Probing the Photochemical Mechanism in Photoactive Yellow Protein (original) (raw)
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Biophysical Journal, 2011
Prior experimental observations, as well as theoretical considerations, have led to the proposal that C 4 -C 7 singlebond rotation may play an important role in the primary photochemistry of photoactive yellow protein (PYP). We therefore synthesized an analog of this protein's 4-hydroxy-cinnamic acid chromophore, (5-hydroxy indan-(1E)-ylidene)acetic acid, in which rotation across the C 4 -C 7 single bond has been locked with an ethane bridge, and we reconstituted the apo form of the wild-type protein and its R52A derivative with this chromophore analog. In PYP reconstituted with the rotation-locked chromophore, 1), absorption spectra of ground and intermediate states are slightly blue-shifted; 2), the quantum yield of photochemistry is~60% reduced; 3), the excited-state dynamics of the chromophore are accelerated; and 4), dynamics of the thermal recovery reaction of the protein are accelerated. A significant finding was that the yield of the transient ground-state intermediate in the early phase of the photocycle was considerably higher in the rotation-locked samples than in the corresponding samples reconstituted with p-coumaric acid. In contrast to theoretical predictions, the initial photocycle dynamics of PYP were observed to be not affected by the charge of the amino acid residue at position 52, which was varied by 1), varying the pH of the sample between 5 and 10; and 2), site-directed mutagenesis to construct R52A. These results imply that C 4 -C 7 single-bond rotation in PYP is not an alternative to C 7 ¼C 8 double-bond rotation, in case the nearby positive charge of R52 is absent, but rather facilitates, presumably with a compensatory movement, the physiological Z/E isomerization of the blue-light-absorbing chromophore.
Journal of the American Chemical Society, 2005
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 polarizationsensitive ultrafast infrared spectroscopy and density functional theory calculations. We identify the νC 8 -C 9 marker modes for solution phase PYP in the P and I 0 states, from which we derive that the first intermediate state I 0 , 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 I 0 correlates with the conversion from the trans to the cis state. For the P and I 0 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 to have a strong influence. The C 7 =C 8 -(C 9 =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 a X-ray diffraction study of PYP crystals. In the case of I 0 , experiment and theory are only in agreement when the C 7 =C 8 -(C 9 =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 in going from the electronic ground P state, via the electronic excited P* state to the intermediate I 0 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.
Physical chemistry chemical physics : PCCP, 2014
The light-activated photoactive yellow protein (PYP) chromophore uses a series of reactions to trigger photo-motility and biological responses, and generate a wide range of structural signals. To provide a comprehensive mechanism of the overall process at the atomic level, we apply a CASPT2//CASSCF/AMBER QM/MM protocol to investigate the relaxation pathways for a variety of possible isomerization and proton transfer reactions upon photoexcitation of the wild-type PYP. The nonadiabatic relay through an S1/S0 conical intersection [CI(S1/S0)] is found to play a decisive major role in bifurcating the excited state relaxation into a complete and short photocycle. Two major and one minor deactivation channels were found starting from the CI(S1/S0)-like intermediate IT, producing the cis isomers pR1, ICP, and ICT through "hula twist", "bicycle pedal" and one-bond flip isomerization reactions. The overall photocycle can be achieved by competitive parallel/sequential reac...
