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Results for “Rhodopsins, Microbial”

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PubMed2002

Methods for study of protein dynamics and protein-protein interaction in protein-ubiquitination by electron paramagnetic resonance spectroscopy.

Ubiquitination is a post-translation modification whereby the C-terminal end of ubiquitin (Ub) is covalently attached to the amino group of a lysine in a target protein. Additional ubiquitin groups are added using Ub-Ub linkages to form a polyubiquitin chain. A 26S protease complex specifically binds polyubiquitinated proteins and degrades them in an ATP-dependent manner. The target lysine in the substrate protein resides in a domain that is recognized by the ubiquitination machinery in a temporally and spatially controlled manner. The accessibility and the molecular dynamics of the target domain for each protein substrate is expected to be distinctive and this article is intended to facilitate investigations in this uncharted research area of ubiquitination mediated protein turnover by means of site-directed spin labeling. Examples illustrate the methodology of electron paramagnetic resonance data acquisition and interpretation in terms of secondary and tertiary structure resolution of proteins and protein complexes. Analysis of the spin labeled side chain mobility, its solvent accessibility, the polarity of the spin label micro-environment and distances between spin labels allow to model protein domains or protein-protein interaction sites and their conformational changes with a spatial resolution at the level of the backbone fold. The structural changes accompanying protein function or protein-protein interaction can be monitored in the millisecond time range. These features make site-directed spin labeling an attractive approach for the study of protein--ubiquitin interaction and protein ubiquitination.

Computer Simulation↗

PubMed2003

Visual and archaeal rhodopsins: similarities, differences and controversy.

Rhodopsins are currently known to belong to two distinct protein families. The visual rhodopsins, found in eyes throughout the animal kingdom, are photosensory pigments. Archaeal rhodopsins, found in extreme halophiles, function as light-driven proton pumps (bacteriorhodopsins), chloride ion pumps (halorhodopsins), or photosensory receptors (sensory rhodopsins). Light absorption by rhodopsins triggers their characteristic photoconversion extending into the (milli)second time range. There are three main paradigms of rhodopsins photoconversion. (1) Initiation of the trans-cis isomerization is the very primary consequence of light absorption. (2) Rhodopsins store light energy via the charge-separation mechanism (the charge of Schiff base is separated from its counterion). (3) Full trans-cis isomerization of the chromophore is a prerequisite for the full biological activity of rhodopsins. These paradigms will be questioned.

Bacteriorhodopsins↗

PubMed2006

Development of the signal in sensory rhodopsin and its transfer to the cognate transducer.

The microbial phototaxis receptor sensory rhodopsin II (NpSRII, also named phoborhodopsin) mediates the photophobic response of the haloarchaeon Natronomonas pharaonis by modulating the swimming behaviour of the bacterium. After excitation by blue-green light NpSRII triggers, by means of a tightly bound transducer protein (NpHtrII), a signal transduction chain homologous with the two-component system of eubacterial chemotaxis. Two molecules of NpSRII and two molecules of NpHtrII form a 2:2 complex in membranes as shown by electron paramagnetic resonance and X-ray structure analysis. Here we present X-ray structures of the photocycle intermediates K and late M (M2) explaining the evolution of the signal in the receptor after retinal isomerization and the transfer of the signal to the transducer in the complex. The formation of late M has been correlated with the formation of the signalling state. The observed structural rearrangements allow us to propose the following mechanism for the light-induced activation of the signalling complex. On excitation by light, retinal isomerization leads in the K state to a rearrangement of a water cluster that partly disconnects two helices of the receptor. In the transition to late M the changes in the hydrogen bond network proceed further. Thus, in late M state an altered tertiary structure establishes the signalling state of the receptor. The transducer responds to the activation of the receptor by a clockwise rotation of about 15 degrees of helix TM2 and a displacement of this helix by 0.9 A at the cytoplasmic surface.

Biological Evolution↗

PubMed2002

Channelrhodopsin-1: a light-gated proton channel in green algae.

Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-type chromophores. We report a complementary DNA sequence in the green alga Chlamydomonas reinhardtii that encodes a microbial opsin-related protein, which we term Channelopsin-1. The hydrophobic core region of the protein shows homology to the light-activated proton pump bacteriorhodopsin. Expression of Channelopsin-1, or only the hydrophobic core, in Xenopus laevis oocytes in the presence of all-trans retinal produces a light-gated conductance that shows characteristics of a channel selectively permeable for protons. We suggest that Channelrhodopsins are involved in phototaxis of green algae.

Amino Acid Sequence↗

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↗

PubMed2003

Hydrogen bonding alteration of Thr-204 in the complex between pharaonis phoborhodopsin and its transducer protein.

Pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronobacterium pharaonis. It forms a 2:2 complex with its transducer protein, pHtrII, in membranes and transmits light signals through the change in the protein-protein interaction. We previously found that the ppR(K) minus ppR spectrum in D(2)O possesses vibrational bands of ppR at 3479 (-)/3369 (+) cm(-1) only in the presence of pHtrII [Furutani, Y., Sudo, Y., Kamo, N., and Kandori, H. (2003) Biochemistry 42, 4837-4842]. A D/H-unexchangeable X-H group appears to form a stronger hydrogen bond upon retinal photoisomerization in the ppR-pHtrII complex. This article aims to identify the group by use of various mutant proteins. According to the crystal structure, Tyr-199 of ppR forms a hydrogen bond with Asn-74 of pHtrII in the complex. Nevertheless, the 3479 (-)/3369 (+) cm(-1) bands were preserved in the Y199F mutant, excluding the possibility that the bands are O-H stretches of Tyr-199. On the other hand, Thr-204 and Tyr-174 form a hydrogen bond between the retinal chromophore pocket and the binding surface of the ppR-pHtrII complex. These FTIR measurements revealed that the bands at 3479 (-)/3369 (+) cm(-1) disappeared in the T204A mutant, while being shifted to 3498 (-) and 3474 (+) cm(-1) in the T204S mutant. They appear at 3430 (-)/3402 (+) cm(-1) in the Y174F mutant. From these results, we concluded that the bands at 3479 (-)/3369 (+) cm(-1) originate from the O-H stretch of Thr-204. A stronger hydrogen bond as shown by a large spectral downshift (110 cm(-1)) suggests that the specific hydrogen bonding alteration of Thr-204 takes place upon retinal photoisomerization, which does not occur in the absence of the transducer protein. Thr-204 has been known as an important residue for color tuning and photocycle kinetics in ppR. The results presented here point to an additional important role of Thr-204 in ppR for the interaction with pHtrII. Specific interaction in the complex that involves Thr-204 presumably affects the decay kinetics and binding affinity in the M intermediate.

Alanine↗

PubMed2003

pH Dependence of the photocycle kinetics of the E46Q mutant of photoactive yellow protein: protonation equilibrium between I1 and I2 intermediates, chromophore deprotonation by hydroxyl uptake, and protonation relaxation of the dark state.

The kinetics of the photocycle of PYP and its mutants E46Q and E46A were investigated as a function of pH. E46 is the putative donor of the chromophore which becomes protonated in the I(2) intermediate. For E46Q we find that I(2) is in a pH-dependent equilibrium with its precursor I(1)' with a pK(a) of 8.15 and n = 1. From this result and from experiments with pH indicator dyes, we conclude that in the I(1)' to I(2) transition one proton is taken up from the external medium. The pK(a) of 8.15 is that of the surface-exposed chromophore in the equilibrium between I(1)' and I(2) and is close to that of the phenolate group of p-hydroxycinnamic acid. The pH-dependent I(1)'/I(2) equilibrium with associated H(+) uptake is reminiscent of the M(I)/M(II) equilibrium in the formation of the signaling state of rhodopsin. Well above this pK(a) no I(2) is formed and I(1)' returns in a pH-independent manner to the initial state P. The decay rate for the return to P via I(2) is between pH 4 and pH 8, exactly proportional to the hydroxide concentration (first order), and the deprotonation of the chromophore in this transition occurs by hydroxide uptake. Well above the pK(a) of 8.15 the apparent rate constant for the return to P is constant due to the branching from I(1)'. Complementary measurements with the pH indicator dye cresol red at pH 8.3 show that the remaining PYP molecules that still cycle via I(2) take up one proton in the formation of I(2). Together, these observations provide compelling evidence that during the photocycle the chromophore in E46Q is protonated and deprotonated from the external medium. For the yellow form of the mutant E46A the apparent rate constant for the return to P is also linear in [OH(-)] below about pH 8.3 and constant above about pH 9.5, with a pK(a) value of 8.8 for I(1)', suggesting a similar mechanism of chromophore protonation/deprotonation as in E46Q. For wild type qualitatively similar observations were made: the amplitude of I(2) decreased at alkaline pH, I(1)' and I(2) were in equilibrium, and I(1)' decayed together with the return to P. Chromophore hydrolysis prevented, however, an accurate determination of the pK(a) of I(1)'. We estimate that its value is above 11. The ground state P is in the dark in a pH-dependent equilibrium with a low-pH bleached form P(bl) with protonated chromophore. The pK(a) values for these equilibria are 4.8 and 7.9 for E46Q and E46A, respectively. When the pH is close to these pK(a)'s, the kinetics of the photocycle contains additional components in the millisecond time range. Using pH-jump stopped-flow experiments, we show that these contributions are due to the relaxation of the P/P(bl) equilibrium which is perturbed by the rapid decrease in the P concentration caused by the flash excitation of P. The condition for the occurrence of this effect is that the relaxation time of the P/P(bl) equilibrium is faster than the photocycle time.

Bacterial Proteins↗

PubMed2004

Environmental genome shotgun sequencing of the Sargasso Sea.

We have applied "whole-genome shotgun sequencing" to microbial populations collected en masse on tangential flow and impact filters from seawater samples collected from the Sargasso Sea near Bermuda. A total of 1.045 billion base pairs of nonredundant sequence was generated, annotated, and analyzed to elucidate the gene content, diversity, and relative abundance of the organisms within these environmental samples. These data are estimated to derive from at least 1800 genomic species based on sequence relatedness, including 148 previously unknown bacterial phylotypes. We have identified over 1.2 million previously unknown genes represented in these samples, including more than 782 new rhodopsin-like photoreceptors. Variation in species present and stoichiometry suggests substantial oceanic microbial diversity.

