RESUMEN
BACKGROUND: Recently, important discoveries regarding the archaeon that functioned as the "host" in the merger with a bacterium that led to the eukaryotes, its "complex" nature, and its phylogenetic relationship to eukaryotes, have been reported. Based on these new insights proposals have been put forward to get rid of the three-domain Model of life, and replace it with a two-domain model. RESULTS: We present arguments (both regarding timing, complexity, and chemical nature of specific evolutionary processes, as well as regarding genetic structure) to resist such proposals. The three-domain Model represents an accurate description of the differences at the most fundamental level of living organisms, as the eukaryotic lineage that arose from this unique merging event is distinct from both Archaea and Bacteria in a myriad of crucial ways. CONCLUSIONS: We maintain that "a natural system of organisms", as proposed when the three-domain Model of life was introduced, should not be revised when considering the recent discoveries, however exciting they may be.
Asunto(s)
Archaea/genética , Bacterias/genética , Evolución Biológica , Eucariontes/genética , Clasificación , FilogeniaRESUMEN
The blue light receptor photoactive yellow protein (PYP) displays rhodopsin-like photochemistry based on the trans to cis photoisomerization of its p-coumaric acid chromophore. Here, we report that protein refolding from the acid-denatured state of PYP mimics the last photocycle transition in PYP. This implies a direct link between transient protein unfolding and photosensory signal transduction. We utilize this link to study general issues in protein folding. Chromophore trans to cis photoisomerization in the acid-denatured state strongly decelerates refolding, and converts the pH dependence of the barrier for refolding from linear to nonlinear. We propose transition state movement to explain this phenomenon. The cis chromophore significantly stabilizes the acid-denatured state, but acidification of PYP results in the accumulation of the acid-denatured state containing a trans chromophore. This provides a clear example of kinetic control in a protein unfolding reaction. These results demonstrate the power of PYP as a light-triggered model system to study protein folding.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Luz , Fotorreceptores Microbianos , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Modelos Químicos , Desnaturalización Proteica , Pliegue de Proteína , Transducción de Señal , Espectrometría de Fluorescencia , Estereoisomerismo , Temperatura , Factores de TiempoRESUMEN
The photoreceptor photoactive yellow protein (PYP) was used as a model system to study receptor activation and protein folding. Refolding was studied by stopped-flow absorbance spectroscopy for PYP with either a trans or a cis chromophore. Chromophore trans to cis isomerization, the mechanism of light detection by PYP, greatly affects the protein folding process. When the cis chromophore is present, refolding from the unfolded state proceeds through the putative signaling state of PYP as an on-pathway intermediate. In addition, moderate denaturant concentrations result in the specific unfolding of the signaling state of PYP. Thus, the signaling state is common to the pathways of folding and signaling. This result provides an avenue for the study of protein folding. We demonstrate how this approach can be used to establish whether a folding intermediate is on-pathway or off-pathway. The results also reveal transient partial unfolding as a molecular mechanism for signaling.
