Stark Spectroscopy on Photoactive Yellow Protein, E46Q, and a Nonisomerizing Derivative, Probes Photo-Induced Charge Motion (original) (raw)
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Initial photoinduced dynamics of the photoactive yellow protein
2005
Abstract 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.
Biophysical Journal, 1994
The photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila was examined by time-resolved difference absorption spectroscopy in the wavelength range of 300-600 nm. Both time-gated spectra and single wavelength traces were measured. Global analysis of the data established that in the time domain between 5 ns and 2 s only two intermediates are involved in the room temperature photocycle of PYP, as has been proposed before (Meyer T. E., E. Yakali, M. . 1991. Biophys. J. 59:988-991). The first, red-shifted intermediate decays biexponentially (60% with T = 0.25 ms and 40% with T = 1.2 ms) to a blue-shifted intermediate. The last step of the photocycle is the biexponential (93% with T = 0.15 s and 7% with T = 2.0 s) recovery to the ground state of the protein. Reconstruction of the absolute spectra of these photointermediates yielded absorbance maxima of about 465 and 355 nm for the red-and blue-shifted intermediate with an Emax at about 50% and 40% relative to the Emax of the ground state. The quantitative analysis of the photocycle in PYP described here paves the way to a detailed biophysical analysis of the processes occurring in this photoreceptor molecule.
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.
The Journal of Physical Chemistry B, 2004
The changes in the electrostatic properties, between the ground and excited state, of thiomethyl p-coumaric acid (TMpCA) and its sterically hindered derivative, thiomethyl-7-hydroxy-coumarin-3-carboxylic acid (TM7HC), have been determined at 77 K using Stark spectroscopy, to better understand the origin of the photoinduced charge motion observed in these chromophores in the native photoactive yellow protein (PYP) environment. Excitation of the anionic chromophores produce changes in the permanent dipole moment (|∆µ b|) of 25 (TMpCA-a) and 15 D (TM7HC-a), which are significantly larger than the |∆µ b|'s measured in the neutral species: 9 (TMpCA-n) and 6 D (TM7HC-n). However, the similarity of the |∆µ b|'s between the anions and the corresponding de-protonated cofactors in the native protein environment implicates the intrinsic electronic properties of the chromophore for the photoreactivity of the initially excited species in the PYP photocycle. Furthermore, the results for the neutral species suggest that, if the cofactor in the protein were to be protonated in the ground state, photon absorption would induce a much smaller degree of charge motion. The implications of these distinct differences in the measured electrostatic properties are discussed in the context of facilitating and/or preventing the twisting of the chromophore and its relevance to the PYP photocycle. Ab initio (time dependent density functional, TDDFT) calculations on these systems yield quite accurate values for the electronic transition energies, and the molecular orbitals that contribute to these transitions provide an insight into the reactivity of the excited-state species. However, the changes in the permanent dipole moments associated with these transitions are underestimated, particularly in the anions, both from Configuration Interactions -Singles and Restricted Open-shell Kohn-Sham calculations.
Biophysical Journal, 2005
Stark (electroabsorption) spectra of the M100A mutant of photoactive yellow protein reveal that the neutral, cis cofactor of the pB intermediate undergoes a strikingly large change in the static dipole moment (jDm mj ¼ 19 Debye) on photon absorption. The formation of this charge-separated species, in the excited state, precedes the cis / trans isomerization of the pB cofactor and the regeneration of pG. The large jDm mj, reminiscent of that produced on the excitation of pG, we propose, induces twisting of the cis cofactor as a result of translocation of negative charge, from the hydroxyl oxygen, O1, toward the C7-C8 double bond. The biological significance of this photoinduced charge transfer reaction underlies the significantly faster regeneration of pG from pB in vitro, on the absorption of blue light.
Biophysical journal, 1995
The photocycle of the photoactive yellow protein (PYP) isolated from Ectothiorhodospira halophila was analyzed by flash photolysis with absorption detection at low excitation photon densities and by temperature-dependent laser-induced optoacoustic spectroscopy (LIOAS). The quantum yield for the bleaching recovery of PYP, assumed to be identical to that for the phototransformation of PYP (pG), to the red-shifted intermediate, pR, was phi R = 0.35 +/- 0.05, much lower than the value of 0.64 reported in the literature. With this value and the LIOAS data, an energy content for pR of 120 kJ/mol was obtained, approximately 50% lower than for excited pG. Concomitant with the photochemical process, a volume contraction of 14 ml/photoconverted mol was observed, comparable with the contraction (11 ml/mol) determined for the bacteriorhodopsin monomer. The contraction in both cases is interpreted to arise from a protein reorganization around a phototransformed chromophore with a dipole moment d...
