Trans Chromophore Research Articles

Power Density Titration of Reversible Photoisomerization of a Fluorescent Protein Chromophore in the Presence of Thermally Driven Barrier Crossing Shown by Quantitative Millisecond Serial Synchrotron X-ray Crystallography.

We present millisecond quantitative serial X-ray crystallography at 1.7 Å resolution demonstrating precise optical control of reversible population transfer from Trans-Cis and Cis-Trans photoisomerization of a reversibly switchable fluorescent protein, rsKiiro. Quantitative results from the analysis of electron density differences, extrapolated structure factors, and occupancy refinements are shown to correspond to optical measurements of photoinduced population transfer and have sensitivity to a few percent in concentration differences. Millisecond time-resolved concentration differences are precisely and reversibly controlled through intense continuous wave laser illuminations at 405 and 473 nm for the Trans-to-Cis and Cis-to-Trans reactions, respectively, while the X-ray crystallographic measurement and laser illumination of the metastable Trans chromophore conformation causes partial thermally driven reconversion across a 91.5 kJ/mol thermal barrier from which a temperature jump between 112 and 128 K is extracted.

Improvement of the Green-Red Förster Resonance Energy Transfer-Based Ca2+ Indicator by Using the Green Fluorescent Protein, Gamillus, with a Trans Chromophore as the Donor.

To monitor the Ca2+ dynamics in cells, various genetically encoded Ca2+ indicators (GECIs) based on Förster resonance energy transfer (FRET) between fluorescent proteins are widely used for live imaging. Conventionally, cyan and yellow fluorescent proteins have been often used as FRET pairs. Meanwhile, bathochromically shifted indicators with green and red fluorescent protein pairs have various advantages, such as low toxicity and autofluorescence in cells. However, it remains difficult to develop them with a similar level of dynamic range as cyan and yellow fluorescent protein pairs. To improve this, we used Gamillus, which has a unique trans-configuration chromophore, as a green fluorescent protein. Based on one of the best high-dynamic-range GECIs, Twitch-NR, we developed a GECI with 1.5-times higher dynamic range (253%), Twitch-GmRR, using RRvT as a red fluorescent protein. Twitch-GmRR had high brightness and photostability and was successfully applied for imaging the Ca2+ dynamics in live cells. Our results suggest that Gamillus with trans-type chromophores contributes to improving the dynamic range of GECIs. Therefore, selection of the cis-trans isomer of the chromophore may be a fundamental approach to improve the dynamic range of green-red FRET indicators, unlimited by GECIs.

Disentangling Chromophore States in a Reversibly Switchable Green Fluorescent Protein: Mechanistic Insights from NMR Spectroscopy.

The photophysical properties of fluorescent proteins, including phototransformable variants used in advanced microscopy applications, are influenced by the environmental conditions in which they are expressed and used. Rational design of improved fluorescent protein markers requires a better understanding of these environmental effects. We demonstrate here that solution NMR spectroscopy can detect subtle changes in the chemical structure, conformation, and dynamics of the photoactive chromophore moiety with atomic resolution, providing such mechanistic information. Studying rsFolder, a reversibly switchable green fluorescent protein, we have identified four distinct configurations of its p-HBI chromophore, corresponding to the cis and trans isomers, with each one either protonated (neutral) or deprotonated (anionic) at the benzylidene ring. The relative populations and interconversion kinetics of these chromophore species depend on sample pH and buffer composition that alter in a complex way the strength of H-bonds that contribute in stabilizing the chromophore within the protein scaffold. We show in particular the important role of histidine-149 in stabilizing the neutral trans chromophore at intermediate pH values, leading to ground-state cis-trans isomerization with a peculiar pH dependence. We discuss the potential implications of our findings on the pH dependence of the photoswitching contrast, a critical parameter in nanoscopy applications.

Infrared spectroscopy reveals multi-step multi-timescale photoactivation in the photoconvertible protein archetype dronpa.