Structure, 2004
goes a fully reversible, light-induced modification of its chromophore . PYP is a bacterial photoreceptor initially isolated from Halorhodospira halophila (Meyer, 1985), which is believed to be involved in a negative phototactic response to blue light . After absorbing a blue light photon, the covalently attached 3 Consortium for Advanced Radiation Sources University of Chicago coumaric acid chromophore undergoes trans to cis isomerization, which initiates a fully reversible photocy-Chicago, Illinois 60637 4 National Institutes of Health cle that lasts on the order of 1 s . The photon energy is transduced into a structural signal as the mole-Bethesda, Maryland 20892 5 European Synchrotron Radiation Facility cule thermally relaxes through a series of spectroscopically distinguishable intermediates, in which the final Grenoble Cedex 9 France two intermediates are denoted pR and pB (alternatively, I1 and I2). The lifetimes of successive intermediates progressively increase throughout the photocycle, with that of pR being 052ف s and of the final intermediate, Summary pB, 051ف ms (Hoff et al., 1994). Although these intermediates are spectroscopically homogeneous, biphasic
Electronic structure and dynamics of torsion-locked photoactive yellow protein chromophores
Physical chemistry chemical physics : PCCP, 2017
The photocycle of photoactive yellow protein (PYP) begins with small-scale torsional motions of the chromophore leading to large-scale movements of the protein scaffold triggering a biological response. The role of single-bond torsional molecular motions of the chromophore in the initial steps of the PYP photocycle are not fully understood. Here, we employ anion photoelectron spectroscopy measurements and quantum chemistry calculations to investigate the electronic relaxation dynamics following photoexcitation of four model chromophores, para-coumaric acid, its methyl ester, and two analogues with aliphatic bridges hindering torsional motions around the single bonds adjacent to the alkene group. Following direct photoexcitation of S1 at 400 nm, we find that both single bond rotations play a role in steering the PYP chromophore through the S1/S0 conical intersection but that rotation around the single bond between the alkene moiety and the phenoxide group is particularly important. F...
Biochemistry, 2002
Photoactive yellow protein (PYP) is a bacterial photoreceptor containing a 4-hydroxycinnamyl chromophore. Photoexcitation of PYP triggers a photocycle that involves at least two intermediate states: an early red-shifted PYP L intermediate and a long-lived blue-shifted PYP M intermediate. In this study, we have explored the active site structures of these intermediates by resonance Raman spectroscopy. Quantum chemical calculations based on a density functional theory are also performed to simulate the observed spectra. The obtained structure of the chromophore in PYP L has cis configuration and no hydrogen bond at the carbonyl oxygen. In PYP M , the cis chromophore is protonated at the phenolic oxygen and forms the hydrogen bond at the carbonyl group. These results allow us to propose structural changes of the chromophore during the photocycle of PYP. The chromophore photoisomerizes from trans to cis configuration by flipping the carbonyl group to form PYP L with minimal perturbation of the tightly packed protein interior. Subsequent conversion to PYP M involves protonation on the phenolic oxygen, followed by rotation of the chromophore as a whole. This large motion of the chromophore is potentially correlated with the succeeding global conformational changes in the protein, which ultimately leads to transduction of a biological signal.
1H, 13C, and 15N resonance assignment of photoactive yellow protein
Biomolecular NMR Assignments, 2012
Photoactive yellow protein (PYP) is involved in the negative phototactic response towards blue light of the bacterium Halorhodospira halophila. Here, we report nearly complete backbone and side chain 1 H, 13 C and 15 N resonance assignments at pH 5.8 and 20°C of PYP in its electronic ground state. Keywords PYP Á Halorhodospira halophila Á paracoumaric acid Á NMR spectroscopy Á Photoactivation Biological context Photoactive yellow protein (PYP) is a 125 amino acid (14 kDa) water-soluble, blue-light sensor protein, first found in the halophilic bacterium Halorhodospira halophila (Meyer 1985). PYP is a photoreceptor, believed to be responsible for the negative phototactic response of its host organism (Sprenger et al. 1993). This kind of response is required for organisms to evade potentially harmful short-wavelength light. Based on this observation, PYP has become a suitable model to understand the signal-transduction mechanism in Per-Arnt-Sim (PAS) domain signaling (Crosthwaite et al. 1997; Nambu et al. 1991). Several PYP-like proteins have meanwhile been found in other organisms, where they are also thought to act as light sensors. In addition, PYP-like proteins found in purple bacteria are involved in cell buoyancy or sensing bacteriophytochromes (Jiang et al. 1999; Kyndt et al. 2004). Understanding of light transduction in PYP requires structural information in atomic detail. A 1.4 Å crystallographic structure was determined in 1995 by Borgstahl et al. and in 1998 Düx and coworkers revealed the solution structure and backbone dynamics of PYP by NMR spectroscopy. The reaction center of PYP is protected from solvent by R52, which is believed to function as a gateway in the photocycle (Borgstahl et al. 1995; Genick et al. 1997). The chromophore, para-coumaric acid (pCA), is covalenty bound to C69 with a thioester bond and pCA participates in two short hydrogen bonds with E46 and Y42 to stabilize the negative charge of pCA in the electronic ground state, pG. Upon blue-light capture, the chromophore undergoes transcis isomerisation and the intermediate pR is formed, which subsequently relaxes to the proposed signaling state, pB. In the latter state, the reaction center is exposed and the two short hydrogen bonds are broken (Borgstahl et al. 1995; Sigala et al. 2009; Yamaguchi et al. 2009). In this paper, we present the nearly complete assignment of the backbone and side chain resonances of the pG state of PYP. Methods and experiments Uniformly 13 C, 15 N-labeled wild type PYP was overexpressed and purified as described previously (Düx et al.
