trans-cis Photoisomerization of a photoactive yellow protein model chromophore in crystalline phase

We have studied the photoinduced trans/cis isomerization of the protonated form of p-hydroxycinnamic thiophenyl ester, a model chromophore of the photoactive yellow protein (PYP), in crystalline phase, by both fluorescence and infrared spectroscopies. The conversion from trans to cis configuration is revealed by a shift of the fluorescence peak and by inspection of the infrared maker bands. The crystal packing apparently stabilizes the cis photoproduct, suggesting different environmental effects from the solvent molecules for this model chromophore in liquid solutions or from the amino acid residues for the PYP chromophore.     

Proton transfer and associated molecular rearrangements in the photocycle of photoactive yellow protein: role of water molecular migration on the proton transfer reaction

The mechanism of the proton transfer and the concomitant molecular structural and hydrogen bond rearrangements after the photoisomerization of the chromophore in the photocycle of photoactive yellow protein are theoretically investigated by using the QM/MM method and molecular dynamics calculations. The free energy surface along this proton-transfer process is determined. This work suggests the important role of the water molecular migration into the moiety of chromophore, which facilitates proton transfer by the hydrogen bond rearrangement and the hydration of the pB' state.     

Spectral tuning of photoactive yellow protein

We report a theoretical study on the optical properties of a small, water-soluble photosensory receptor, photoactive yellow protein (PYP). A hierarchical ab initio molecular orbital calculation accurately evaluated the optical absorption maximum of the wild-type, as well as the lambda(max) values of 12 mutants. Electronic excitation of the chromophore directly affects the electronic state of nearby atoms in the protein environment. This effect is explicitly considered in the present study. Furthermore, the spectral tuning mechanism of PYP was investigated at the atomic level. The static disorder of a protein molecule is intimately related to the complex nature of its energy landscape. By using molecular dynamics simulation and quantum mechanical structure optimization, we obtained multiple minimum energy conformations of PYP. The statistical distribution of electronic excitation energies of these minima was compared with the hole-burning experiment (Masciangioli, T. [2000] Photochem. Photobiol. 72, 639), a direct observation of the distribution of excitation energies.     

Probing the photochemical mechanism in photoactive yellow protein

Selectively bridged model compounds related to the chromophore in photoactive yellow protein have been synthesized where the single bond adjacent to the benzene ring (bond 1) and where both bond 1 and the adjacent double bond (bond 2) are bridged. They were compared to the nonbridged reference compound regarding their photophysical properties using steady-state and time-resolved fluorescence at various temperatures. Quantum chemical calculations were additionally performed and showed that several conformers are populated in the ground state. The neutral model compounds show that the nonradiative deactivation channel is linked to both single- and double-bond twisting. The relative importance of single-bond twisting is increased for the corresponding deprotonated hydroxy compounds with an enhanced donor character. The simultaneous photochemical activity of both single and double bonds explains the ease of photochemical isomerization in the confined environment of the natural PYP protein and also of the primary step in the vision process in rhodopsin.     

Coherent oscillations in ultrafast fluorescence of photoactive yellow protein

The ultrafast photoinduced dynamics of photoactive yellow protein in aqueous solution were studied at room temperature by femtosecond fluorescence spectroscopy using an optical Kerr-gate technique. Coherent oscillations of the wave packet were directly observed in the two-dimensional time-energy map of ultrafast fluorescence with 180 fs time resolution and 5 nm spectral resolution. The two-dimensional map revealed that four or more oscillatory components exist within the broad bandwidth of the fluorescence spectrum, each of which is restricted in the respective narrow spectral region. Typical frequencies of the oscillatory modes are 50 and 120 cm(-1). In the landscape on the map, the oscillatory components were recognized as the ridges which were winding and descending with time. The amplitude of the oscillatory and winding behaviors is a few hundred cm(-1), which is the same order as the frequencies of the oscillations. The mean spectral positions of the oscillatory components in the two-dimensional map are well explained by considering the vibrational energies of intramolecular modes in the electronic ground state of the chromophore. The entire view of the wave packet oscillations and broadening in the electronic excited state, accompanied by fluorescence transitions to the vibrational sublevels belonging to the electronic ground state, was obtained.     

