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At least 19 recordsLinked to original sources
PubMed1996

PHH1, a novel gene from Arabidopsis thaliana that encodes a protein similar to plant blue-light photoreceptors and microbial photolyases.

A cDNA from Arabidopsis thaliana similar to microbial photolyase genes, and designated AT-PHH1, was isolated using a photolyase-like cDNA from Sinapsis alba (SA-PHR1) as a probe. Multiple isolations yielded only PHH1 cDNAs, and a few blue-light-receptor CRY1 (HY4) cDNAs (also similar to microbial photolyase genes), suggesting the absence of any other highly similar Arabidopsis genes. The AT-PHH1 and SA-PHR1 cDNA sequences predict 89% identity at the protein level, except for an AT-PHH1 C-terminal extension (111 amino acids), also not seen in microbial photolyases. AT-PHH1 and CRY1 show less similarity (54% p4erein identity), including respective C-terminal extensions that are themselves mostly dissimilar. Analysis of fifteen AT-PHH1 genomic isolates reveals a single gene, with three introns in the coding sequence and one in the 5'-untranslated leader. Full-length AT-PHH1, and both AT-PHH1 and AT-PHH1 delta C-513 (truncated to be approximately the size of microbial photolyase genes) cDNAs, were overexpressed, respectively, in yeast and Escherichia coli mutants hypersensitive to ultraviolet light. The absence of significant effects on resistance suggests either that any putative AT-PHH1 DNA repair activity requires cofactors/chromophores not present in yeast or E. coli, or that AT-PHH1 encodes a blue-light/ultraviolet-A receptor rather than a DNA repair protein.

Amino Acid Sequence↗
PubMed2005

Phylogenetic analysis of the phytochrome superfamily reveals distinct microbial subfamilies of photoreceptors.

Phys (phytochromes) are a superfamily of photochromic photoreceptors that employ a bilin-type chromophore to sense red and far-red light. Although originally thought to be restricted to plants, accumulating genetic and genomic analyses now indicate that they are also prevalent among micro-organisms. By a combination of phylogenetic and biochemical studies, we have expanded the Phy superfamily and organized its members into distinct functional clades which include the phys (plant Phys), BphPs (bacteriophytochromes), Cphs (cyanobacterial Phys), Fphs (fungal Phys) and a collection of Phy-like sequences. All contain a signature GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain, which houses the bilin lyase activity. A PHY domain (uppercase letters are used to denote the PHY domain specifically), which helps stabilize the Pfr form (far-red-light-absorbing form of Phy), is downstream of the GAF region in all but the Phy-like sequences. The phy, Cph, BphP and Fph families also include a PLD [N-terminal PAS (Per/Arnt/Sim)-like domain] upstream of the GAF domain. Site-directed mutagenesis of conserved residues within the GAF and PLD motifs supports their importance in chromophore binding and/or spectral activity. In agreement with Lamparter, Carrascal, Michael, Martinez, Rottwinkel and Abian [(2004) Biochemistry 43, 3659-3669], a conserved cysteine within the PLD of several BphPs was found to be necessary for binding the chromophore via the C-3 vinyl side chain on the bilin A ring. Phy-type sequences were also discovered in the actinobacterium Kineococcus radiotolerans and collections of microorganisms obtained from marine and extremely acidic environments, thus expanding further the range of these photoreceptors. Based on their organization and distribution, the evolution of the Phy superfamily into distinct photoreceptor types is proposed.

Amino Acid Sequence↗
PubMed1998

Molecular dynamics study of femtosecond events in photoactive yellow protein after photoexcitation of the chromophore.

Molecular dynamics simulations were carried out to study what happens in a photoreceptor protein, photoactive yellow protein (PYP), immediately after the vertical transition of the chromophore from the ground to the excited state. A photon absorption simulation was performed to investigate the movement of amino acid residues upon photoexcitation. To calculate the excited state of the chromophore, SCF-CI calculation was carried out with INDO/S Hamiltonian. We observed that some amino acid residues have strong interactions with the chromophore. Most of these amino acid residues are conserved in PYPs from three different species of bacteria. This observation indicates the biological importance of these residues.

