Subpicosecond Excited-State Proton Transfer Preceding Isomerization During the Photorecovery of Photoactive Yellow Protein

The ultrafast excited-state dynamics underlying the receptor state photorecovery is resolved in the M100A mutant of the photoactive yellow protein (PYP) from Halorhodospira halophila. The M100A PYP mutant, with its distinctly slower photocycle than wt PYP, allows isolation of the pB signaling state for study of the photodynamics of the protonated chromophore cis-p-coumaric acid. Transient absorption signals indicate a subpicosecond excited-state proton-transfer reaction in the pB state that results in chromophore deprotonation prior to the cis-trans isomerization required in the photorecovery dynamics of the pG state. Two terminal photoproducts are observed, a blue-absorbing species presumed to be deprotonated trans-p-coumaric acid and an ultraviolet-absorbing protonated photoproduct. These two photoproducts are hypothesized to originate from an equilibrium of open and closed folded forms of the signaling state, I(2) and I(2)'.     

Photoisomerization and proton transfer in photoactive yellow protein

The photoactive yellow protein (PYP) is a bacterial photosensor containing a para-coumaryl thioester chromophore that absorbs blue light, initiating a photocycle involving a series of conformational changes. Here, we present computational studies to resolve uncertainties and controversies concerning the correspondence between atomic structures and spectroscopic measurements on early photocycle intermediates. The initial nanoseconds of the PYP photocycle are examined using time-dependent density functional theory (TDDFT) to calculate the energy profiles for chromophore photoisomerization and proton transfer, and to calculate excitation energies to identify photocycle intermediates. The calculated potential energy surface for photoisomerization matches key, experimentally determined, spectral parameters. The calculated excitation energy of the photocycle intermediate cryogenically trapped in a crystal structure by Genick et al. [Genick, U. K.; Soltis, S. M.; Kuhn, P.; Canestrelli, I. L.; Getzoff, E. D. Nature 1998, 392, 206-209] supports its assignment to the PYP(B) (I(0)) intermediate. Differences between the time-resolved room temperature (298 K) spectrum of the PYP(B) intermediate and its low temperature (77 K) absorbance are attributed to a predominantly deprotonated chromophore in the former and protonated chromophore in the latter. This contrasts with the widely held belief that chromophore protonation does not occur until after the PYP(L) (I(1) or pR) intermediate. The structure of the chromophore in the PYP(L) intermediate is determined computationally and shown to be deprotonated, in agreement with experiment. Calculations based on our PYP(B) and PYP(L) models lead to insights concerning the PYP(BL) intermediate, observed only at low temperature. The results suggest that the proton is more mobile between Glu46 and the chromophore than previously realized. The findings presented here provide an example of the insights that theoretical studies can contribute to a unified analysis of experimental structures and spectra.     

Structural Heterogeneity of Cryotrapped Intermediates in the Bacterial Blue Light Photoreceptor,Photoactive Yellow Protein¶

We investigate by X‐ray crystallographic techniques the cryotrapped states that accumulate on controlled illumination of the blue light photoreceptor, photoactive yellow protein (PYP), at 110 K in both the wild‐type species and its E46Q mutant. These states are related to those that occur during the chromophore isomerization process in the PYP photocycle at room temperature. The structures present in such states were determined at high resolution, 0.95–1.05Å. In both wild type and mutant PYP, the cryotrapped state is not composed of a single, quasitransition state structure but rather of a heterogeneous mixture of three species in addition to the ground state structure. We identify and refine these three photoactivated species under the assumption that the structural changes are limited to simple isomerization events of the chromophore that otherwise retains chemical bonding similar to that in the ground state. The refined chromophore models are essentially identical in the wild type and the E46Q mutant, which implies that the early stages of their photocycle mechanisms are the same.     

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.     

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.     

Structural changes during the photocycle of photoactive yellow protein monitored by ultraviolet resonance raman spectra of tyrosine and tryptophan