Characterization of photocycle intermediates in crystalline photoactive yellow protein

The photocycle in photoactive yellow protein (PYP) crystals was studied by single-crystal absorption spectroscopy with experimental setups for low-temperature and time-resolved measurements. Thin and flat PYP crystals, suitable for light absorption studies, were obtained using special crystallization conditions. Illumination of PYP crystals at 100 K led to the formation of a photostationary state, which includes at least one hypsochromic and one bathochromic photoproduct that resemble PYP(H) and PYP(B), respectively. The effect of temperature, light color and light pulse duration on the occupancy of these low-temperature photoproducts was determined and appeared similar to that observed in solution. At room temperature a blueshifted photocycle intermediate was identified that corresponds to the blueshifted state of PYP (pB). Kinetic studies show that the decay of this blueshifted intermediate is biphasic at -12 degrees C and 15-fold faster than that observed in solution at room temperature. These altered pB decay kinetics confirm a model that holds that the photocycle in crystals takes place in a shortcut version. In this version the key structural events of the photocycle, such as photoisomerization and reversible protonation of the chromophore, take place, but large conformational changes in the surrounding protein are limited by constraints imposed by the crystal lattice.     

Protonation of the chromophore in the photoactive yellow protein

The photoactive yellow protein (PYP) acts as a light sensor to its bacterial host: it responds to light by changing shape. After excitation by blue light, PYP undergoes several transformations, to partially unfold into its signaling state. One of the crucial steps in this photocycle is the protonation of p-coumaric acid after excitation and isomerization of this chromophore. Experimentalists still debate on the nature of the proton donor and on whether it donates the hydrogen directly or indirectly. To obtain better knowledge of the mechanism, we studied this proton transfer using Car-Parrinello molecular dynamics, classical molecular dynamics, and computer simulations combining these two methods (quantum mechanics/molecular mechanics, QMMM). The simulations reproduce the chromophore structure and hydrogen-bond network of the protein measured by X-ray crystallography and NMR. When the chromophore is protonated, it leaves the assumed proton donor, glutamic acid 46, with a negative charge in a hydrophobic environment. We show that the stabilization of this charge is a very important factor in the mechanism of protonation. Protonation frequently occurs in simplified ab initio simulations of the chromophore binding pocket in vacuum, where amino acids can easily hydrogen bond to Glu46. When the complete protein environment is incorporated in a QMMM simulation on the complete protein, no proton transfer is observed within 14 ps. The hydrogen-bond rearrangements in this time span are not sufficient to stabilize the new protonation state. Force field molecular dynamics simulations on a much longer time scale have shown which internal rearrangements of the protein are needed. Combining these simulations with more QMMM calculations enabled us to check the stability of protonation states and clarify the initial requirements for the proton transfer in PYP.     

Interaction between N-terminal loop and beta-scaffold of photoactive yellow protein

During the photoreaction cycle of photoactive yellow protein (PYP), a physiologically active intermediate (PYP(M)) is formed as a consequence of global protein conformational change. Previous studies have demonstrated that the photocycle of PYP is regulated by the N-terminal loop region, which is located across the central beta-sheet from the p-coumaric acid chromophore. In this paper, the hydrophobic interaction between N-terminal loop and beta-sheet was studied by characterizing PYP mutants of the hydrophobic residues. The rate constants and structural changes of the photocycle of L15A and L23A possibly participating in such an interaction were more similar to wild-type than F6A, showing that the CH/pi interaction between Phe6 and Lys123 is the most essential as reported previously. To better understand the interactions between N-terminal tail and beta-sheet of PYP, Phe6 and Phe121 were replaced by Cys and linked by a disulfide bond. Since the photocycle kinetics, structural change and thermal stability of F6C/F121C were similar to F6A, the CH/pi interaction between Phe6 and Lys123 is not substitutable. It is likely that the detachment of position 6 from position 123 substantially alters the nature of PYP.     

Initial photoinduced dynamics of the photoactive yellow protein.

