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.     

Unraveling the similarity of the photoabsorption of deprotonated p-coumaric acid in the gas phase and within the photoactive yellow protein

Using advanced QM/MM methods, the surprisingly negligible shift of the lowest-lying bright electronic excitation of the deprotonated p-coumaric acid (pCA(-)) within the photoactive yellow protein (PYP) is shown to stem from a subtle balance between hypsochromic and bathochromic effects. More specifically, it is found that the change in the excitation energy as a consequence of the disruption of the planarity of pCA(-) inside PYP is nearly canceled out by the shift induced by the intermolecular interactions of the chromophore and the protein as a whole. These results provide important insights about the primary absorption and the tuning of the chromophore by the protein environment in PYP.     

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.     

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.     

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.     

Electrochemistry, spectroscopy, and electrogenerated chemiluminescence of silole-based chromophores

We studied the electrochemical and spectroscopic properties of a series of extended silole-based chromophores to understand the effect of structure on behavior. By changing the substituents attached to the chromophore, we observed large variations in luminescence quantum efficiency (ca. 0-0.6), lambdamax for absorbance and photoluminescence (PL), and radical ion stability. The differences are related to the motion in the 2,5-substituents and the steric protection of both the chromophore and the reactive parts of the substituents. For several compounds the electrogenerated chemiluminescence (ECL) spectrum was also compared to the photoluminescence spectrum. In all cases, the ECL lambdamax and the PL lambdamax were about the same.     

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.     

Structure and photoreaction of photoactive yellow protein, a structural prototype of the PAS domain superfamily

Photoactive yellow protein (PYP) is a water-soluble photosensor protein found in purple photosynthetic bacteria. Unlike bacterial rhodopsins, photosensor proteins composed of seven transmembrane helices and a retinal chromophore in halophilic archaebacteria, PYP is a highly soluble globular protein. The alpha/beta fold structure of PYP is a structural prototype of the PAS domain superfamily, many members of which function as sensors for various kinds of stimuli. To absorb a photon in the visible region, PYP has a p-coumaric acid chromophore binding to the cysteine residue via a thioester bond. It exists in a deprotonated trans form in the dark. The primary photochemical event is photo-isomerization of the chromophore from trans to cis form. The twisted cis chromophore in early intermediates is relaxed and finally protonated. Consequently, the chromophore becomes electrostatically neutral and rearrangement of the hydrogen-bonding network triggers overall structural change of the protein moiety, in which local conformational change around the chromophore is propagated to the N-terminal region. Thus, it is an ideal model for protein conformational changes that result in functional change, responding to stimuli and expressing physiological activity. In this paper, recent progress in investigation of the photoresponse of PYP is reviewed.     

Mechanistic pathways for the photoisomerization reaction of the anchored,tethered chromophore of the photoactive yellow protein and its mutants

We show by way of physical organic reasoning that the currently known photochemical results of the chromophore of photoactive yellow protein (PYP) are consistent with that expected of a least volume-demanding process for an anchored, tethered chromophore. The primary photoreaction, interestingly, does not appear to involve a hula-twist process. However, the latter might be involved during subsequent transition of dark intermediates. Absorption data of intermediates obtained from a microsecond time-resolved spectroscopic study of three PYP mutants (E46Q, T50V and R52Q) are consistent with the above analyses.     

非对称环氧乙烷的区域选择性亲核开环反应

本文总结了常用亲核试剂对非对称环氧乙烷的亲核开环反应及其区域选择性。强亲核性的亲核试剂通常只受空间效应影响,进攻非对称环氧乙烷位阻小的碳原子,对于烯基取代环氧乙烷还可以进攻烯基的β-碳原子发生S_N2'开环反应,其他亲核试剂同时受空间效应和电子效应的影响,对于烷基环氧乙烷通常进攻其取代少的碳原子, 空间效应起主导作用,而对芳基和烯基取代环氧乙烷开环反应通常发生在环氧乙烷芳甲位和烯丙位的碳原子上, 电子效应起主导作用。在质子酸或强Lewis酸存在下,虽然单烷基环氧乙烷的开环仍然发生在其取代少的碳原子上,但对于芳基、烯基和同碳双取代环氧乙烷,亲核开环反应将主要受电子效应控制,一般亲核试剂倾向于进攻环氧乙烷的芳甲位、烯丙位或多取代的碳原子。分子内的亲核开环反应主要受成环时环大小的控制, 成环时的倾向是五元环> 六元环> 七元环。环氧乙烷亲核开环的区域选择性是环氧乙烷和亲核试剂空间效应和电子效应平衡的结果。     

Identification of six new photoactive yellow proteins--diversity and structure-function relationships in a bacterial blue light photoreceptor

Photoactive yellow proteins (PYP) are bacterial photoreceptors with a Per-Arnt-Sim (PAS) domain fold. We report the identification of six new PYPs, thus nearly doubling the size of this protein family. This extends the taxonomic diversity of PYP-containing bacteria from photosynthetic to nonphotosynthetic bacteria, from aquatic to soil-dwelling organisms, and from Proteobacteria to Salinibacter ruber from the phylum Bacteriodetes. The new PYPs greatly increase the sequence diversity of the PYP family, reducing the most prevalent pair-wise identity from 45% to 25%. Sequence alignments and analysis indicate that all 14 PYPs share a common structure with 13 highly conserved residues that form the chromophore binding pocket. Nevertheless, the functional properties of the PYPs vary greatly--the absorbance maximum extends from 432 to 465 nm, the pK(a) of the chromophore varies from pH 2.8 to 10.2, and the lifetime of the presumed PYP signaling state ranges from 1 ms to 1 h. Thus, the PYP family offers an excellent opportunity to investigate how functional properties are tuned over a wide range, while maintaining the same overall protein structural fold. We discuss the implications of these results for structure-function relationships in the PYP family.     

