Structure Of Photoactive Yellow Protein Research Articles

Photoactive Yellow Protein Adsorption at Hydrated Polyethyleneimine and Poly-l-Glutamic Acid Interfaces.

Chiral and achiral vibrational sum-frequency generation (VSFG) spectroscopy was performed in the 1400-1700 and 2800-3800 cm-1 range to study the interfacial structure of photoactive yellow protein (PYP) adsorbed on polyethyleneimine (PEI) and poly-l-glutamic acid (PGA) surfaces. Nanometer-thick polyelectrolyte layers served as the substrate for PYP adsorption, with 6.5-pair layers providing the most homogeneous surfaces. When the topmost material was PGA, it acquired a random coil structure with a small number of β2-fibrils. Upon adsorption on oppositely charged surfaces, PYP yielded similar achiral spectra. However, the VSFG signal intensity increased for PGA surfaces with a concomitant redshift of the chiral Cα-H and N-H stretching bands, suggesting increased adsorption for PGA compared to PEI. At low wavenumbers, both the backbone and the side chains of PYP induced drastic changes to all measured chiral and achiral VSFG spectra. Decreasing ambient humidity led to the loss of tertiary structure with a re-orientation of α-helixes, evidenced by a strongly blue-shifted chiral amide I band of the β-sheet structure with a shoulder at 1654 cm-1. Our observations indicate that chiral VSFG spectroscopy is not only capable of determining the main type of secondary structure of PYP, i.e., β-scaffold, but is also sensitive to tertiary protein structure.

Analysis of Electronic Structure of Photoactive Yellow Protein by Canonical Molecular Orbitals Calculation

Analysis of Electronic Structure of Photoactive Yellow Protein by Canonical Molecular Orbitals Calculation

Visualization of H atoms in the X-ray crystal structure of photoactive yellow protein: Does it contain low-barrier hydrogen bonds?

The hydrogen bond (HB) between 4-hydroxycinnamic acid (HC4) and glutamic acid E46 of photoactive yellow protein is exceptionally strong. In the 0.82-å resolution X-ray structure for this protein (PDB ID: 1NWZ), the OH…O distance is only 2.57 å. The position of the H atom between these two O atoms has not been determined in that structure, and in the absence of that information, it is impossible to determine whether or not this HB is a low-barrier HB (LBHB), as was proposed recently based on neutron structures of this protein (Yamaguchi et al., Proceedings of the National Academy of Sciences of the United States of America, 2009, 106: 440-444). Residual electron density maps computed using the 1NWZ data reveal that this H atom is 0.92 å from the Oε2 atom of E46 and 1.67 å from the O4 ' of HC4, and that the OH…O bond angle is 167°. These observations indicate that E46 is protonated, and HC4 is deprotonated, as was originally suggested, and that the HB in question is not an LBHB.

Neutron crystallography of photoactive yellow protein reveals unusual protonation state of Arg52 in the crystal

Because of its high pKa, arginine (Arg) is believed to be protonated even in the hydrophobic environment of the protein interior. However, our neutron crystallographic structure of photoactive yellow protein, a light sensor, demonstrated that Arg52 adopts an electrically neutral form. We also showed that the hydrogen bond between the chromophore and Glu46 is a so-called low barrier hydrogen bond (LBHB). Because both the neutral Arg and LBHB are unusual in proteins, these observations remain controversial. To validate our findings, we carried out neutron crystallographic analysis of the E46Q mutant of PYP. The resultant structure revealed that the proportion of the cationic form is higher in E46Q than in WT, although the cationic and neutral forms of Arg52 coexist in E46Q. These observations were confirmed by the occupancy of the deuterium atom bound to the Nη1 atom combined with an alternative conformation of the N(η2)D2 group comprising sp2 hybridisation. Based on these results, we propose that the formation of the LBHB decreases the proton affinity of Arg52, stabilizing the neutral form in the crystal.