The photoactive yellow protein (PYP) is the photoreceptor protein responsible for initiating the blue-light repellent response of the Halorhodospira halophila bacterium. Optical excitation of the intrinsic chromophore in PYP, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates. The dynamical processes responsible for the initiation of the PYP photocycle have been explored with several time-resolved techniques, which include ultrafast electronic and vibrational spectroscopies. Ultrafast electronic spectroscopies, such as pump-visible probe, pump-dump-visible probe, and fluorescence upconversion, are useful in identifying the timescales and connectivity of the transient intermediates, while ultrafast vibrational spectroscopies link these intermediates to dynamic structures. Herein, we present the use of these techniques for exploring the initial dynamics of PYP, and show how these techniques provide the basis for understanding the complex relationship between protein and chromophore, which ultimately results in biological function.     

Transient vibronic structure in ultrafast fluorescence spectra of photoactive yellow protein

The ultrafast photo-induced dynamics of wild-type photoactive yellow protein and its site-directed mutant of E46Q in aqueous solution was studied at room temperature by femtosecond fluorescence spectroscopy using the optical Kerr-gate method. The vibronic structure appears, depending on the excitation photon energy, in the time-resolved fluorescence spectra just after photoexcitation, which winds with time and disappears on a time scale of sub-picoseconds. This result indicates that the wavepacket is localized in the electronic excited state followed by dumped oscillations and broadening, and also that the initial condition of the wavepacket prepared depending on the excitation photon energy affects much the following ultrafast dynamics in the electronic excited state.     

Vibrational assignment of the 4-hydroxycinnamyl chromophore in photoactive yellow protein

Photoactive yellow protein (PYP) is a bacterial photoreceptor containing a 4-hydroxycinnamyl chromophore. We report the Raman spectra for the dark state of PYP whose chromophore is isotopically labeled with 13C at the carbonyl carbon atom or at the ring carbon atoms. Spectra have been also measured with PYP in D2O where the exchangeable protons are deuterated. Most of the observed Raman bands are assigned on the basis of the observed isotope shifts and normal mode calculations using a density functional theory. We discuss the implication for the analysis of the infrared spectra of PYP. The comprehensive assignment provides a satisfactory framework for future investigations of the photocycle mechanism in PYP by vibrational spectroscopy.     

Ab initio excited-state dynamics of the photoactive yellow protein chromophore

The photoisomerization mechanism of the neutral form of the photoactive yellow protein (PYP) chromophore is investigated using ab initio quantum chemistry and first-principles nonadiabatic molecular dynamics (ab initio multiple spawning or AIMS). We identify the nature of the two lowest-lying excited states, characterize the short-time behavior of molecules excited directly to S2, and explain the origin of the experimentally observed wavelength-dependent photoisomerization quantum yield.     

Gas-phase photochemistry of the photoactive yellow protein chromophore trans-p-coumaric acid

The photoisomerization of trans-p-coumaric acid (trans-CA) triggers a photocycle in photoactive yellow protein that ultimately mediates a phototactic response to blue light in certain purple bacteria. We have used fluorescence excitation and dispersed emission methods in a supersonic jet to investigate the nature of the electronic excited states involved in the initial photoexcitation and subsequent photoisomerization of trans-CA. We observed three distinct regions in the fluorescence excitation spectrum of trans-CA. Region I is characterized by sharp features that upon excitation exhibit trans-CA S(1) emission. In region II, features increase in width and decrease in intensity with increasing excitation energy. Upon excitation, we observed dual emission from the S(1) state of trans-CA and what may be the S(1) state of cis-CA. The onset of dual emission corresponds to an isomerization barrier of about 3.4 kcal/mol. Finally, the extremely broad absorption feature in region III is excitation to the S(2) electronic excited state and excitation results in trans-CA S(1) emission. Furthermore, we collected CA from the molecular beam after laser excitation in each of the three regions as further evidence of the photoisomerization process. The relative amounts of trans- and cis-CA in the collected molecules were measured with high-pressure liquid chromatography. Although trans-CA was excited in all three regions, a significant cis-CA peak appeared only in region II, though a small cis peak was observed in region III.     

Ultrafast dynamics of photoactive yellow protein via the photoexcitation and emission processes

Pump-dump fluorescence spectroscopy was performed for photoactive yellow protein (PYP) at room temperature. The effect of the dump pulse on the population of the potential energy surface of the electronic excited state was examined as depletion in the stationary fluorescence intensity. The dynamic behavior of the population in the electronic excited state was successfully probed in the various combinations of the pump-dump delay, the dump-pulse wavelength, the dump-pulse energy and the observation wavelength. The experimental results were compared with the results obtained by the femtosecond time-resolved fluorescence spectroscopy.     