Photochromic fluorescent proteins play key roles in super-resolution microscopy and optogenetics. The light-driven structural changes that modulate the fluorescence involve both trans-to-cis isomerization and proton transfer. The mechanism, timescale and relative contribution of chromophore and protein dynamics are currently not well understood. Here, the mechanism of off-to-on-state switching in dronpa is studied using femtosecond-to-millisecond time-resolved infrared spectroscopy and isotope labelling. Chromophore and protein dynamics are shown to occur on multiple timescales, from picoseconds to hundreds of microseconds. Following excitation of the trans chromophore, a ground-state primary product is formed within picoseconds. Surprisingly, the characteristic vibrational spectrum of the neutral cis isomer appears only after several tens of nanoseconds. Further fluctuations in protein structure around the neutral cis chromophore are required to form a new intermediate, which promotes the final proton-transfer reaction. These data illustrate the interplay between chromophore dynamics and the protein environment underlying fluorescent protein photochromism.

Role of Gln222 in Photoswitching of Aequorea Fluorescent Proteins: A Twisting and H-Bonding Affair?

Reversibly photoswitchable fluorescent proteins (RSFPs) admirably combine the genetic encoding of fluorescence with the ability to repeatedly toggle between a bright and dark state, adding a new temporal dimension to the fluorescence signal. Accordingly, in recent years RSFPs have paved the way to novel applications in cell imaging that rely on their reversible photoswitching, including many super-resolution techniques such as F-PALM, RESOLFT, and SOFI that provide nanoscale pictures of the living matter. Yet many RSFPs have been engineered by a rational approach only to a limited extent, in the absence of clear structure-property relationships that in most cases make anecdotic the emergence of the photoswitching. We reported [ Bizzarri et al. J. Am Chem Soc. 2010 , 102 , 85 ] how the E222Q replacement is a single photoswitching mutation, since it restores the intrinsic cis-trans photoisomerization properties of the chromophore in otherwise nonswitchable Aequorea proteins of different color and mutation pattern (Q-RSFPs). We here investigate the subtle role of Q222 on the excited-state photophysics of the two simplest Q-RSFPs by a combined experimental and theoretical approach, using their nonswitchable anacestor EGFP as benchmark. Our findings link indissolubly photoswitching and Q222 presence, by a simple yet elegant scenario: largely twisted chromophore structures around the double bond (including hula-twist configurations) are uniquely stabilized by Q222 via H-bonds. Likely, these H-bonds subtly modulate the electronic properties of the chromophore, enabling the conical intersection that connects the excited cis to ground trans chromophore. Thus, Q222 belongs to a restricted family of single mutations that change dramatically the functional phenotype of a protein. The capability to distinguish quantitatively T65S/E222Q EGFP ("WildQ", wQ) from the spectrally identical EGFP by quantitative Optical Lock-In Detection (qOLID) witnesses the relevance of this mutation for cell imaging.