Asunto(s)
Células Fotorreceptoras/química , Pliegue de Proteína , Transducción de Señal , Isomerismo , Cinética , Sondas Moleculares , FotoquímicaRESUMEN
Photoactive yellow protein (PYP) is a eubacterial photoreceptor and a structural prototype of the PAS domain superfamily of receptor and regulatory proteins. We investigate the activation mechanism of PYP using time-resolved Fourier transform infrared (FTIR) spectroscopy. Our data provide structural, kinetic, and energetic evidence that the putative signaling state of PYP is formed during a large-amplitude protein quake that is driven by the formation of a new buried charge, COO(-) of the conserved Glu46, in a highly hydrophobic pocket at the active site. A protein quake is a process consisting of global conformational changes that are triggered and driven by a local structural "fault". We show that large, global structural changes take place after Glu46 ionization via intramolecular proton transfer to the anionic p-coumarate chromophore, and are suppressed by the absence of COO(-) formation in the E46Q mutant. Our results demonstrate the significance of buried charge formation in photoreceptor activation. This mechanism may serve as one of the general themes in activation of a range of receptor proteins. In addition, we report the results of time-resolved FTIR spectroscopy of PYP crystals. The direct comparison of time-resolved FTIR spectroscopic data of PYP in aqueous solution and in crystals reveals that the structure of the putative signaling state is not developed in P6(3) crystals. Therefore, when the structural developments during the functional process of a protein are experimentally determined to be very different in crystals and solutions, one must be cautious in drawing conclusions regarding the functional mechanism of proteins based on time-resolved X-ray crystallography.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Ácidos Cumáricos/metabolismo , Cristalización , Cristalografía por Rayos X , Transferencia de Energía , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Glutamina/genética , Cinética , Mutagénesis Sitio-Dirigida , Propionatos , Conformación Proteica , Protones , Electricidad Estática , Relación Estructura-Actividad , TermodinámicaRESUMEN
Biological signaling generally involves the activation of a receptor protein by an external stimulus followed by protein-protein interactions between the activated receptor and its downstream signal transducer. The current paradigm for the relay of signals along a signal transduction chain is that it occurs by highly specific interactions between fully folded proteins. However, recent results indicate that many regulatory proteins are intrinsically unstructured, providing a serious challenge to this paradigm and to the nature of structure-function relationships in signaling. Here we study the structural changes that occur upon activation of the blue light receptor photoactive yellow protein (PYP). Activation greatly reduces the tertiary structure of PYP but leaves the level secondary structure largely unperturbed. In addition, activated PYP exposes previously buried hydrophobic patches and allows significant solvent penetration into the core of the protein. These traits are the distinguishing hallmarks of molten globule states, which have been intensively studied for their role in protein folding. Our results show that receptor activation by light converts PYP to a molten globule and indicate stimulus-induced unfolding to a partially unstructured molten globule as a novel theme in signaling.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Fotorreceptores Microbianos , Acrilamida/farmacología , Naftalenosulfonatos de Anilina , Dicroismo Circular , Escherichia coli , Colorantes Fluorescentes , Cinética , Luz , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , EspectrofotometríaRESUMEN
Light-dependent pH changes were measured in unbuffered solutions of wild type photoactive yellow protein (PYP) and its H108F and E46Q variants, using two independent techniques: transient absorption changes of added pH indicator dyes and direct readings with a combination pH electrode. Depending on the absolute pH of the sample, a reversible protonation as well as a deprotonation can be observed upon formation of the transient, blue-shifted photocycle intermediate (pB) of this photoreceptor protein. The latter is observed at very alkaline pH, the former at acidic pH values. At neutral pH, however, the formation of the pB state is not paralleled by significant protonation/deprotonation of PYP, as expected for concomitant protonation of the chromophore and deprotonation of Glu-46 during pB formation. We interpret these results as further evidence that a proton is transferred from Glu-46 to the coumaric acid chromophore of PYP, during pB formation. One cannot exclude the possibility, however, that this transfer proceeds through the bulk aqueous phase. Simultaneously, an amino acid side chain(s) (e.g. His-108) changes from a buried to an exposed position. These results, therefore, further support the idea that PYP significantly unfolds in the pB state and resolve the controversy regarding proton transfer during the PYP photocycle.