Active Site Structure of Photoactive Yellow Protein with a Locked Chromophore Analogue Revealed by Near-Infrared Raman Optical Activity

Many biological cofactors, such as light-absorbing chromophores in photoreceptors, are intrinsically planar molecules. A protein environment, however, causes structural distortions of the cofactor, and such structural changes can lead to a modulation of chemical properties of the cofactor to maximize its biological activity. Here, we investigate the active site structure of photoactive yellow protein (PYP), a blue light photoreceptor that contains a p-coumaric acid (pCA) chromophore, by a near-infrared excited Raman optical activity (ROA). Specifically, we measured the ROA spectra of PYP, whose chromophore is replaced with a locked pCA analogue. Furthermore, we show that a spectral analysis based on quantum mechanical/molecular mechanical (QM/MM) calculations of the whole protein molecule is useful to obtain structural information from the observed ROA spectra. The use of the near-infrared ROA combined with QM/MM calculations is a novel and generally applicable spectroscopic tool to study the chromophore distortions within a protein environment.

The Solution Structure of a Transient Photoreceptor Intermediate: Δ25 Photoactive Yellow Protein

The N-terminally truncated variant of photoactive yellow protein (Delta25-PYP) undergoes a very similar photocycle as the corresponding wild-type protein (WT-PYP), although the lifetime of its light-illuminated (pB) state is much longer. This has allowed determination of the structure of both its dark- (pG) as well as its pB-state in solution by nuclear magnetic resonance (NMR) spectroscopy. The pG structure shows a well-defined fold, similar to WT-PYP and the X-ray structure of the pG state of Delta25-PYP. In the long-lived photocycle intermediate pB, the central beta sheet is still intact, as well as a small part of one alpha helix. The remainder of pB is unfolded and highly flexible, as evidenced by results from proton-deuterium exchange and NMR relaxation studies. Thus, the partially unfolded nature of the presumed signaling state of PYP in solution, as suggested previously, has now been structurally demonstrated.

Structure of photoactive yellow protein (PYP) E46Q mutant at 1.2 Å resolution suggests how Glu46 controls the spectroscopic and kinetic characteristics of PYP

Photoactive yellow protein from Ectothiorhodospira halophila is a photoreceptor protein involved in the negative phototaxis of this bacterium. Its chromophore (p-coumaric acid) is deprotonated in the ground state, which is stabilized by a hydrogen-bond network between Tyr42, Glu46 and Thr50. Glu46 is a key residue as it has been suggested that the proton at Glu46 is transferred to the chromophore during its photoconversion from the dark state to the signalling state. The structure of E46Q mutant protein was determined at 1.2 A resolution, revealing that the phenolic O atom of p-coumaric acid is hydrogen bonded to NH(2) of Gln46 in E46Q with a longer distance (2.86 +/- 0.02 A) than its distance (2.51 A) to Glu46 OH in the wild type. This and the decreased thermal stability of E46Q relative to the wild type show that this hydrogen bond is weakened in the E46Q mutant compared with the corresponding bond in the wild type. Several characteristic features of E46Q such as an alkali shift in the pK(a) and the rapid photocycle can be explained by this weakened hydrogen bond. Furthermore, the red shift in the absorption maximum in E46Q can be explained by the delocalization of the electron on the phenolic oxygen of p-coumaric acid owing to the weakening of this hydrogen bond.

Measuring and modelling dynamical changes in the structure of photoactive yellow protein