Amino Acid Sequence↗
PubMed2001

Examples of hula-twist in photochemical cis- trans isomerization.

ALiu and Hammond recently reasoned that the hula-twist (HT), a volume-conserving cis-trans isomerization mechanism, is involved in reactions of confined systems. We now show that HT can be applied to various reported photochemical isomerization of chromophores (small organic systems as well as photoactive bio-pigments). The results, when taken as a whole, argue powerfully that HT is a common supramolecular photoisomerization reaction mechanism.

Bacterial Proteins↗
PubMed2006

The trans-cis isomerization reaction dynamics in sensory rhodopsin II by femtosecond time-resolved midinfrared spectroscopy: chromophore and protein dynamics.

Transient infrared (IR) vibrational spectroscopy at subpicosecond time resolution on sensory rhodopsin II from Natronomonas pharaonis, NpSRII, has been performed for the first time. The experiments yield three time constants for the description of the primary photoinduced reaction dynamics, i.e. 0.5, 3.7-4.4, and 11 ps. The data are consistent with a sequential reaction scheme, with the isomerization taking place within 0.5 ps, succeeded by an electronic ground state relaxation. The 11 ps component, observed at 1550 and 1530 cm(-1), is attributed to dynamics of protein vibrational bands, possibly amide II bands of the protein backbone, perturbed by the ultrafast retinal photoisomerization. Similar observations, yet not as strongly expressed, have been made earlier in bacteriorhodopsin and halorhodopsin.

Bacteriorhodopsins↗
PubMed2005

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.

Absorption↗
PubMed1988

Isolation of a prokaryotic photoreceptor: sensory rhodopsin from halobacteria.

The photoreceptor sensory rhodopsin was isolated from halobacterial cell membranes solubilized in laurylmaltoside. In the presence of retinal, detergent and salt the native protein was obtained in pure form by sucrose density gradient centrifugation, hydroxyapatite chromatography and gel filtration. The apparent mol. wt of the molecule was 24 kd if analyzed by SDS gel electrophoresis, and 49 kd by sedimentation and size-exclusion chromatographic analysis. The chromoprotein had an absorption maximum at 580 nm which was 8 nm blue-shifted compared to the membrane-bound state. The molecule was photochemically active and the action spectrum for formation of SR380, the long-lived intermediate, coincided with the absorption spectrum.

Centrifugation, Density Gradient↗
PubMed1993

Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila, with evidence for the mass and the binding site of the chromophore.

The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP+ ++ CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/- 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.

Amino Acid Sequence↗
PubMed2002

Signal transduction in the photoactive yellow protein. II. Proton transfer initiates conformational changes.

Molecular dynamics simulation techniques, together with semiempirical PM3 calculations, have been used to investigate the effect of photoisomerization of the 4-hydroxy-cinnamic acid chromophore on the structural properties of the photoactive yellow protein (PYP) from Ectothiorodospira halophila. In this bacteria, exposure to blue light leads to a negative photoactic response. The calculations suggest that the isomerization does not directly destabilize the protein. However, because of the isomerization, a proton transfer from a glutamic acid residue (Glu46) to the phenolate oxygen atom of the chromophore becomes energetically favorable. The proton transfer initiates conformational changes within the protein, which are in turn believed to lead to signaling.

Bacterial Proteins↗
PubMed2002

Signal transduction in the photoactive yellow protein. I. Photon absorption and the isomerization of the chromophore.

Molecular dynamics simulation techniques together with time-dependent density functional theory calculations have been used to investigate the effect of photon absorption by a 4-hydroxy-cinnamic acid chromophore on the structural properties of the photoactive yellow protein (PYP) from Ectothiorodospira halophila. The calculations suggest that the protein not only modifies the absorption spectrum of the chromophore but also regulates the subsequent isomerization of the chromophore by stabilizing the isomerization transition state. Although signaling from PYP is thought to involve partial unfolding of the protein, the mechanical effects accompanying isomerization do not appear to directly destabilize the protein.