SORCI for photochemical and thermal reaction paths: A benchmark study

Dynamic electron correlation is very important for the correct description of potential energy surfaces, especially surface with differential dynamic electron correlation. Moreover, an accurate description of both the ground and excited state potential energy surfaces is essential to understand thermal and photochemical reactions of molecular systems. In the present work we test the performance of the Spectroscopy ORiented Configuration Interaction (SORCI) method that was originally designed to account for the dynamic electron correlation in an efficient manner at fixed geometry. Here, we assess the performance of SORCI along several paths on the ground and excited state potential energy surfaces that are related to the thermal and photochemical isomerization of a reduced retinal chromophore model. The ground state mapping involves three paths. Two of them are minimum energy paths that lead through different transitions states from one cis/trans stereoisomer to the other. One transition state has a diradical (open-shell) character while the other one has a charge-transfer character. The third ground state path connects these two transition states via a conical intersection. In addition, we assess the performance of SORCI for three excited state paths that comprise a minimum energy path starting from each of the cis and from the trans chromophore. These two pathways arrive at different conical intersections that are part of one seam of conical intersections. The third excited state path presents a profile along this seam.Despite the high complexity and varying mixture of different electronic structures involved in this benchmark, SORCI produces energy differences and energy profiles in good agreement with the multireference configuration interaction plus quadruples correction (MRCISD+Q) reference along all six paths. We find that SORCI is capable of producing smooth energy profiles, which require tighter thresholds than the present default. A further tightening of these thresholds by one order of magnitude yields converged SORCI values as compared to the limit of the method obtained by setting the three cutoffs to zero. However, we note also some deficiencies relative to MRCISD+Q in the description of relative energy differences. When comparing energies of structures that have very different geometries on the same potential energy surface we find that SORCI underestimates the reference values. Here SORCI underestimates the corresponding MRCISD+Q reference values thus indicating a non-negligible effect of the inactive double excitations. In addition we observe some artifacts at conical intersections due to the Davidson correction for higher excitations. Ways to overcome these drawbacks are discussed.

Primary Events of Photodynamics in Reversible Photoswitching Fluorescent Protein Dronpa

Reversible photoswitching fluorescent protein Dronpa can reversibly switch between fluorescent on-state and nonfluorescent off-state by two radiations. The primary events of photodynamics in Dronpa were elucidated by nonadiabatic ONIOM (CASSCF:AMBER) molecular dynamics simulations. All radiationless decay processes are found mainly to result from one bond flip of a bridge C−C bond of the chromophore in the protein, regardless of its protonation state or conformation, rather than hula twisting. In the off-state protein, trans−cis photoisomerization of the neutral trans chromophore takes place via a rotation around the imidazolinone ring. In the wild-type on-state protein, the anionic cis chromophore mostly remains planar for at least 20 ps. In contrast, in the H193T mutant on-state, faster decay via a rotation of the phenoxy ring or imidazolinone ring of the anionic cis chromophore was found, suggesting that flexibility of the chromophore and its immediate protein environment is the key to radiationless decays.

Photophysics of the Red Chromophore of HcRed: Evidence for Cis−Trans Isomerization and Protonation-State Changes

HcRed is a dimeric intrinsically fluorescent protein with origins in the sea anemone Heteractis crispa. This protein exhibits deep red absorption and emission properties. Using a combination of ensemble and single molecule methods and by varying environmental parameters such as temperature and pH, we found spectroscopic evidence for the presence of two ground state conformers, trans and cis chromophores that are in thermal equilibrium and that follow different excited-state pathways upon exposure to light. The photocycle of HcRed appears to be a combination of both kindling proteins and bright emitting GFP/GFP-like proteins: the trans chromophore undergoes light driven isomerization followed by radiative relaxation with a fluorescence lifetime of 0.5 ns. The cis chromophore exhibits a photocycle similar to bright GFPs and GFP-like proteins such as enhanced GFP, enhanced YFP or DsRed, with radiative relaxation with a fluorescence lifetime of 1.5 ns, singlet-triplet deactivation on a microsecond time scale and solvent controlled protonation/deprotonation in tens of microseconds. Using single molecule spectroscopy, we identify trans and cis conformers at the level of individual moieties and show that it is possible that the two conformers can coexist in a single protein due to the dimeric nature of HcRed.