Asunto(s)
Proteínas Bacterianas/química , Fotorreceptores Microbianos/química , Rhodospirillaceae/metabolismo , Proteínas Bacterianas/genética , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fotoquímica , Conformación Proteica , Pliegue de Proteína , Protones , EspectrofotometríaRESUMEN
Biological signal transduction starts with the activation of a receptor protein. Two central questions in signaling are the mechanism of activation by a stimulus and the nature and extent of the protein conformational changes involved. We report extensive evidence for the occurrence of large structural changes upon the light activation of photoactive yellow protein (PYP), a eubacterial photosensor. Absorption of a blue photon by the p-coumaric acid (pCA) chromophore in pG, the initial state of PYP, results in the formation of pB, a putative signaling state. In the presence of an adequate hydration shell, large structural changes in the protein backbone, involving both solvent accessible and core regions, were detected using Fourier transform infrared (FTIR) difference spectroscopy. A significant part (23%) of the amide groups which are buried in pG become exposed to the solvent in pB, as measured through light-induced H/D exchange, using both electrospray ionization mass spectrometry and FTIR spectroscopy. Exposure of previously buried hydrophobic sites would lead to an increase in heat capacity during pB formation and a decrease in heat capacity during pB decay. Thermodynamic studies indeed show that the heat capacity change of pB activation is -2.35 +/- 0.08 kJ/(mol/K), independent of pH from pH 2.4-7.5. A model for photoactivation of PYP is proposed, which provides a framework for a deeper understanding of receptor activation in general.
Asunto(s)
Bacterias/química , Proteínas Bacterianas/química , Fotorreceptores Microbianos/metabolismo , Proteínas Bacterianas/fisiología , Luz , Espectrometría de Masas , Modelos Químicos , Oxidación-Reducción , Fotorreceptores Microbianos/fisiología , Conformación Proteica , Transducción de Señal , Espectroscopía Infrarroja por Transformada de Fourier , Termodinámica , AguaRESUMEN
Molecular dynamics simulations have been performed with the aim of identifying concerted backbone motions in the photoactive yellow protein. Application of the essential dynamics method revealed large, chromophore-linked fluctuations of the protein in the ground state, as well as in a form containing the isomerized chromophore. Various loops become more mobile upon isomerization of the chromophore, including a loop which is part of the PAS domain motif, found in light perception proteins. The hinge points identified in these fluctuations correlate with the positions of evolutionary conserved glycines. The results derived from the simulations directly correlate with available experimental data, provide a framework for understanding the dynamic behaviour of the yellow protein and give clues to subsequent steps in the signal transduction pathway.
Asunto(s)
Proteínas Bacterianas/química , Fotorreceptores Microbianos , Pliegue de Proteína , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Colorantes/metabolismo , Simulación por Computador , Secuencia Conservada , Cristalización , Glicina/metabolismo , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Alineación de Secuencia , Factores de TiempoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Fotorreceptores Microbianos , Conformación Proteica , Termodinámica , Chromatiaceae/química , Cristalización , Cristalografía por Rayos X , Deuterio , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Pliegue de Proteína , Protones , SolucionesRESUMEN
Among the signal transfer systems in bacteria two types predominate: two-component regulatory systems and quorum sensing systems. Both types of system can mediate signal transfer across the bacterial cell envelope; however, the signalling molecule typically is not taken up into the cells in the former type of system, whereas it usually is in the latter. The Two-component systems include the recently described (eukaryotic) phosphorelay systems; quorum sensing systems can be based upon autoinducers of the N-acylated homoserine lactones, and on autoinducers of a peptidic nature. A single bacterial cell contains many signalling modules that primarily operate in parallel. This may give rise to neural-network behaviour. Recently, however, for both types of basic signal transfer modules, it has been demonstrated that they also can be organised in series (i.e. in a hierarchical order). Besides their hierarchical position in the signal transduction network of the cell, the spatial distribution of individual signalling modules may also be an important factor in their efficiency in signal transfer. Many challenges lie hidden in future work to understand these signal transfer processes in more detail. These are discussed here, with emphasis on the mutual interactions between different signal transfer processes. Successful contributions to this work will require rigorous mathematical modelling of the performance of signal transduction components, and -networks, as well as studies on light-sensing signal transduction systems, because of the unsurpassed time resolution obtainable in those latter systems, the opportunity to apply repeated reproducible stimuli, etc. The increased understanding of bacterial behaviour that already has resulted--and may further result--from these studies, can be used to fine-tune the beneficial activities of bacteria and/or more efficiently inhibit their deleterious ones.
