Photoactive Yellow Protein Chromophore Research Articles

Controlling electron emission from the photoactive yellow protein chromophore by substitution at the coumaric acid group.

Understanding how the interactions between a chromophore and its surrounding protein control the function of a photoactive protein remains a challenge. Here, we present the results of photoelectron spectroscopy measurements and quantum chemistry calculations aimed at investigating how substitution at the coumaryl tail of the photoactive yellow protein chromophore controls competing relaxation pathways following photoexcitation of isolated chromophores in the gas phase with ultraviolet light in the range 350-315 nm. The photoelectron spectra are dominated by electrons resulting from direct detachment and fast detachment from the 2(1)ππ* state but also have a low electron kinetic energy component arising from autodetachment from lower lying electronically excited states or thermionic emission from the electronic ground state. We find that substituting the hydrogen atom of the carboxylic acid group with a methyl group lowers the threshold for electron detachment but has very little effect on the competition between the different relaxation pathways, whereas substituting with a thioester group raises the threshold for electron detachment and appears to 'turn off' the competing electron emission processes from lower lying electronically excited states. This has potential implications in terms of tuning the light-induced electron donor properties of photoactive yellow protein.

Solvent effects on de-excitation channels in the p-coumaric acid methyl ester anion, an analogue of the photoactive yellow protein (PYP) chromophore.

In an attempt to shed light on the environmental effects on the deactivation channels of the PYP chromophore, radiative and non-radiative deactivation mechanisms of the anionic p-coumaric acid methyl ester (pCE-) in the gas phase and water solution are compared at the CASPT2//CASSCF/cc-pVDZ level and, when necessary, at the CASPT2//CASPT2/cc-pVDZ level. We find that the solvent produces dramatic modifications on the free energy profile of the S1 state. Two twisted structures that are minima in the gas phase could not be localized in aqueous solution. Furthermore, the relative stability of minima and conical intersections (CIs) is reverted with respect to the gas phase values, affecting the prevalent de-excitation paths. As a consequence of these changes, three competitive de-excitation channels are open in aqueous solution: the fluorescence emission from a planar minimum on S1, the trans-cis photoisomerization through a CI that involves the rotation of the vinyl double bond and the non-radiative, non-reactive, de-excitation through the CI associated with the rotation of the single bond adjacent to the phenyl group. In the gas phase, the minima are the structures with lower energy, while in solution the CIβ structure, characterized by a large charge separation, is strongly stabilized by interactions with water molecules and becomes the structure with the lowest energy on S1. These facts explain the low fluorescence signal of pCE- in aqueous solution and the presence of partial trans-cis photoisomerization in this system.

In situ SERS monitored photoactive yellow protein (PYP) chromophore model elimination, nano-catalyzed phenyl redox and I2 addition reactions

PhS nitration products absorbed on Ag/Au NPs underwent nitro- and amino-group redox cycle reactions monitored using SERS spectra. I2 addition to the CC double bond was also confirmed with SERS.

A PYP chromophore acts as a 'photoacid' in an isolated hydrogen bonded complex.

The light-induced response of a neutral Photoactive Yellow Protein chromophore in a hydrogen-bonded complex with a proton acceptor has been studied by dual-detection action absorption spectroscopy and density functional theory. We show that the chromophore is a 'photoacid' and that ultrafast excited-state proton transfer might be operative in an isolated complex.

Photo Control of Protein Function Using Photoactive Yellow Protein.

Photoswitchable proteins are becoming increasingly common tools for manipulating cellular processes with high spatial and temporal precision. Photoactive yellow protein (PYP) is a small, water-soluble protein that undergoes a blue light induced change in conformation. It can serve as a scaffold for designing new tools to manipulate biological processes, but with respect to other protein scaffolds it presents some technical challenges. Here, we present practical information on how to overcome these, including how to synthesize the PYP chromophore, how to express and purify PYP, and how to screen for desired activity.

Influence of intermolecular hydrogen bonding and solvent effects on the excited-state properties of a photoactive yellow protein chromophore compound

The time-dependent density functional theory (TDDFT) method has been carried out to investigate the hydrogen bonding dynamics of a series of hydrogen-bonded complexes of PYP chromophore formed with water molecules both in vacuum and aqueous solution. We demonstrate that the intermolecular hydrogen bonds formed between carbonyl oxygen and hydrogen of water are strengthened in the lowest bright state as well as one other charge transfer state and result in spectral redshift. Whereas, a totally opposite result happens to the site of phenolate oxygen, which can be confirmed based on the excited-state geometric optimizations by TDDFT method. In addition, the frontier molecular orbital analysis reveals the nature of the lowest bright state and the CT state as well as the understanding of the electronic excited-state hydrogen bonding dynamics. Moreover, two approaches are adopted to consider the solvation effect.