Spectral Tuning of Deep Red Cone Pigments

Visual pigments are G-protein-coupled receptors that provide a critical interface between organisms and their external environment. Natural selection has generated vertebrate pigments that absorb light from the far-UV (360 nm) to the deep red (630 nm) while using a single chromophore, in either the A1 (11- cis-retinal) or A2 (11- cis-3,4-dehydroretinal) form. The fact that a single chromophore can be manipulated to have an absorption maximum across such an extended spectral region is remarkable. The mechanisms of wavelength regulation remain to be fully revealed, and one of the least well-understood mechanisms is that associated with the deep red pigments. We investigate theoretically the hypothesis that deep red cone pigments select a 6- s- trans conformation of the retinal chromophore ring geometry. This conformation is in contrast to the 6- s- cis ring geometry observed in rhodopsin and, through model chromophore studies, the vast majority of visual pigments. Nomographic spectral analysis of 294 A1 and A2 cone pigment literature absorption maxima indicates that the selection of a 6- s- trans geometry red shifts M/LWS A1 pigments by approximately 1500 cm (-1) ( approximately 50 nm) and A2 pigments by approximately 2700 cm (-1) ( approximately 100 nm). The homology models of seven cone pigments indicate that the deep red cone pigments select 6- s- trans chromophore conformations primarily via electrostatic steering. Our results reveal that the generation of a 6- s- trans conformation not only achieves a significant red shift but also provides enhanced stability of the chromophore within the deep red cone pigment binding sites.

Structural Characterization of a Blue Chromoprotein and Its Yellow Mutant from the Sea Anemone Cnidopus Japonicus

Green fluorescent protein (GFP) and its relatives (GFP protein family) have been isolated from marine organisms such as jellyfish and corals that belong to the phylum Cnidaria (stinging aquatic invertebrates). They are intrinsically fluorescent proteins. In search of new members of the family of green fluorescent protein family, we identified a non-fluorescent chromoprotein from the Cnidopus japonicus species of sea anemone that possesses 45% sequence identity to dsRed (a red fluorescent protein). This newly identified blue color protein has an absorbance maximum of 610 nm and is hereafter referred to as cjBlue. Determination of the cjBlue 1.8 A crystal structure revealed a chromophore comprised of Gln(63)-Tyr(64)-Gly(65). The ring stacking between Tyr(64) and His(197) stabilized the cjBlue trans chromophore conformation along the Calpha2-Cbeta2 bond of 5-[(4-hydroxyphenyl)methylene]-imidazolinone, which closely resembled that of the "Kindling Fluorescent Protein" and Rtms5. Replacement of Tyr(64) with Leu in wild-type cjBlue produced a visible color change from blue to yellow with a new absorbance maximum of 417 nm. Interestingly, the crystal structure of the yellow mutant Y64L revealed two His(197) imidazole ring orientations, suggesting a flip-flop interconversion between the two conformations in solution. We conclude that the dynamics and structure of the chromophore are both essential for the optical appearance of these color proteins.

Photochromicity of Anabaena Sensory Rhodopsin, an Atypical Microbial Receptor with a cis-Retinal Light-adapted Form

We characterize changes in isomeric states of the retinylidene chromophore during light-dark adaptation and photochemical reactions of Anabaena (Nostoc) sp. PCC7120 sensory rhodopsin (ASR). The results show that ASR represents a new type of microbial rhodopsin with a number of unusual characteristics. The three most striking are: (i) a primarily all-trans configuration of retinal in the dark-adapted state and (ii) a primarily 13-cis light-adapted state with a blue-shifted and lower extinction absorption spectrum, opposite of the case of bacteriorhodopsin; and (iii) efficient reversible light-induced interconversion between the 13-cis and all-trans unphotolyzed states of the pigment. The relative amount of ASR with cis and trans chromophore forms depends on the wavelength of illumination, providing a mechanism for single-pigment color sensing analogous to that of phytochrome pigments. In addition ASR exhibits unusually slow formation of L-like and M-like intermediates, with a dominant accumulation of M during the photocycle. Co-expression of ASR with its putative cytoplasmic transducer protein shifts the absorption maximum and strongly decreases the rate of dark adaptation of ASR, confirming interaction between the two proteins. Thus ASR, the first non-haloarchaeal sensory rhodopsin characterized, demonstrates the diversity of photochemistry of microbial rhodopsins. Its photochromic properties and the position of its two ground state absorption maxima suggest it as a candidate for controlling differential photosynthetic light-harvesting pigment synthesis (chromatic adaptation) or other color-sensitive physiological responses in Anabaena cells.