Asunto(s)
Bacterias , Transducción de Señal/fisiología , Homoserina/análogos & derivados , Péptidos/metabolismo , Feromonas/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
Two sensory rhodopsins (SRI and SRII) mediate color-sensitive phototaxis responses in halobacteria. These seven-helix receptor proteins, structurally and functionally similar to animal visual pigments, couple retinal photoisomerization to receptor activation and are complexed with membrane-embedded transducer proteins (HtrI and HtrII) that modulate a cytoplasmic phosphorylation cascade controlling the flagellar motor. The Htr proteins resemble the chemotaxis transducers from Escherichia coli. The SR-Htr signaling complexes allow studies of the biophysical chemistry of signal generation and relay, from the photobiophysics of initial excitation of the receptors to the final output at the level of the flagellar motor switch, revealing fundamental principles of sensory transduction and more broadly the nature of dynamic interactions between membrane proteins. We review here recent advances that have led to new insights into the molecular mechanism of signaling by these membrane complexes.
Asunto(s)
Proteínas Arqueales , Bacteriorodopsinas/fisiología , Carotenoides , Halobacterium/fisiología , Halorrodopsinas , Rodopsinas Sensoriales , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Datos de Secuencia MolecularRESUMEN
Photoactive yellow proteins (PYPs) constitute a new class of eubacterial photoreceptors, containing a deprotonated thiol ester-linked 4-hydroxycinnamic acid chromophore. Interactions with the protein dramatically change the (photo)chemical properties of this cofactor. Here we describe the reconstitution of apoPYP with anhydrides of various chromophore analogues. The resulting hybrid PYPs, their acid-denatured states, and corresponding model compounds were characterized with respect to their absorption spectrum, pK for chromophore deprotonation, fluorescence quantum yield, and Stokes shift. Three factors contributing to the tuning of the absorption of the hybrid PYPs were quantified: (i) thiol ester bond formation, (ii) chromophore deprotonation, and (iii) specific chromophore-protein interactions. Analogues lacking the 4-hydroxy substituent lack both contributions (chromophore deprotonation and specific chromophore-protein interactions), confirming the importance of this substituent in optical tuning of PYP. Hydroxy and methoxy substituents in the 3- and/or 5-position do not disrupt strong interactions with the protein but increase their pK for protonation and the fluorescence quantum yield. Both deprotonation and binding to apoPYP strongly decrease the Stokes shift of chromophore fluorescence. Therefore, coupling of the chromophore to the apoprotein not only reduces the energy gap between its ground and excited state but also the extent of reorganization between these two states. Two of the PYP hybrids show photoactivity comparable with native PYP, although with retarded recovery of the initial state.
Asunto(s)
Proteínas Bacterianas/química , Chromatiaceae/química , Histidina , Fotorreceptores Microbianos , Cinamatos , Concentración de Iones de Hidrógeno , Péptidos , Espectrometría de Fluorescencia , Espectrofotometría AtómicaRESUMEN
Photoactive yellow protein (PYP) is a photoreceptor containing a unique 4-hydroxycinnamic acid (pCA) chromophore. The trans to cis photoisomerization of this chromophore activates a photocycle involving first a short-lived red-shifted intermediate (pR), then a long-lived blue-shifted intermediate (pB), and finally recovery of the original receptor state (pG). The pCA chromophore is deprotonated in pG and protonated in pB, but the proton donor for this process has not yet been identified. Here we report the first FTIR spectroscopic data on pG, pR, and pB. The IR difference signals in the carbonyl stretching region of COOH groups (1700-1800 cm-1) reveal that a buried carboxylic group close to the chromophore (i) is protonated in pG, (ii) develops a stronger hydrogen bonding in pR, and (iii) becomes deprotonated in pB. These signals are unambiguously assigned to Glu46, on the basis of the IR data and the 1.4 A X-ray structure of PYP [Borgstahl et al. (1995) Biochemistry 34, 6278-6287]. Our data demonstrate that in pR Glu46 remains in hydrogen bonding contact with the negatively charged phenolic oxygen of pCA after chromophore photoisomerization. This strongly implies that the chromophore is isomerized to the 7-cis 9-s-trans conformation in pR, resulting from co-isomerization of both the C7 = C8 and C9-C10 bonds. In the pR to pB transition, Glu46 becomes deprotonated, concomitant with chromophore protonation. Therefore, we conclude that Glu46 functions as the proton donor for the protonation of pCA during the PYP photocycle. We propose a molecular mechanism in which intramolecular proton transfer in PYP leads to global protein conformational changes involved in signal transduction.