Photoactive yellow protein (PYP) is a bacterial blue light photoreceptor, and photoexcitation of dark-state PYP (PYP(dark)) triggers a photocycle that involves several intermediate states. We report the ultraviolet resonance Raman spectra of PYP with 225-250 nm excitations and investigate protein structural changes accompanying the formation of the putative signaling state denoted PYP(M). The PYP(M)-PYP(dark) difference spectra show several features of tyrosine and tryptophan, indicating environmental changes for these amino acid residues. The tyrosine difference signals show small upshifts with intensity changes in Y8a and Y9a bands. Although there are five tyrosine residues in PYP, Tyr42 and Tyr118 are suggested to be responsible for the difference signals on the basis of a global fitting analysis of the difference spectra at different excitation wavelengths and the crystal structure of PYP(dark). A further experiment on the Thr50-->Val mutant supports environmental changes in Tyr42. The observed upshift of the Y8a band suggests a weaker or broken hydrogen bond between Tyr42 and the chromophore in PYP(M). In addition, a reorientation of the OH group in Tyr42 is suggested from the upshift of the Y9a band. For tryptophan, the Raman bands of W3, W16, and W18 modes diminish in intensity upon formation of PYP(M). The loss of intensities is attributable to an exposure of tryptophan in PYP(M). PYP contains only one tryptophan (Trp119) that is located more than 10 A from the active site. Thus the observed changes are indicative of global conformational changes in protein during the transition from PYP(dark) to PYP(M). These results are in line with the currently proposed photocycle mechanism of PYP.     

Time-resolved thermodynamics: heat capacity change of transient species during photoreaction of PYP

Heat capacity changes of short-lived transient species in different time ranges were measured for the first time by using the thermal component of the transient grating and transient lens signals at various temperatures. This method was applied to the transient intermediates of Photoactive Yellow Protein (PYP). The temperature dependence of the enthalpy change shows that the heat capacity of the short-lived intermediate pR2 (also called I1 or PYP(L)) species is the same as that of the ground state (pG) species within our experimental accuracy, whereas that of the long-lived intermediate pB (I2 or PYP(M)) is much larger (2.7 +/- 0.4 kJ/mol K) than that of pG. The larger heat capacity is interpreted in terms of the conformational change of the pB species such as melted conformation and/or exposure of the nonpolar residues to the aqueous phase. This technique can be used for photochemical reactions in general to investigate the conformational change and the hydrophobic interaction in a time domain.     

The photoreaction of the photoactive yellow protein domain in the light sensor histidine kinase Ppr is influenced by the C-terminal domains

To study the role of the C-terminal domains in the photocycle of a light sensor histidine kinase (Ppr) having a photoactive yellow protein (PYP) domain as the photosensor domain, we analyzed the photocycles of the PYP domain of Ppr (Ppr-PYP) and full-length Ppr. The gene fragment for Ppr-PYP was expressed in Escherichia coli, and it was chemically reconstituted with p-coumaric acid; the full-length gene of Ppr was coexpressed with tyrosine ammonia-lyase and p-coumaric acid ligase for biosynthesis in cells. The light/dark difference spectra of Ppr-PYP were pH sensitive. They were represented as a linear combination of two independent difference spectra analogous to the PYP(L)/dark and PYP(M)/dark difference spectra of PYP from Halorhodospira halophila, suggesting that the pH dependence of the difference spectra is explained by the equilibrium shift between the PYP(L)- and PYP(M)-like intermediates. The light/dark difference spectrum of Ppr showed the equilibrium shift toward PYP(L) compared with that of Ppr-PYP. Kinetic measurements of the photocycles of Ppr and Ppr-PYP revealed that the C-terminal domains accelerate the recovery of the dark state. These observations suggest an interaction between the C-terminal domains and the PYP domain during the photocycle, by which light signals captured by the PYP domain are transferred to the C-terminal domains.     

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.     

Photokinetic,Biochemical and Structural Features of Chimeric Photoactive Yellow Protein Constructs

Of the 10 photoactive yellow protein (PYPs) that have been characterized, the two from Rhodobacter species are the only ones that have an additional intermediate spectral form in the resting state (λ_max = 375 nm), compared to the prototypical Halorhodospira halophila PYP. We have constructed three chimeric PYP proteins by replacing the first 21 residues from the N‐terminus (Hyb1PYP), 10 from the β4–β5 loop (Hyb2PYP) and both (Hyb3PYP) in Hhal PYP with those from Rb. capsulatus PYP. The N‐terminal chimera behaves both spectrally and kinetically like Hhal PYP, indicating that the Rcaps N‐terminus folds against the core of Hhal PYP. A small fraction shows dimerization and slower recovery, possibly due to interaction at the N‐termini. The loop chimera has a small amount of the intermediate spectral form and a photocycle that is 20 000 times slower than Hhal PYP. The third chimera, with both regions exchanged, resembles Rcaps PYP with a significant amount of intermediate spectral form (λ_max = 380 nm), but has even slower kinetics. The effects are not strictly additive in the double chimera, suggesting that what perturbs one site, affects the other as well. These chimeras suggest that the intermediate spectral form has its origins in overall protein stability and solvent exposure.     

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.     