Sensitive Circular Dichroism Marker for the Chromophore Environment of Photoactive Yellow Protein: Assignment of the 307 and 318 nm Bands to then→ π* Transition of the Carbonyl

The absorption and CD spectra of wild-type PYP, apo-PYP, and the mutants, E46Q and M100A, were measured between 250 and 550 nm. At neutral pH, the two very weak absorption bands of wild-type PYP at 307 and 318 nm (epsilon(max) = 600 +/- 100 M(-1) cm(-1) at 318 nm) are associated with quite strong positive CD bands (Deltaepsilon(max) approximately 6.8 M(-1) cm(-1)). Both sets of bands are absent in the apoprotein. On the basis of this evidence, we assign these optical signals to the n --> pi* transition of the oxygen of the carbonyl group of the 4-hydroxycinnamic acid chromophore, which is expected to be electric dipole forbidden but magnetic dipole allowed. The progression of narrow bands at 307 and 318 nm with a shoulder in the CD around 329 nm is due to vibrational fine structure with a frequency of about 1050 +/- 50 cm(-1). This is the carbonyl stretch frequency in the electronically excited state and is well-known from the vibrational structure in the CD spectra of carbonyl compounds. The positive sign of the CD in the near UV is in accordance with the octant rule and the high-resolution X-ray structure, if we assume that the NH group of cysteine 69 to which the carbonyl is hydrogen bonded is the principle perturbant. Similar absorption and CD spectra were observed in the range of 300-340 nm for the mutants E46Q and M100A at neutral pH. Protonation of the trans chromophore by lowering the pH in the dark (without photoisomerization) broadens the 307 and 318 nm CD bands in the mutant E46Q but does not significantly affect their positions or alter their sign. For the long-lived I(2) photointermediate of the mutant M100A with protonated cis chromophore, we observed that the sign of the rotational strength in the 310-320 nm range is negative (i.e., opposite to that in the dark state with trans chromophore). This suggests that the light-induced isomerization of the chromophore, which leads to breaking of the hydrogen bond with the backbone amide of C69, brings the carbonyl into a new protein environment with different asymmetry than in the unbleached protein. The observed change in sign is mainly due to this effect, but a change in chromophore twist may also contribute. Thus, the 318 nm CD signal is a sensitive marker for the environment of the chromophore carbonyl, which samples various environments and configurations during the photocycle.

Anabaena sensory rhodopsin: a photochromic color sensor at 2.0 A.

Microbial sensory rhodopsins are a family of membrane-embedded photoreceptors in prokaryotic and eukaryotic organisms. Structures of archaeal rhodopsins, which function as light-driven ion pumps or photosensors, have been reported. We present the structure of a eubacterial rhodopsin, which differs from those of previously characterized archaeal rhodopsins in its chromophore and cytoplasmic-side portions. Anabaena sensory rhodopsin exhibits light-induced interconversion between stable 13-cis and all-trans states of the retinylidene protein. The ratio of its cis and trans chromophore forms depends on the wavelength of illumination, thus providing a mechanism for a single protein to signal the color of light, for example, to regulate color-sensitive processes such as chromatic adaptation in photosynthesis. Its cytoplasmic half channel, highly hydrophobic in the archaeal rhodopsins, contains numerous hydrophilic residues networked by water molecules, providing a connection from the photoactive site to the cytoplasmic surface believed to interact with the receptor's soluble 14-kilodalton transducer.