Asunto(s)
Proteínas Bacterianas/química , Ácido Glutámico/química , Fotorreceptores Microbianos , Escherichia coli , Bacterias Gramnegativas/genética , Enlace de Hidrógeno , Fotoquímica , Protones , Proteínas Recombinantes , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Photobiological processes are relevant for microorganisms for energy generation, protection against excess and/or damaging radiation, and for signalling. In this review we give an overview of the knowledge on the functioning of photosensors in microorganisms, with special emphasis on the conformational changes that lead to signal generation and transduction. Light is absorbed by specific chromophores, which are tuned, by their proteinaceous environment, to function optimally. These chromophores belong to three classes: tetrapyrroles, polyenes and aromatics. The chemical structure of photosensing pigment/protein complexes has been resolved for many of the photobiological processes that have a characteristic sensitivity in the visible and infrared part of the spectrum of (solar) radiation. However, knowledge about the structure of photoreceptors responsible for several physiologically well-characterized photoresponses to UV- and blue light is still lacking. For a limited number of phototransduction processes, the details of light-induced signal transfer are beginning to be understood in atomic detail. This applies particularly to two photosensors involved in phototactic responses in bacteria: sensory rhodopsin I (SR-I) from Halobacterium salinarium and photoactive yellow protein (PYP) from Ectothiorhodospira halophila. The SR-1 system is of special interest because the transducer accepting the signal from SR-1 was recently identified as Htr-1, a homologue of the methyl-accepting chemotaxis proteins which have been characterized in Escherichia coli. PYP, on the other hand, may be the first photosensor to actually reveal all relevant details of the kinetics, thermodynamics, and molecular motion of light-induced signal generation, through an understanding of how the photo-isomerization of the chromophore forces the sensor protein into the signalling state. Here we compare these photosensors and discuss common themes in the initiation of photosensory signal transduction in microorganisms in terms of the molecular properties of photosensors and their signalling state.
Asunto(s)
Bacterias/química , Fotones , Pigmentos Biológicos/química , Transducción de Señal/fisiología , Eucariontes/química , Hongos/química , FotoquímicaRESUMEN
Photoactive yellow protein (PYP) is a photoreceptor that has been isolated from three halophilic phototrophic purple bacteria. The PYP from Ectothiorhodospira halophila BN9626 is the only member for which the sequence has been reported at the DNA level. Here we describe the cloning and sequencing of the genes encoding the PYPs from E.halophila SL-1 (type strain) and Rhodospirillum salexigens. The latter protein contains, like the E.halophila PYP, the chromophore trans p-coumaric acid, as we show here with high performance capillary zone electrophoresis. Additionally, we present evidence for the presence of a gene encoding a PYP homolog in Rhodobacter sphaeroides, the first genetically well-characterized bacterium in which this photoreceptor has been identified. An ORF downstream of the pyp gene from E.halophila encodes an enzyme, which is proposed to be involved in the biosynthesis of the chromophore of PYP. The pyp gene from E.halophila was used for heterologous overexpression in both Escherichia coli and R.sphaeroides, aimed at the development of a holoPYP overexpression system (an intact PYP, containing the p-coumaric acid chromophore and displaying the 446 nm absorbance band). In both organisms the protein could be detected immunologically, but its yellow color was not observed. Molecular genetic construction of a histidine-tagged version of PYP led to its 2500-fold overproduction in E.coli and simplified purification of the heterologously produced apoprotein. HoloPYP could be reconstituted by the addition of p-coumaric anhydride to the histidine-tagged apoPYP (PYP lacking its chromophore). We propose to call the family of photoactive yellow proteins the xanthopsins, in analogy with the rhodopsins.