Bacterial Proteins↗
PubMed2004

Role of protein in the primary step of the photoreaction of yellow protein.

We show the unexpectedly important role of the protein environment in the primary step of the photoreaction of the yellow protein after light illumination. The driving force of the trans-to-cis isomerization reaction was analyzed by a computational method. The force was separated into two different components: the term due to the protein-chromophore interaction and the intrinsic term of the chromophore itself. As a result, we found that the contribution from the interaction term was much greater than that coming from the intrinsic term. This accounts for the efficiency of the isomerization reaction in the protein environment in contrast to that in solution environments. We then analyzed the relaxation process of the chromophore on the excited-state energy surface and compared the process in the protein environment and that in a vacuum. Based on this analysis, we found that the bond-selectivity of the isomerization reaction also comes from the interaction between the chromophore and the protein environment.

Bacterial Proteins↗
PubMed2004

Direct measure of functional importance visualized atom-by-atom for photoactive yellow protein: application to photoisomerization reaction.

Photoreceptor proteins serve as efficient nano-machines for the photoenergy conversion and the photosignal transduction of living organisms. For instance, the photoactive yellow protein derived from a halophilic bacterium has the p-coumaric acid chromophore, which undergoes an ultrafast photoisomerization reaction after light illumination. To understand the structure-function relationship at the atomic level, we used a computational method to find functionally important atoms for the photoisomerization reaction of the photoactive yellow protein. In the present study, a "direct" measure of the functional significance was quantitatively evaluated for each atom by calculating the partial atomic driving force for the photoisomerization reaction. As a result, we revealed the reaction mechanism in which the specific role of each functionally important atom has been well characterized in a systematic manner. In addition, we observed that this mechanism is strongly conserved during the thermal fluctuation of the photoactive yellow protein. We compared the experimental data of fluorescence decay constant of several different mutants and the present analysis. As a result, we found that the reaction rate constant is decreased when a large positive driving force is missing.

Bacterial Proteins↗
PubMed1993

Transient proton uptake and release is associated with the photocycle of the photoactive yellow protein from the purple phototrophic bacterium Ectothiorhodospira halophila.

Upon excitation by a laser flash at 445 nm, the photoactive yellow protein (PYP) undergoes a bleach and red-shift occurring in less than 10 ns, undergoes a further bleach in approximately 200 microseconds, and then recolors in approximately 200 ms. A conformational change occurs during photobleaching which exposes a hydrophobic site. We have now shown that this photocycle also involves a net uptake of one proton during formation of the fully bleached second intermediate, followed by an equivalent proton release upon return of PYP to the ground state. Proton uptake lags slightly behind PYP bleaching and is first-order, indicating that the protein conformational change occurs in two steps. The results suggest that a basic residue which is normally buried in the protein interior is transiently exposed to solvent during the PYP photocycle and, as a consequence, undergoes a change in pK. On the basis of the crystal structure of PYP, we propose that this basic residue is lysine 111.

Bacterial Proteins↗
PubMed1997

Preparation and properties of a 3,4-dihydroxycinnamic acid chromophore variant of the photoactive yellow protein.

