectra of the complexes were characterized by time-dependent DFT calculations, suggesting a change of color upon Co ion binding which can in fact be observed with the naked eye. Hybrid TiO₂–PH photoelectrodes were impregnated with Co(II) ions from aqueous cobalt nitrate solutions. Optical absorption data suggest that Co(II) ions are predominantly present as single ions coordinated within the nitrogen cavities of TiO₂–PH, and any undesired blocking of light absorption is negligible. The cobalt-induced cocatalytic sites can efficiently couple to the holes photogenerated by visible light in TiO₂–PH, leading to complete oxidation of water to dioxygen. Our results indicate that coordinative incorporation of metal ions into well-designed surface sites in the light absorber is sufficient to drive complex multielectron transformations in artificial photosynthetic systems.     

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.     

A comparison of the fluorescence dynamics of single molecules of a green fluorescent protein: one- versus two-photon excitation.

We report on the dynamics of fluorescence from individual molecules of a mutant of the wild-type green fluorescent protein (GFP) from Aequorea victoria, super folder GFP (SFGFP). SFGFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. It shows increased thermal stability and is able to retain its fluorescence when fused to poorly folding proteins. We use a recently developed single-molecule technique which combines fluorescence-fluctuation spectroscopy and time-correlated single photon counting in order to characterize the photophysical properties of SFGFP under one- (OPE) and two- (TPE) photon excitation conditions. We use Rhodamine 110 as a model chromophore to validate the methodology and to explain the single-molecule results of SFGFP. Under OPE, single SFGFP molecules undergo fluorescence flickering on the time scale of micros and tens of micros due to triplet formation and ground-state protonation-deprotonation, respectively, as demonstrated by excitation intensity- and pH-dependent experiments. OPE single-molecule fluorescence lifetimes indicate heterogeneity in the population of SFGFP, indicating the presence of the deprotonated I and B forms of the SFGFP chromophore. TPE of single SFGFP molecules results in the photoconversion of the chromophore. TPE of single SFGFP molecules show fluorescence flickering on the time scale of micros due to triplet formation. A flicker connected with protonation-deprotonation of the SFGFP chromophore is detected only at low pH. Our results show that SFGFP is a promising fusion reporter for intracellular applications using OPE and TPE microscopy.