Proceedings of the National Academy of Sciences, 1998
The chromophore of photoactive yellow protein (PYP) (i.e., 4-hydroxycinnamic acid) has been replaced by an analogue with a triple bond, rather than a double bond (by using 4-hydroxyphenylpropiolic acid in the reconstitution, yielding hybrid I) and by a ''locked'' chromophore (through reconstitution with 7-hydroxycoumarin-3-carboxylic acid, in which a covalent bridge is present across the vinyl bond, resulting in hybrid II). These hybrids absorb maximally at 464 and 443 nm, respectively, which indicates that in both hybrids the deprotonated chromophore does fit into the chromophorebinding pocket. Because the triple bond cannot undergo cis͞trans (or E͞Z) photoisomerization and because of the presence of the lock across the vinyl double bond in hybrid II, it was predicted that these two hybrids would not be able to photocycle. Surprisingly, both are able. We have demonstrated this ability by making use of transient absorption, low-temperature absorption, and Fourier-transform infrared (FTIR) spectroscopy. Both hybrids, upon photoexcitation, display authentic photocycle signals in terms of a red-shifted intermediate; hybrid I, in addition, goes through a blueshifted-like intermediate state, with very slow kinetics. We interpret these results as further evidence that rotation of the carbonyl group of the thioester-linked chromophore of PYP, proposed in a previous FTIR study and visualized in recent time-resolved x-ray diffraction experiments, is of critical importance for photoactivation of PYP.
Structural and dynamic changes of photoactive yellow protein during its photocycle in solution
Nature structural biology, 1998
Light irradiation of photoactive yellow protein (PYP) induces a photocycle, in which red-shifted (pR) and blue-shifted (pB) intermediates have been characterized. An NMR study of the long-lived pB intermediate now reveals that it exhibits a large degree of disorder and exists as a family of multiple conformers that exchange on a millisecond time scale. This shows that the behavior of PYP in solution is different from what has been observed in the crystalline state. Furthermore, differential refolding to ground state pG is observed, whereby the central beta-sheet and parts of the helical structure are formed first and the region around the chromophore at a later stage.