Biological photoreceptors are very suitable for studies of the structure–function relationship in proteins. Of the many possible candidates, from at least six different families, we discuss here photoactive yellow protein (PYP, a member of the Xanthopsin family). The excellent physical and photochemical stability of PYP has allowed recording of a large amount of data relevant for this characteristic. The timescales relevant for functional transitions in PYP are most easily studied with transient spectroscopy. Application of the combination of UV/Vis and FTIR spectroscopy has revealed (besides multiple electronically excited states) the subsequent involvement of the following transient intermediates: I0, pR1, pR2, pB′, pB and pBdeprot. The structures of these transient intermediates have been characterized with general structural techniques like X-ray diffraction and NMR spectroscopy. Most extensive results have been obtained with X-ray diffraction, applied both in a time-resolved mode and by making use of low-temperature trapping. This has led to a detailed description of the reversible spatial changes in PYP that follow light activation. The structure of the transient intermediates, however, is exquisitely sensitive to constraints from the molecular environment of the protein, like the absence/presence of a crystalline lattice. Extrapolating these results to the signalling state of PYP in vivo is extremely complicated but also very challenging. Rather than measure, one can also simulate and/or calculate the spatial structures of transient intermediates of PYP. This challenge is most straightforwardly tackled with a detailed analysis of the intrinsic dynamics of the stable receptor state of PYP. Such studies have revealed striking similarities in the dynamics of all PAS domain proteins of which a spatial structure has been determined. The next challenge is to properly describe the change in configuration of the chromophore, induced by light, and to expand such simulations to the timescale relevant for completion of this photocycle.

Solution structure and backbone dynamics of the photoactive yellow protein.

The solution structure of photoactive yellow protein (PYP), a photosensory protein from Ectothiorhodospira halophila, has been determined by multidimensional NMR spectroscopy. The structure consists of an open, twisted, 6-stranded, antiparallel beta-sheet, which is flanked by four alpha-helices on both sides. The final set of 26 selected structures is well-defined for the regions spanning residues Phe6-Ala16, Asp24-Ala112, and Tyr118-Val125 and displays a root-mean-square deviation, versus the average, of 0.45 A for the backbone and 0.88 A for all heavy atoms. Comparison of the solution structure with an earlier published 1.4 A crystal structure (Borgstahl, G. E. O., Williams, D. R., and Getzoff, E. D. (1995) Biochemistry 34, 6278-6287) reveals a similarity with a root-mean-square deviation of 1.77 A for the backbone for the well-defined regions. The most distinct difference in the backbone with the crystal structure is found near the N-terminus, for residues Asp19-Leu23, which corresponds to an alpha-helix in the crystal structure and to one of the poorest defined regions in the solution structure. To characterize the dynamic behavior of PYP in solution, we undertook a 15N relaxation study and measurements of hydrogen/deuterium exchange. Determination of order parameters through the model-free Lipari-Szabo approach enabled the identification of several regions of enhanced dynamics. The comparison of atomic displacements in the backbone traces of the ensemble structures, with mobility measurements from NMR, show that the poorly defined regions feature fast internal motions in the nanosecond to picosecond time scale.

1.4 .ANG. Structure of Photoactive Yellow Protein, a Cytosolic Photoreceptor: Unusual Fold, Active Site, and Chromophore

A photosensing protein directs light energy captured by its chromophore into a photocycle. The protein's structure must accommodate the photocycle and promote the resulting chemical or conformational changes that lead to signal transduction. The 1.4 A crystallographic structure of photoactive yellow protein, determined by multiple isomorphous replacement methods, provides the first view at atomic resolution of a protein with a photocycle. The alpha/beta fold, which differs from the original chain tracing, shows striking similarity to distinct parts of the signal transduction proteins profilin and the SH2 domain. In the dark state structure of photoactive yellow protein, the novel 4-hydroxycinnamyl chromophore, covalently attached to Cys69, is buried within the major hydrophobic core of the protein and is tethered at both ends by hydrogen bonds. In the active site, the yellow anionic form of the chromophore is stabilized by hydrogen bonds from the side chains of Tyr42 and buried Glu46 to the phenolic oxygen atom and by electrostatic complementarity with the positively charged guanidinium group of Arg52. Thr50 further interlocks Tyr42, Glu46, and Arg52 through a network of active site hydrogen bonds. Arg52, located in a concavity of the protein surface adjacent to the dominant patch of negative electrostatic potential, shields the chromophore from solvent and is positioned to form a gateway for the phototactic signal. Overall, the high-resolution structure of photoactive yellow protein supports a mechanism whereby electrostatic interactions create an active site poised for photon-induced rearrangements and efficient protein-mediated signal transduction.