Photoexcitation of the O-Intermediate in Bacteriorhodopsin Mutant L93A

During the extended lifetime of the O-state in bacteriorhodopsin (bR) mutant L93A, two substates have been distinguished. The first O-intermediate (OI) is in rapid equilibrium with N and apparently still has a 13- cis chromophore. OI undergoes a photoreaction with a small absorbance change, positive charge transport in the pumping direction, and proton release and uptake. None of these effects was detected after photoexcitation of the late O (OII). The most likely interpretation of the effects seen is an accelerated return of the molecule from the OI- to the bR-state. However, with a lifetime ≈140 ms, the reaction cannot account for the observed high pumping efficiency of the mutant under continuous illumination. We suggest that OII corresponds to the O-intermediate with a twisted all- trans chromophore seen in the photocycle of wild-type bR, where the 13- cis OI-intermediate under the usual conditions does not accumulate in easily detectable amounts and, therefore, has generally been overlooked. Both the OI- and OII-decays are apparently strongly inhibited in the mutant.

Mimic of Photocycle by a Protein Folding Reaction in Photoactive Yellow Protein

The blue light receptor photoactive yellow protein (PYP) displays rhodopsin-like photochemistry based on the trans to cis photoisomerization of its p-coumaric acid chromophore. Here, we report that protein refolding from the acid-denatured state of PYP mimics the last photocycle transition in PYP. This implies a direct link between transient protein unfolding and photosensory signal transduction. We utilize this link to study general issues in protein folding. Chromophore trans to cis photoisomerization in the acid-denatured state strongly decelerates refolding, and converts the pH dependence of the barrier for refolding from linear to nonlinear. We propose transition state movement to explain this phenomenon. The cis chromophore significantly stabilizes the acid-denatured state, but acidification of PYP results in the accumulation of the acid-denatured state containing a trans chromophore. This provides a clear example of kinetic control in a protein unfolding reaction. These results demonstrate the power of PYP as a light-triggered model system to study protein folding.

Static and Time-Resolved Step-Scan Fourier Transform Infrared Investigations of the Photoreaction of Halorhodopsin from Natronobacterium Pharaonis: Consequences for Models of the Anion Translocation Mechanism

The molecular changes during the photoreaction of halorhodopsin from Natronobacterium pharaonis have been monitored by low-temperature static and by time-resolved step-scan Fourier transform infrared difference spectroscopy. In the low-temperature L spectrum anions only influence a band around 1650 cm −1, tentatively assigned to the C N stretch of the protonated Schiff base of L. The analysis of the time-resolved spectra allows to identify the four states: K, L 1, L 2, and O. Between L 1 and L 2, only the apoprotein undergoes alterations. The O state is characterized by an all- trans chromophore and by rather large amide I spectral changes. Because in our analysis the intermediate containing O is in equilibrium with a state indistinguishable from L 2, we are unable to identify an N-like state. At very high chloride concentrations (>5 M), we observe a branching of the photocycle from L 2 directly back to the dark state, and we provide evidence for direct back-isomerization from L 2. This branching leads to the reported reduction of transport activity at such high chloride concentrations. We interpret the L 1 to L 2 transition as an accessibility change of the anion from the extracellular to the cytosolic side, and the large amide I bands in O as an indication for opening of the cytosolic channel from the Schiff base toward the cytosolic surface and/or as indication for changes of the binding constant of the release site.

Absorption and circular dichroism spectra of the c i s- and t r a n s-butadiene chromophores α- and β-phellandrene

The absorption and circular dichroism spectra of gas-phase (−) -α-phellandrene and (−) -β-phellandrene have been measured in the spectral region 300–135 nm. Solution spectra of these compounds were measured in perfluoro-n-hexane to 160 nm. The spectra were interpreted in terms of the excited electronic states of the cis- and trans-butadiene chromophores. The overlapping valence and Rydberg states were unambiguously differentiated by comparison of the vapor and solution phase spectra. Comparison between absorption and CD spectra made possible the symmetry assignments of magnetic dipole allowed (as opposed to electric dipole allowed) excited states of butadiene. For both molecular systems the first observed excited singlet state corresponds to 1Bu for the trans chromophore and to 1B2 for the cis chromophore. No evidence was found for a ’’lowest-lying’’ singlet state of Ag or A1 symmetry.