Substituent and solvent effects on the UV-vis absorption spectrum of the photoactive yellow protein chromophore.

Solvent effects on the UV-vis absorption spectra and molecular properties of four models of the photoactive yellow protein (PYP) chromophore have been studied with ASEP/MD, a sequential quantum mechanics/molecular mechanics method. The anionic trans-p-coumaric acid (pCA(-)), thioacid (pCTA(-)), methyl ester (pCMe(-)), and methyl thioester (pCTMe(-)) derivatives have been studied in gas phase and in water solution. We analyze the modifications introduced by the substitution of sulfur by oxygen atoms and hydrogen by methyl in the coumaryl tail. We have found some differences in the absorption spectra of oxy and thio derivatives that could shed light on the different photoisomerization paths followed by these compounds. In solution, the spectrum substantially changes with respect to that obtained in the gas phase. The n → π1* state is destabilized by a polar solvent like water, and it becomes the third excited state in solution displaying an important blue shift. Now, the π → π1* and π → π2* states mix, and we find contributions from both transitions in S1 and S2. The presence of the sulfur atom modulates the solvent effect and the first two excited states become practically degenerate for pCA(-) and pCMe(-) but moderately well-separated for pCTA(-) and pCTMe(-).

Controlling Radical Formation in the Photoactive Yellow Protein Chromophore

AbstractTo 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.

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.

Unveiling the mechanism of photoinduced isomerization of the photoactive yellow protein (PYP) chromophore.

A detailed theoretical analysis, based on extensive ab initio second-order approximate coupled cluster calculations, has been performed for the S1 potential energy surface (PES) of four photoactive yellow protein (PYP) chromophore derivatives that are hydrogen bonded with two water molecules and differ merely in the carbonyl substituent. The main focus is put on contrasting the isomerization properties of these four species in the S1 excited state, related to torsion around the chromophore's single and double carbon-carbon bonds. The analysis provides evidence of the different isomerization behavior of these four chromophore complexes, which relates to the difference in their carbonyl substituents. While a stable double-bond torsion pathway exists on the S1 PES of the chromophores bearing the -O-CH3 and -NH2 substituents, this is not the case for the -S-CH3 and -CH3 substituted species. The presence of the -S-CH3 group leads to a strong instability of the chromophore with respect to the single-bond twist, whereas in the case of the -CH3 substituent a crossing of the S1 and S2 PESs occurs, which perturbs the pathway. Based on this analysis, the key factors that support the double-bond torsion have been identified. These are (i) the hydrogen bonds at the phenolic oxygen of the chromophore, (ii) the weak electron-acceptor character of the carbonyl group, and (iii) the ethylene-like pattern of the torsion in the beginning of the process. Our results suggest that the interplay between these factors determines the chromophore's isomerization in the solvent environment and in the native PYP environment.

Molecular Beam and ab Initio Studies of Photoactive Yellow Protein Chromophores: Influence of the Thioester Functionality and Single Bond Rotation

We report on experimental high-resolution spectroscopic studies in combination with ab initio computational studies that investigate the excited-state dynamics of methyl-4-hydroxycinnamate thioester and (5-hydroxyindan-(1E)-ylidene)acetic acid, derivatives of the photoactive yellow protein (PYP) chromophore. These studies aim to elucidate (a) how the thioester moiety influences the photophysics and photochemistry of the p-coumaric acid chromophore and (b) to what extent rotation of the single bond adjacent to the phenyl ring is involved in the decay dynamics of the electronically excited states. The experimental studies show that sulfur substitution leads to broad, unstructured excitation spectra that contrast sharply with the well-resolved spectra of compounds with an oxygen-based ester. Furthermore, internal conversion to the lower-lying nπ* state is absent. The absence of this decay channel is rationalized by quantum-chemical calculations that reveal that in the nπ* state of the thio compounds the molecule exhibits a large out-of-plane "kink" at the sulfur atom. Franck-Condon simulations of the excitation spectra of the V(ππ*) state highlight the activity of various vibrational modes in the neutral chromophore and indicate that upon sulfur substitution internal conversion to the ground state occurs at a significantly higher rate. The similarities observed in the excitation spectra and decay dynamics of the locked and unlocked compounds suggest that in the present experiments single-bond torsion does not show up prominently. The conclusion that for the isolated molecule double-bond torsion is dominating the excited-state dynamics is tentatively confirmed by the quantum-chemical calculations.