Ultrafast dynamics of isolated model photoactive yellow protein chromophores: "Chemical perturbation theory" in the laboratory

Pump-probe and pump-dump probe experiments have been performed on several isolated model chromophores of the photoactive yellow protein (PYP). The observed transient absorption spectra are discussed in terms of the spectral signatures ascribed to solvation, excited-state twisting, and vibrational relaxation. It is observed that the protonation state has a profound effect on the excited-state lifetime of p-coumaric acid. Pigments with ester groups on the coumaryl tail end and charged phenolic moieties show dynamics that are significantly different from those of other pigments. Here, an unrelaxed ground-state intermediate could be observed in pump-probe signals. A similar intermediate could be identified in the sinapinic acid and in isomerization-locked chromophores by means of pump-dump probe spectroscopy; however, in these compounds it is less pronounced and could be due to ground-state solvation and/or vibrational relaxation. Because of strong protonation-state dependencies and the effect of electron donor groups, it is argued that charge redistribution upon excitation determines the twisting reaction pathway, possibly through interaction with the environment. It is suggested that the same pathway may be responsible for the initiation of the photocycle in native PYP.     

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.     

LOW TEMPERATURE ABSORBANCE AND FLUORESCENCE SPECTROSCOPY OF THE PHOTOACTIVE YELLOW PROTEIN FROM Ectothiorhodospira halophila

A simplified procedure was developed to purify the photoactive yellow protein (PYP) from Ecrorhiorhodospira halophila. Specific antibodies were used to follow the distribution of PYP through the separate purification steps. Low temperature absorbance and fluorescence characteristics of this photoactive protein were investigated. The absorbance spectrum of PYP in 67% (vol/vol) glycerol peaked at 449 and 447 nm, at room-and liquid nitrogen temperatures, respectively. It sharpened significantly upon cooling to 77 K and displayed fine-structure on the blue side of its absorbance maximum, with a spacing of 25 nm. At room temperature PYP fluoresced with a quantum yield of approximately 3.5 times 10^?-³ an emission maximum of 495 nm. Maximal excitation occurred at 457 nm, 10 nm red-shifted with respect to the absorbance maximum. At -low temperature the excitation maximum remained unaltered but maximal emission shifted significantly to the blue (to 482 nm). The quantum yield of fluorescence increased to 0.07 at this temperature. Illumination of PYP at low temperature with light from the visible part of the spectrum of electromagnetic radiation induced pronounced changes in its absorbance and fluorescence characteristics. At least two new stable intermediates were formed: one highly fluorescent, with an excitation maximum at 430 nm; additionally, a non-fluorescent red-shifted intermediate with an absorbance maximum at 490 nm. The amount formed of these two intermediates depended strongly on the wavelength of actinic illumination. In combination, these data underline the spectroscopic similarities between PYP and the retinal-containing chromoproteins that are present in Halobacterium halobium.     

Structural evolution of the chromophore in the primary stages of trans/cis isomerization in photoactive yellow protein

We have studied the structural changes induced by optical excitation of the chromophore in wild-type photoactive yellow protein (PYP) in liquid solution with a combined approach of polarization-sensitive ultrafast infrared spectroscopy and density functional theory calculations. We identify the nuC8-C9 marker modes for solution phase PYP in the P and I0 states, from which we derive that the first intermediate state I0 that appears with a 3 ps time constant can be characterized to have a cis geometry. This is the first unequivocal demonstration that the formation of I0 correlates with the conversion from the trans to the cis state. For the P and I0 states we compare the experimentally measured vibrational band patterns and anisotropies with calculations and find that for both trans and cis configurations the planarity of the chromophore has a strong influence. The C7=C8-(C9=O)-S moiety of the chromophore in the dark P state has a trans geometry with the C=O group slightly tilted out-of-plane, in accordance with the earlier reported structure obtained in an X-ray diffraction study of PYP crystals. In the case of I0, experiment and theory are only in agreement when the C7=C8-(C9=O)-S moiety has a planar configuration. We find that the carboxylic side group of Glu46 that is hydrogen-bonded to the chromophore phenolate oxygen does not alter its orientation on going from the electronic ground P state, via the electronic excited P state to the intermediate I0 state, providing conclusive experimental evidence that the primary stages of PYP photoisomerization involve flipping of the enone thioester linkage without significant relocation of the phenolate moiety.     