Asunto(s)
Proteínas Bacterianas/genética , Chromatiaceae/genética , Fotorreceptores Microbianos , Rhodospirillum/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Two complementary aspects of the thermodynamics of the photoactive yellow protein (PYP), a new type of photoreceptor that has been isolated from Ectothiorhodospira halophila, have been investigated. First, the thermal denaturation of PYP at pH 3.4 has been examined by global analysis of the temperature-induced changes in the UV-VIS absorbance spectrum of this chromophoric protein. Subsequently, a thermodynamic model for protein (un)folding processes, incorporating heat capacity changes, has been applied to these data. The second aspect of PYP that has been studied is the temperature dependence of its photocycle kinetics, which have been reported to display an unexplained deviation from normal Arrhenius behavior. We have extended these measurements in two solvents with different hydrophobicities and have analyzed the number of rate constants needed to describe these data. Here we show that the resulting temperature dependence of the rate constants can be quantitatively explained by the application of a thermodynamic model which assumes that heat capacity changes are associated with the two transitions in the photocycle of PYP. This result is the first example of an enzyme catalytic cycle being described by a thermodynamic model including heat capacity changes. It is proposed that a strong link exists between the processes occurring during the photocycle of PYP and protein (un)folding processes. This permits a thermodynamic analysis of the light-induced, physiologically relevant, conformational changes occurring in this photoreceptor protein.
Asunto(s)
Proteínas Bacterianas/química , Fotorreceptores Microbianos , 1-Butanol , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/efectos de la radiación , Fenómenos Biofísicos , Biofisica , Butanoles/farmacología , Chromatiaceae , Concentración de Iones de Hidrógeno , Cinética , Modelos Químicos , Fotoquímica , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Espectrofotometría , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
Analysis of the chromophore p-coumaric acid, extracted from the ground state and the long-lived blue-shifted photocycle intermediate of photoactive yellow protein, shows that the chromophore is reversibly converted from the trans to the cis configuration, while progressing through the photocycle. The detection of the trans and cis isomers was carried out by high performance capillary zone electrophoresis and further substantiated by 1H NMR spectroscopy. The data presented here establish the photo-isomerization of the vinyl double bond in the chromophore as the photochemical basis for the photocycle of photoactive yellow protein, a eubacterial photosensory protein. A similar isomerization process occurs in the structurally very different sensory rhodopsins, offering an explanation for the strong spectroscopic similarities between photoactive yellow protein and the sensory rhodopsins. This is the first demonstration of light-induced isomerization of a chromophore double bond as the photochemical basis for photosensing in the domain of Bacteria.
Asunto(s)
Proteínas Bacterianas/química , Ácidos Cumáricos/química , Fotorreceptores Microbianos , Proteínas Bacterianas/efectos de la radiación , Chromatiaceae/química , Ácidos Cumáricos/efectos de la radiación , Electroforesis Capilar , Isomerismo , Espectroscopía de Resonancia Magnética , Fotoquímica , Propionatos , Protones , Rayos UltravioletaRESUMEN
The photoactive yellow proteins (PYP) have been found to date only in three species of halophilic purple phototrophic bacteria. They have photochemical activity remarkably similar to that of the bacteria rhodopsins. In contrast to rhodopsins, however, the PYPs are small water-soluble proteins. We now report the complete amino acid sequences of Rhodospirillum salexigens and Chromatium salexigens PYP which allow comparison with the known sequence and three-dimensional structure of the prototypic protein from Ectothiorhodospira halophila. Although isolated from three different families of bacteria, the PYP sequences are 70-76% identical. All three contain 125 amino acid residues, and no insertions or deletions are necessary for alignment. This is a remarkable result when it is considered that electron transfer proteins from these purple bacterial species are only 25-40% identical and that insertions and deletions are needed for their proper alignment. It thus appears that PYP has the same important function in each of the purple bacteria and that most of the amino acid residues are necessary to maintain structure and function. By most standards, PYP would be called a "slowly evolving protein". R. salexigens PYP is uniquely degraded by proteolysis at low ionic strength, probably as a consequence of unfolding due to electrostatic repulsion of the excess negative charge. Therefore it may also be classified as a "halophilic protein".