Short hydrogen bonds and negative charge in photoactive yellow protein promote fast isomerization but not high quantum yield.

Biological signal transduction by photoactive yellow protein (PYP) in halophilic purple sulfur bacteria is initiated by trans-to-cis isomerization of the p-coumaric acid chromophore (pCa) of PYP. pCa is engaged in two short hydrogen bonds with protein residues E46 and Y42, and it is negatively charged at the phenolate oxygen. We investigated the role in the isomerization process of the E46 short hydrogen bond and that of the negative charge on the anionic phenolate moiety of the chromophore. We used wild-type PYP and the mutant E46A, in protonated and deprotonated states (referred to as pE46A and dpE46A, respectively), to reduce the number of hydrogen bond interactions between the pCa phenolate oxygen and the protein and to vary the negative charge density in the chromophore-binding pocket. Their effects on the yield and rate of chromophore isomerization were determined by ultrafast spectroscopy. Molecular dynamics simulations were used to relate these results to structural changes in the mutant protein. We found that deprotonated pCa in E46A has a slower isomerization rate as the main part of this reaction was associated with time constants of 1 and 6 ps, significantly slower than the 0.6 ps time constant in wild-type PYP. The quantum yield of isomerization in dpE46A was estimated to be 30 ± 2%, and that of pE46A was 32 ± 3%, very close to the value determined for wtPYP of 32 ± 2%. Relaxation of the isomerized product state I0 to I1 was faster in dpE46A. We conclude that the negative charge on pCa stabilized by the short hydrogen bonds with E46 and Y42 affects the rate of isomerization but not the quantum yield of isomerization. With this information, we propose a scheme for the potential energy surfaces involved in the isomerization and suggest protein motions near the pCa backbone as key events in successful isomerization.

Steering the excited state dynamics of a photoactive yellow protein chromophore analogue with external electric fields

The first excited state of the Photoactive Yellow Protein chromophore exhibits a strong charge transfer character and the dipole moments of the excited and ground states differ significantly. Furthermore, the excited state charge distribution changes during the isomerization of this chromophore. These observations suggest that external electric fields can be used to control photo-isomerization, providing a new concept for developing photochromic devices, such as e-paper or optical memory. To test this idea, we performed excited state dynamics simulations and static calculations of a PYP chromophore analogue (pCK−) in an external electric field. By adjusting direction and strength of the field, we were able to control both selectivity (i.e., which bond isomerizes) and efficiency (excited state lifetime) of the isomerization process. Simulations like these could potentially be useful in validating whether a given chromophore could be suitable for electrochromic applications.

On the use of reduced active space in CASSCF calculations

The complete active space self-consistent field (CASSCF) method is one of the most used approaches to explore theoretically non-adiabatic photochemical processes in medium-size molecular systems. It is also frequently utilized in direct dynamics studies because this method offers a reliable description of the electronic state mixing in the regions of surface crossings (conical intersections). In this context, however, the large computational demand required to perform such on-the-fly molecular dynamics simulations using CASSCF as the electronic structure method means that only relatively small active space can be handled (typically not more than 10 electrons distributed in 10 orbitals). In this article, we present some guidelines to reduce the active space size in CASSCF calculations in order to decrease the computational cost necessary to perform, for example, molecular dynamics simulations. Three important criteria are considered: (i) the degradation of the accuracy in the description of the potential energy surface following the reduction of the active space, (ii) the stability of the reduced active space across the global potential energy surface, and (iii) the cost of the CASSCF calculation using the reduced active space. Two illustrations are given. The first one is the benzene to benzvalene S1 photochemical pathway, the second one is the photoisomerization of thiomethyl para-coumaric acid, the chromophore in photoactive yellow protein.