Bacteriorhodopsin photocycle kinetics analyzed by the maximum entropy method

A maximum entropy method (MEM) was developed for the study of the bacteriorhodopsin photocycle kinetics. The method can be applied directly to experimental kinetic absorption data without any assumption for the number of the intermediate states taking part in the photocycle. Though this method does not give a specific kinetics, its result is very useful for selection between possible photocycle kinetics. Using simulated data, it is shown that MEM gives correct results for the number of the intermediate states and the amplitude distributions around the characteristic lifetimes. Analyzing experimental absorption data at five different wavelengths, MEM gives seven or eight characteristic lifetimes, which means that at least so many distinct intermediate states exist during the photocycle. Many possible photocycle kinetic models were studied and compared with the MEM result. The best agreement was found with a branching photocycle model of eight intermediate states (K, L, M(1), M(2), M(3), M(4), N, O). The branching occurs at the L intermediate state (M(1) and M(2) being in one branch and M(3) and M(4) in the other branch), but at high pH it occurs already at the K state.     

Kinetics of photo-active bacteriorhodopsin analog 3,4-didehydroretinal

Kinetics of the photo-induced processes of the transient states of the 3,4-didehydroretinal (3,4-dhr) modified bacteriorhodopsin (bR) was studied by a flash photolysis method in a water suspension at room temperature. The excitation initiated a photocycle with several transient intermediates similar to the trans photocycle of native bR. The main observation of the study was that although major part (80%) of the population of the M state relaxed via the O intermediate as in natural bR, 20% relaxed directly to the bR ground state in 200 ms.     

Array of aromatic amino acid side chains located near the chromophore of photoactive yellow protein

The role of the array of aromatic amino acid side chains located close to the chromophore binding loop of photoactive yellow protein (PYP) was studied using the alanine-substitution mutagenesis. Phe92, Tyr94, Phe96 and Tyr98 were replaced with alanine (F92A, Y94A, F96A and Y98A, respectively), then these mutants were characterized by UV-visible absorption spectra, circular dichroism (CD) spectra, thermal stability and photocycle kinetics. Absorption maxima of F92A, Y94A, F96A and Y98A were 444, 442, 439 and 447 nm, respectively, different to wild type (WT) at 446 nm. Far-UV CD spectra of mutants other than F92A were different from WT, indicating that Tyr94, Phe96 and Tyr98 maintain the native secondary structure of PYP. Mid-point temperatures of thermal denaturation of F92A, Y94A and F96A, estimated by the CD signal at 222 nm, were 5-10 degrees C lower than WT. Time constants of the photocycle estimated by flash-induced absorbance change were 0.36 s for WT and 1.4 s for Y98A, however, 100, 30 and 3000 times slower than WT for F92A, Y94A and F96A, respectively. Tyr98 is located in the loop region, whereas Phe92, Tyr94 and Phe96 are incorporated in the beta4 strand, showing that aromatic amino acid residues in the beta-sheet regulate the absorption spectrum, thermal stability and photocycle of PYP. Aromatic rings of Phe92, Tyr94 and Phe96 lie nearly perpendicular to the aromatic ring of Phe75 or chromophore. Possible weak hydrogen bonds between the aromatic ring hydrogen and pi-electrons of these residues are discussed.     

Anharmonic vibrational calculations modeling the raman spectra of intermediates in the photoactive yellow protein (PYP) photocycle

The role of anharmonic effects in the vibrational spectroscopy of the dark state and two major chromophore intermediates of the photoactive yellow protein (PYP) photocycle is examined via ab initio vibrational self-consistent field (VSCF) calculations and time-resolved resonance Raman spectroscopy. For the first time, anharmonicity is considered explicitly in calculating the vibrational spectra of an ensemble consisting of the PYP chromophore surrounded by model compounds used as mimics of the important active-site residues. Predictions of vibrational frequencies on an ab initio corrected semiempirical potential energy surface show remarkable agreement with experimental frequencies for all three states, thus shedding light on the potential along the reaction path. For example, calculated frequencies for vibrational modes of the red-shifted intermediate, PYPL, exhibit an overall average error of 0.82% from experiment. Upon analysis of anharmonicity patterns in the PYP modes we observe a decrease in anharmonicity in the C8-C9 stretching mode nu29 (trans-cis isomerization marker mode) with the onset of the cis configuration in PYPL. This can be attributed to the loss of the hydrogen-bonding character of the adjacent C9-O2 to the methylamine (Cys69 backbone). For several of the modes, the anharmonicity is mostly due to mode-mode coupling, while for others it is mostly intrinsic. This study shows the importance of the inclusion of anharmonicity in theoretical spectroscopic calculations, and the sensitivity of experiments to anharmonicity. The characterization of protein active-site residues by small molecular mimics provides an acceptable chemical structural representation for biomolecular spectroscopy calculations.