Native photoactive yellow protein (PYP) is reversibly bleached by laser excitation at the 446-nm wavelength maximum, during which the trans-4-hydroxycinnamic acid chromophore (covalently bound via a thioester to Cys 69) is isomerized, causing the protein to undergo a conformational change. We have reconstituted the holoprotein from recombinant apoprotein plus thiophenol thioester-activated chromophore and have also successfully attached a synthetic 3,4-dihydroxycinnamic acid chromophore and purified the resultant variant. The reconstituted recombinant protein has the same spectral and photochemical properties as the native protein. However, the absorption maximum of the protein with the dihydroxy chromophore variant is red-shifted to 458 nm, with an additional shoulder at about 342 nm. Following a laser flash, the rate constants for the first phase of bleaching in both the native and the variant proteins are too large to measure with the present apparatus. The second bleaching phase is only marginally accessible in the variant and has a rate constant (k approximately 2.3 x 10(4) s-1) at least an order of magnitude larger than that of the native PYP. In contrast, the rate constant for recovery of absorbance in the variant (k approximately 0.15 s-1) is about 40-fold smaller than for native PYP and is insensitive to pH (the native protein has a biphasic 16-fold variation in rate constant with pH). We previously observed similar changes in kinetic rate constants for protein denatured by urea or alcohols, which suggests that the dihydroxy protein is less stable than the native PYP. This was confirmed by measurement of protein unfolding in guanidine hydrochloride. We conclude from these results that the binding site is too small to accommodate the dihydroxybenzene ring of the variant chromophore without introducing strain into the protein, which is then reflected in the kinetic properties of the photocycle.

Apoproteins↗
PubMed2001

Excluded volume in protein side-chain packing.

The excluded volume occupied by protein side-chains and the requirement of high packing density in the protein interior should severely limit the number of side-chain conformations compatible with a given native backbone. To examine the relationship between side-chain geometry and side-chain packing, we use an all-atom Monte Carlo simulation to sample the large space of side-chain conformations. We study three models of excluded volume and use umbrella sampling to effectively explore the entire space. We find that while excluded volume constraints reduce the size of conformational space by many orders of magnitude, the number of allowed conformations is still large. An average repacked conformation has 20 % of its chi angles in a non-native state, a marked reduction from the expected 67 % in the absence of excluded volume. Interestingly, well-packed conformations with up to 50 % non-native chi angles exist. The repacked conformations have native packing density as measured by a standard Voronoi procedure. Entropy is distributed non-uniformly over positions, and we partially explain the observed distribution using rotamer probabilities derived from the Protein Data Bank database. In several cases, native rotamers that occur infrequently in the database are seen with high probability in our simulation, indicating that sequence-specific excluded volume interactions can stabilize rotamers that are rare for a given backbone. In spite of our finding that 65 % of the native rotamers and 85 % of chi(1) angles can be predicted correctly on the basis of excluded volume only, 95 % of positions can accommodate more than one rotamer in simulation. We estimate that, in order to quench the side-chain entropy observed in the presence of excluded volume interactions, other interactions (hydrophobic, polar, electrostatic) must provide an additional stabilization of at least 0.6 kT per residue in order to single out the native state.

Algorithms↗
PubMed2000

Control strategies for the polarotactic orientation of the microorganism Euglena gracilis.

A simple mathematical model for the signal received by the dichroic photoreceptor molecules in the motile alga, Euglena gracilis, when irradiated by polarized light, is described and used to test hypotheses for the control strategies employed by the microorganism during negative phototaxis. The model is used to analyse and explain the experimental results of Häder (1987. Arch. Microbiol.147, 179-183).

Animals↗
PubMed2000

Bacteriophytochromes: new tools for understanding phytochrome signal transduction.

The recent discovery of phytochrome-like photoreceptors, collectively called bacteriophytochromes, in a number of bacteria has greatly expanded our understanding of the origins and modes of action of phytochromes in higher plants. These primitive receptors contain an N-terminal domain homologous to the chromophore-binding pocket of phytochromes, and like phytochromes, they bind a variety of bilins to generate photochromic holoproteins. Following the chromophore pocket is a domain similar to two-component histidine kinases, suggesting that these bacterial photoreceptors function in phosphorelay cascades that respond to the light environment. Their organization and distribution support the views that higher-plant phytochromes evolved from a cyanobacterial precursor and that they act as light-regulated kinases. With the ability to exploit bacterial genetics, these bacteriophytochromes now offer simple models to help unravel the biochemical and biophysical events that initiate phytochrome signal transmission.

Amino Acid Sequence↗