Protein-chromophore interactions in photoreceptors often shift the chromophore absorbance maximum to a biologically relevant spectral region. A fundamental question regarding such spectral tuning effects is how the electronic ground state S 0 and excited state S 1 are modified by the protein. It is widely assumed that changes in energy gap between S 0 and S 1 are the main factor in biological spectral tuning. We report a generally applicable approach to determine if a specific residue modulates the energy gap, or if it alters the equilibrium nuclear geometry or width of the energy surfaces. This approach uses the effects that changes in these three parameters have on the absorbance and fluorescence emission spectra of mutants. We apply this strategy to a set of mutants of photoactive yellow protein (PYP) containing all 20 side chains at active site residue 46. While the mutants exhibit significant variation in both the position and width of their absorbance spectra, the fluorescence emission spectra are largely unchanged. This provides strong evidence against a major role for changes in energy gap in the spectral tuning of these mutants and reveals a change in the width of the S 1 energy surface. We determined the excited state lifetime of selected mutants and the observed correlation between the fluorescence quantum yield and lifetime shows that the fluorescence spectra are representative of the energy surfaces of the mutants. These results reveal that residue 46 tunes the absorbance spectrum of PYP largely by modulating the width of the S 1 energy surface. photoreceptor | protein-chromophore interactions | wavelength regulation L ight-driven proteins, consisting of a protein-chromophore complex, employ two general strategies to optimize the biological effectiveness of the position of their absorbance spectra. First, covalent modifications of a chromophore can shift its absorbance maximum λ abs max. An important example is the strong red-shift in the absorbance spectrum of bacteriochlorophyll compared to plant chlorophyll (1). Second, specific protein-chromo-phore interactions can shift the absorbance spectrum of the protein-bound chromophore. A classic example of this spectral tuning phenomenon is color vision in vertebrates (2, 3): the same retinal chromophore can absorb in the blue, green, and red, depending on the amino acid sequence of the rhodopsin to which it is bound. Spectral tuning provides an example of protein-ligand interactions that tune the properties of the cofactor to biologically relevant values. Two main approaches have been used to unravel the factors involved in spectral tuning: determining (i) which amino acids in the protein contribute to spectral tuning, and (ii) what type of protein-chromophore interactions cause spectral tuning. Interactions that alter the degree of charge delocalization over the chromophore are of particular importance. These two issues have been explored extensively in animal visual rhodopsins and the ar-chaeal rhodopsins (4-7). Here we study spectral tuning in a more recently discovered photoreceptor, photoactive yellow protein (PYP) (8, 9), and use it to develop and explore a third approach to investigate spectral tuning. Central in this approach are the effects of an individual residue on the shape of the S 0 ground state and S 1 excited state energy surfaces. PYP offers a highly accessible system (10) to study spectral tuning. We use it here to test a widely used assumption that has dominated the field of biological spectral tuning: that changes in the energy gap ΔE between the S 0 and S 1 energy surfaces are the major factor in spectral tuning. PYP is a blue light receptor from the halophilic photosynthetic eubacterium Halorhodospira halophila (8, 9). It functions as the photoreceptor for negative phototaxis in H. halophila (11). The purified protein exhibits a photocycle (9, 12) initiated by the photoisomerization (13) of its p-coumaric acid (pCA) chromo-phore (14, 15) by a flip in the position of its carbonyl group (16). PYP consists of an antiparallel 6-stranded β-sheet flanked by five α-helices (17). Its pCA chromophore is fully buried within the protein and its deprotonated phenolic oxygen forms short hydrogen bonds with the side chains of Tyr42 and Glu46 (Fig. 1A). Site-directed mutagenesis studies (summarized in ref. 18) have identified Glu46 as a key factor in the spectral tuning of PYP (19-21). Three major factors contribute to spectral tuning in PYP (22) (Fig. 1B). First, the formation of the thioester bond between the pCA and Cys69 results in a red-shift from 284 to 335 nm. Second, the deprotonation of the phenolic oxygen of the pCA causes a further red-shift to 400 nm. The strong downshift in the pK a of the pCA from 8.8 in solution (22) to 2.8 in PYP (8, 23) ensures that the chromophore in PYP is deprotonated under physiological conditions. Finally, specific protein-chromophore interactions result in the final red-shift to 446 nm. These interactions have been studied by both site-directed mutagenesis (18-21) and computational approaches (24-27) (SI Text). These studies have revealed Tyr42 and Glu46 as the two dominant side chains in the spectral tuning of PYP (18-21, 24-28). Weakening of the Glu46-pCA hydrogen bond (29, 30) in the E46Q mutant results in a red-shift to 460 nm, while complete disruption of the hydrogen bond in the E46V mutant results in a further red-shift to 478 nm (21). Based on computational studies it has been proposed that hydrogen bonding, charge-charge interactions, and burial of the pCA in the hydrophobic protein interior are factors in the spectral tuning of PYP (24, 25). Here we propose a straightforward approach to probe if spectral tuning alters not only the ΔE but also the equilibrium nuclear geometry R e or the width W of the S 0 and S 1 energy surfaces. We