Functional Tuning of Photoactive Yellow Protein by Active Site Residue 46
Protein-ligand interactions alter the properties of active site groups to achieve specific biological functions. The active site of photoactive yellow protein (PYP) provides a model system for studying such functional tuning. PYP is a small bacterial photoreceptor with photochemistry based on its p-coumaric acid (pCA) chromophore. The absorbance maximum and pK a of the pCA in the active site of native PYP are shifted from 400 nm and 8.8 in water to 446 nm and 2.8 in the native protein milieu, respectively, by protein-ligand interactions. We report high-throughput microscale methods for the purification and spectroscopic investigation of PYP and use these to examine the role of active site residue Glu46 in PYP, which is hydrogen bonded to the pCA anion. The functional and structural attributes of the 19 substitution mutants of PYP at critical active site position 46 vary widely, with absorbance maxima from 441 to 478 nm, pCA fluorescence quantum yields from 0.19 to 1.4%, pCA pK a values from 3.0 to 9.0, and protein folding stabilities from 6.5 to 12.9 kcal/mol. The kinetics of the last photocycle transition vary by more than 4 orders of magnitude and are often strongly biphasic. Only E46Q PYP exhibits a greatly accelerated photocycling rate. All substitutions yield a folded, photoactive PYP, illustrating the robustness of protein structure and function. Correlations between side chain and mutant properties establish the importance of residue 46 in tuning the function of PYP and the significance of the strength of its hydrogen bond to the pCA. Native PYP exhibits the lowest values for pCA fluorescence quantum yield and pK a , indicating their functional relevance. These results demonstrate the value of quantitative high-throughput biophysical studies of proteins. Photoactive yellow protein (PYP) 1 from Halorhodospira halophila is a cytosolic photoreceptor (1, 2) that mediates the host organism's negative phototaxis in response to blue light (3). The biochemical and biophysical properties of PYP have been studied extensively (4-6) due to the rich biophys-ics of its light-triggered function and its extraordinary ease of handling, including its solubility (1), thermostability (2), and excellent overexpression in Escherichia coli (7, 8). The protein interacts with light via a p-coumaric acid (pCA) chromophore covalently bound to Cys69 (9, 10). Absorption of a photon by the initial pG state of PYP causes pCA trans-cis isomerization that triggers a complex photocycle (2, 11), resulting in the formation a photoactivated state, pB, that presumably has a downstream effect on cell motility. In the pG state, the pCA is deprotonated and in the trans conformation (10, 12). At neutral pH, the pB state decays back to the initial pG state in approximately 250 ms (2, 8, 11). The crystal structure of PYP (13) provides detailed information about the side chains interacting with the pCA chromophore (Figure 1). The side chains Glu46 and Tyr42 form hydrogen bonds to the phenolic oxygen of the pCA, thus stabilizing its buried charge. Mutagenesis of Glu46 and Tyr42 significantly alters the properties of PYP, confirming the importance of these two side chains. Residue 46 is known to be involved in tuning the absorbance maximum (λ max) and pK a of the pCA chromophore, and the photocycle kinetics in PYP, based on studies of the E46Q, E46A, and E46D mutants (8, 14-16). In addition, it has a very short hydrogen bond to the pCA (17) (Figure 1b), has been identified as the proton donor for the ionized pCA during the PYP photocycle (18-20), and has been uncovered as a key factor in triggering protein conformational changes upon pB formation (18, 20, 21). The importance of Glu46 is also supported by the high degree of conservation of this residue within the PYP family of proteins (7, 22). The properties of the pCA chromophore in PYP are greatly shifted compared to their values in water. In solution, pCA has a λ max at 286 nm (at neutral pH) and a pK a of 8.8 (10, 23). Within PYP, the pCA has its λ max shifted to 446 nm and its pK a shifted to 2.8 (1, 24, 25). In addition, the photochemical quantum yield is high (approximately 50%) (26, 27), while its fluorescence quantum yield is low (0.19%) (28), indicating that the PYP binding pocket promotes photochemistry over fluorescence. These tuning phenomena illustrate a common † λ max , absorbance maximum; Φ fl , fluorescence quantum yield; E46Q Ht , mutant of His-tagged PYP in which Glu46 is substituted with glutamine; E46X, library of 20 PYP derivatives in which residue 46 is substituted with each of the 20 side chains; ∆G°U, free energy difference between the folded and unfolded state; Gdm-HCl, guanidinium hydrochloride; pB, blue-shifted long-lived photocycle intermediate; pB dark , blue-shifted acid-denatured state of PYP; pCA, p-coumaric acid; PYP, photoactive yellow protein; pG, ground state of PYP; wtPYP nHt , wild-type PYP without a His tag.