Low-temperature spectroscopy of Met100Ala mutant of photoactive yellow protein

The trans-to-cis photoisomerization of the p-coumaroyl chromophore of photoactive yellow protein (PYP) triggers the photocycle. Met100, which is located in the vicinity of the chromophore, is a key residue for the cis-to-trans back-isomerization of the chromophore, which is a rate-determining reaction of the PYP photocycle. Here we characterized the photocycle of the Met100Ala mutant of PYP (M100A) by low temperature UV-visible spectroscopy. Irradiation of M100A at 80 K yielded a 380 nm species (M100A(BL)), while the corresponding intermediate of wild type (WT; PYP(BL)) is formed above 90 K. The amounts of redshifted intermediates produced from M100A (M100A(B') and M100A(L)) were substantially less than those from WT. While the near-UV intermediate (PYP(M)) is not formed from WT in glycerol samples at low temperature, M100A(M) was clearly observed above 190 K. These alterations of the photocycle of M100A were explained by the shift in the equilibrium between the intermediates. The carbonyl oxygen of the thioester linkage of the cis-chromophore in the photocycle intermediates is close to the phenyl ring of Phe96 (<3.5 A), which would be displaced by the mutation of Met100. These findings imply that the interaction between chromophore and amino acid residues near Met100 is altered during the early stage of the PYP photocycle.     

Probing the primary event in the photocycle of photoactive yellow protein using photochemical hole-burning technique

Photochemical hole-burning spectroscopy was used to study the excited-state electronic structure of the 4-hydroxycinnamyl chromophore in photoactive yellow protein (PYP). This system is known to undergo a trans-to-cis isomerization process on a femtosecond-to-picosecond time scale, similar to membrane-bound rhodopsins, and is characterized by a broad featureless absorbance at 446 nm. Resolved vibronic structure was observed for the hole-burned spectra obtained when PYP in phosphate buffer at pH 7 was frozen at low temperature and irradiated with narrow bandwidth laser light at 431 nm. The approximate homogeneous width of 752 cm-1 could be calculated from the deconvolution of the hole-burned spectra leading to an estimated dephasing time of approximately 14 fs for the PYP excited-state structure. The resolved vibronic structure also enabled us to obtain an estimated change in the C=C stretching frequency, from 1663 cm-1 in the ground state to approximately 1429 cm-1 upon photoexcitation. The results obtained allowed us to speculate about the excited-state structure of PYP. We discuss the data for PYP in relation to the excited-state model proposed for the photosynthetic membrane protein bacteriorhodopsin, and use it to explain the primary event in the function of photoactive biological protein systems. Photoexcitation was also carried out at 475 nm. The vibronic structure obtained was quite different both in terms of the frequencies and Franck-Condon envelope. The origin of this spectrum was tentatively assigned.     

Protein structural dynamics of photoactive yellow protein in solution revealed by pump-probe X-ray solution scattering

Photoreceptor proteins play crucial roles in receiving light stimuli that give rise to the responses required for biological function. However, structural characterization of conformational transition of the photoreceptors has been elusive in their native aqueous environment, even for a prototype photoreceptor, photoactive yellow protein (PYP). We employ pump-probe X-ray solution scattering to probe the structural changes that occur during the photocycle of PYP in a wide time range from 3.16 μs to 300 ms. By the analysis of both kinetics and structures of the intermediates, the structural progression of the protein in the solution phase is vividly visualized. We identify four structurally distinct intermediates and their associated five time constants and reconstructed the molecular shapes of the four intermediates from time-independent, species-associated difference scattering curves. The reconstructed structures of the intermediates show the large conformational changes such as the protrusion of N-terminus, which is restricted in the crystalline phase due to the crystal contact and thus could not be clearly observed by X-ray crystallography. The protrusion of the N-terminus and the protein volume gradually increase with the progress of the photocycle and becomes maximal in the final intermediate, which is proposed to be the signaling state. The data not only reveal that a common kinetic mechanism is applicable to both the crystalline and the solution phases, but also provide direct evidence for how the sample environment influences structural dynamics and the reaction rates of the PYP photocycle.