Asunto(s)
Proteínas Bacterianas/genética , Chromatium/genética , Fotorreceptores Microbianos , Rhodospirillum/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Chromatium/química , Secuencia Conservada , Electroquímica , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Conformación Proteica , Rhodospirillales/química , Rhodospirillales/genética , Rhodospirillum/química , Homología de Secuencia de AminoácidoRESUMEN
We have recently identified p-coumaric acid as the chromophore of the photoactive yellow protein (PYP) from the purple sulfur bacterium Ectothiorhodospira halophila, a blue-light photoreceptor with rhodopsin-like photochemistry [Hoff, W. D., Düx, P., Hård, K., Nugteren-Roodzant, I. M., Crielaard, W., Boelens, R., Kaptein, R., Van Beeumen, J., & Hellingwerf, K. J. (1994) Biochemistry 33, 13959-13962]. Here we report on the chemistry of the linkage of this new photoactive cofactor to apoPYP: (i) Analysis of chromophore-peptide conjugates of PYP by high-resolution mass spectrometry unambiguously shows that the p-coumaric acid molecule is bound to Cys 69 via a thiol ester bond. The PYP chromophore is the first cofactor known to be stably thiol ester-linked to its apoprotein. (ii) The chemical reactivity of this thiol ester bond with respect to dithiothreitol, performic acid, and high pH is similar to that of disulfide bridges. These treatments result in the cleavage of the thiol ester bond, concomitant with strong shifts in the UV/vis absorbance band of the chromophore. (iii) The spectral properties of the PYP chromophore under different conditions are related to the structural integrity of the protein, the presence of the thiol ester bond, and the ionization state of the phenolic proton of the chromophore. These results are important for the general problem of spectral tuning in photoreceptor proteins.
Asunto(s)
Proteínas Bacterianas/química , Chromatiaceae/química , Ácidos Cumáricos/química , Fotorreceptores Microbianos , Pigmentos Biológicos/química , Compuestos de Sulfhidrilo/química , Secuencia de Aminoácidos , Apoproteínas/química , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Cromatografía Líquida de Alta Presión , Ditiotreitol/farmacología , Endopeptidasa K , Ésteres , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Pepsina A/metabolismo , Péptidos/química , Propionatos , Serina Endopeptidasas/metabolismo , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , EspectrofotometríaRESUMEN
Resonance Raman spectra of the ground state of photoactive yellow protein (PYP), a photoactive pigment found in Ectothiorhodospira halophila, have been obtained with excitation at 413.1 nm using a microspinning sample cell. The resonance Raman spectra of the thioester-linked 4-hydroxycinnamyl chromophore in the protein are compared with the preresonance Raman spectra of the 4-hydroxycinnamyl phenyl thioester and 4-hydroxycinnamic acid model compounds at various pH values. Bands at 1568, 1542, 1500, 1434, and 1166 cm-1 in the Raman spectrum of the anionic form of the 4-hydroxycinnamyl phenyl thioester are shown to be characteristic for the deprotonation of the chromophore. The observation of bands in PYP exhibiting very similar frequency and intensity patterns provides strong evidence that the chromophore in PYP is stabilized as a phenolate anion at pH 7.4, in support of conclusions from crystallographic studies. Furthermore, the insensitivity of the PYP Raman spectrum to placement of the protein in D2O buffer is consistent with the absence of the exchangeable phenolic proton on the cinnamyl chromophore. These results establish the feasibility of elucidating the molecular mechanism of light-to-information transduction by this new photosensory pigment with resonance Raman spectroscopy.