Probing Anisotropic Structure Changes in Proteins with Picosecond Time-Resolved Small-Angle X-ray Scattering

We have exploited the principle of photoselection and the method of time-resolved small-angle X-ray scattering (SAXS) to investigate protein size and shape changes following photoactivation of photoactive yellow protein (PYP) in solution with ∼150 ps time resolution. This study partially overcomes the orientational average intrinsic to solution scattering methods and provides structural information at a higher level of detail. Photoactivation of the p-coumaric acid (pCA) chromophore in PYP produces a highly contorted, short-lived, red-shifted intermediate (pR0), and triggers prompt, protein compaction of approximately 0.3% along the direction defined by the electronic transition dipole moment of the chromophore. Contraction along this dimension is accompanied by expansion along the orthogonal directions, with the net protein volume change being approximately -0.25%. More than half the strain arising from formation of pR0 is relieved by the pR0 to pR1 structure transition (1.8 ± 0.2 ns), with the persistent strain presumably contributing to the driving force needed to generate the spectroscopically blue-shifted pB signaling state. The results reported here are consistent with the near-atomic resolution structural dynamics reported in a recent time-resolved Laue crystallography study of PYP crystals and suggest that the early time structural dynamics in the crystalline state carry over to proteins in solution.

Ultrafast Carbonyl Motion of the Photoactive Yellow Protein Chromophore Probed by Femtosecond Circular Dichroism

Motions of the trans-p-coumaric acid carbonyl group following the photoexcitation of the R52Q mutant of photoactive yellow protein (PYP) are investigated, for the first time, by ultrafast time-resolved circular dichroism (TRCD) spectroscopy. TRCD is monitored in the near-ultraviolet, over a time scale of 10 ps. Immediately after excitation, TRCD is found to exhibit a large negative peak, which decays within a few picoseconds. A quantitative analysis of the signals shows that, upon excitation, the carbonyl group undergoes a fast (≪0.8 ps) and unidirectional flipping motion in the excited state with an angle of ca. 17-53°. For the subset of proteins that do not enter the signaling photocycle, TRCD provides strong evidence that the carbonyl group moves back to its initial position, leading to the formation of a nonreactive ground-state intermediate of trans conformation. The initial ground state is then restored within ca. 3 ps. Comparative study of R52Q and wild-type PYP provides direct evidence that the absence of Arg52 has no effect on the conformational changes of the chromophore during those steps.

Photo-isomerization upshifts the pKa of the Photoactive Yellow Protein chromophore to contribute to photocycle propagation

a b s t r a c t The influence of chromophore structure on the protonation constant of the Photoactive Yellow Protein chromophore is explored with isolated para-coumaric acid (pCA) and thiomethyl-para-coumaric acid model chromophores in solution. pH titration coupled with visible absorption spectra of the trans and photogenerated cis conformer of isolated pCA demonstrates that the isomerization of the chromophore increases the pKa of the phenolate group by 0.6 units (to 10.1 ± 0.22). Formation of the pCA thioester reduces the pKa of the phenolic group by 0.3 units (from 9.5 ± 0.15 to 9.2 ± 0.16). Unfortunately, a macro- scopic cis-TMpCA population was not achieved via photoexcitation. Both trends were explained with electronic structure calculations including a Natural Bond Orbital analysis that resolves that the pKa upshift for the cis configuration is attributed to increased Columbic repulsion between the coumaryl tail and the phenolate moieties. This structurally induced pKa upshift after isomerization is argued to aid in the protonation of the chromophore within the PYP protein environment and the subsequent propagation of the photocycle response and in vivo photo-activity. © 2013 Elsevier B.V. All rights reserved.

Cover Picture: Visible-Light-Induced Photodimerization of a Photoactive Yellow Protein (PYP) Chromophore Model in a Single Crystal (Chem. Eur. J. 25/2013)

Cover Picture: Visible-Light-Induced Photodimerization of a Photoactive Yellow Protein (PYP) Chromophore Model in a Single Crystal (Chem. Eur. J. 25/2013)

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.

Raman Optical Activity Probing Structural Deformations of the 4-Hydroxycinnamyl Chromophore in Photoactive Yellow Protein.

Many biological cofactors, such as light-absorbing chromophores in photoreceptors, contain a π-electron system and are planar molecules. These cofactors are, however, usually nonplanar within a protein environment, and such structural distortions have been shown to be functionally important. Because the nonplanar structure makes the molecule chiral, Raman optical activity (ROA) provides a wealth of stereochemical information about the structural and conformational details of cofactors. The present study applied a near-infrared excited ROA to photoactive yellow protein, a blue light receptor. We successfully obtained the ROA spectra of the 4-hydroxycinnamyl chromophore embedded in a protein environment. Furthermore, calculations of the ROA spectra utilizing density functional theory provide detailed structural information, such as data on out-of-plane distortions of the chromophore. The structural information obtained from the ROA spectra includes the positions of hydrogen atoms, which are usually not detected in the crystal structures of biological samples.