非对称氮杂环丙烷的亲核开环反应及其区域选择性

本文系统地总结了各类亲核试剂对非对称氮杂环丙烷(吖丙啶)的亲核开环反应及开环的区域选择性.氮杂环丙烷亲核开环的区域选择性是一种空间效应和电子效应平衡的结果,非芳基和非烯基取代的氮杂环丙烷的亲核开环通常发生在氮杂环丙烷取代少的碳原子上,空间效应起主导作用;而芳基和烯基取代的氮杂环丙烷的亲核开环通常发生在氮杂环丙烷芳甲位和烯丙位的碳原子上,电子效应起主导作用,烯基取代的氮杂环丙烷的亲核开环还可以发生在烯基的β-碳原子上;分子内的亲核开环反应主要受成环时环大小的控制,成环时的倾向是五元环>六元环>七元环.对于亲核试剂,一般的亲核试剂也同时受电子效应和空间效应的影响; 而亲核性强的亲核试剂通常只受空间效应的影响.容易生成稳定自由基的亲核试剂容易发生单电子转移机理的开环反应,生成相当于亲核试剂进攻氮杂环丙烷中取代多的碳原子得到的开环产物.     

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.     

(Sub)-picosecond spectral evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system

The spectral evolution of three photoactive proteins has been investigated by measuring the fluorescence with good temporal and wavelength resolution and a high signal-to-noise ratio. Upon excitation at 400 nm wild-type (wt) PYP both at neutral pH and in the low-pH blueshifted pBdark state exhibited a strong quenching of the fluorescence, the major part of which could be described by lifetimes of about 1.7 and 7.7 ps. The remaining fluorescence decay occurred multiexponentially with lifetimes between 30 and 125 ps. Additionally, in wtPYP at neutral pH, a dynamic Stokes shift was found to occur with a time constant of about 0.25 ps. In a PYP preparation that was reconstituted with the chromophore 7-hydroxy-coumarin-3- carboxylic acid rather than the native coumaric acid, and which is therefore not capable of performing the cis-trans-isomerization that initiates the photocycle in wtPYP, the fluorescence was found to decay multiexponentially with lifetimes of 51 ps, 0.33 and 3.77 ns. Additionally, dynamic Stokes shifts were observed with time constants of about 0.1 and 3.5 ps. Upon comparison of the dynamics of this preparation with that of wtPYP the multiexponential decay with lifetimes of 1.7 and 7.7 ps found in wtPYP was attributed to photochemistry of the p-coumaric-acid chromophore. The emission from bacteriorhodopsin mutant D85S upon excitation at 635 nm decays biexponentially with estimated lifetimes of 5.2 and 19.1 ps. No dynamic Stokes shift was observed here. Four lifetimes were needed to describe the decay of the emission from the A* state in the green fluorescent protein. From a target analysis it was concluded that the longer lifetimes are accompanied by a decreasing probability of forming I*, which approaches zero with the longest A* lifetime of 1.5 ns. These observations may be explained by heterogeneity of A and by relaxation of A*. In all three systems studied, multiexponential decay of emission was present, suggesting that heterogeneity is a common feature of these chromophore protein complexes.     

Photoinduced isomerization of the photoactive yellow protein (PYP) chromophore: interplay of two torsions, a HOOP mode and hydrogen bonding

We report on a detailed theoretical analysis, based on extensive ab initio calculations at the CC2 level, of the S(1) potential energy surface (PES) of the photoactive yellow protein (PYP) chromophore. The chromophore's photoisomerization pathway is shown to be fairly complex, involving an intimate coupling between single-bond and double-bond torsions. Furthermore, these torsional modes are shown to couple to a third coordinate of hydrogen out-of-plane (HOOP) type whose role in the isomerization is here identified for the first time. In addition, it is demonstrated that hydrogen bonding at the phenolate moiety of the chromophore can hinder the single-bond torsion and thus facilitates double-bond isomerization. These results suggest that the interplay between intramolecular factors and H-bonding determines the isomerization in native PYP.     

Solvation of p-coumaric acid in water

The photoactive yellow protein (PYP) is an important model protein for many (photoactive) signaling proteins. Key steps in the PYP photocycle are the isomerization and protonation of its chromophore, p-coumaric acid (pCA). In the ground state of the protein, this chromophore is in the trans configuration with its phenolic oxygen deprotonated. For this paper, we studied four different configurations of pCA solvated in water with ab initio molecular dynamics simulations as implemented in CP2K/Quickstep. We researched the influence of the protonation and isomerization state of pCA on its hydrogen-bonding properties and on the Mulliken charges of the atoms in the simulation. The chromophore isomerization state influenced the hydrogen-bonding less than its protonation state. In general, more charge yielded a higher hydrogen-bond coordination number. Where deprotonation increases both the coordination number and the residence time of the water molecules around the chromophore, protonation showed a somewhat lower coordination number on two of the three pCA oxygens but much higher residence times on all of them. This could be explained by the increased polarization of the OH groups of the molecule. The presence of the chromophore also influenced the charge and polarization of the water molecules around it. This effect was different in the four systems studied and mainly localized in the first solvation shell. We also performed a proton-transfer reaction from hydronium through various other water molecules to the chromophore. In this small charge-separated system, the protonation occurred within 6.5 ps. We identified the transition state for the final step in this protonation series.     

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.     

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.     

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.