Archaea↗

PubMed2001

Proteorhodopsin phototrophy in the ocean.

Proteorhodopsin, a retinal-containing integral membrane protein that functions as a light-driven proton pump, was discovered in the genome of an uncultivated marine bacterium; however, the prevalence, expression and genetic variability of this protein in native marine microbial populations remain unknown. Here we report that photoactive proteorhodopsin is present in oceanic surface waters. We also provide evidence of an extensive family of globally distributed proteorhodopsin variants. The protein pigments comprising this rhodopsin family seem to be spectrally tuned to different habitats--absorbing light at different wavelengths in accordance with light available in the environment. Together, our data suggest that proteorhodopsin-based phototrophy is a globally significant oceanic microbial process.

Amino Acid Sequence↗

PubMed2005

Rhodopsin-mediated photoreception in cryptophyte flagellates.

We show that phototaxis in cryptophytes is likely mediated by a two-rhodopsin-based photosensory mechanism similar to that recently demonstrated in the green alga Chlamydomonas reinhardtii, and for the first time, to our knowledge, report spectroscopic and charge movement properties of cryptophyte algal rhodopsins. The marine cryptophyte Guillardia theta exhibits positive phototaxis with maximum sensitivity at 450 nm and a secondary band above 500 nm. Variability of the relative sensitivities at these wavelengths and light-dependent inhibition of phototaxis in both bands by hydroxylamine suggest the involvement of two rhodopsin photoreceptors. In the related freshwater cryptophyte Cryptomonas sp. two photoreceptor currents similar to those mediated by the two sensory rhodopsins in green algae were recorded. Two cDNA sequences from G. theta and one from Cryptomonas encoding proteins homologous to type 1 opsins were identified. The photochemical reaction cycle of one Escherichia-coli-expressed rhodopsin from G. theta (GtR1) involves K-, M-, and O-like intermediates with relatively slow (approximately 80 ms) turnover time. GtR1 shows lack of light-driven proton pumping activity in E. coli cells, although carboxylated residues are at the positions of the Schiff base proton acceptor and donor as in proton pumping rhodopsins. The absorption spectrum, corresponding to the long-wavelength band of phototaxis sensitivity, makes this pigment a candidate for one of the G. theta sensory rhodopsins. A second rhodopsin from G. theta (GtR2) and the one from Cryptomonas have noncarboxylated residues at the donor position as in known sensory rhodopsins.

Amino Acid Sequence↗

PubMed2005

Reflection on medium effects on photochemical reactivity.

In reexamining medium effects on photochemical reactions, we have emphasized those on unequilibrated excited species such as the Franck-Condon species. Despite recent advances in femtochemistry, such a discussion in molecular photochemistry is uncommon, and the problem remains challenging on account of the extremely short-lived excited species. However, in such cases, a small perturbation resulting from, for example, weak guest-host interactions could turn into a determining factor in dictating the course of a photochemical channel of deactivation. Examples of medium-directed diabatic processes have been examined with this idea in mind. A modified view on rhodopsin photoisomerization is presented along with the consideration that confinement does not necessarily lead to inhibition of reactions of the trapped substrate.

Bacterial Proteins↗

PubMed2004

FTIR spectroscopy of the K photointermediate of Neurospora rhodopsin: structural changes of the retinal, protein, and water molecules after photoisomerization.

Neurospora rhodopsin (NR, also known as NOP-1) is the first rhodopsin of the haloarchaeal type found in eucaryotes. NR demonstrates a very high degree of conservation of the amino acids that constitute the proton-conducting pathway in bacteriorhodopsin (BR), a light-driven proton pump of archaea. Nevertheless, NR does not appear to pump protons, suggesting the absence of the reprotonation switch that is necessary for the active transport. The photocycle of NR is much slower than that of BR, similar to the case of pharaonis phoborhodopsin (ppR), an archaeal photosensory protein. The functional and photochemical differences between NR and BR should be explained in the structural context. In this paper, we studied the structural changes of NR following retinal photoisomerization by means of low-temperature Fourier transform infrared (FTIR) spectroscopy and compared the obtained spectra with those for BR. For the spectroscopic analysis, we established the light-adaptation procedure for NR reconstituted into 1,2-dimyristoyl-sn-glycero- 3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphate (DMPC/DMPA) liposomes, which takes approximately 2 orders of magnitudes longer than in BR. The structure of the retinal chromophore and the hydrogen-bonding strength of the Schiff base in NR are similar to those in BR. Unique spectral features are observed for the S-H stretching vibrations of cysteine and amide-I vibrations for NR before and after retinal isomerization. In NR, there are no spectral changes assignable to the amide bands of alpha helices. The most prominent difference between NR and BR was seen for the water O-D stretching vibrations (measured in D(2)O). Unlike for haloarchaeal rhodopsins such as BR and ppR, no O-D stretches of water under strong hydrogen-bonded conditions (<2400 cm(-1)) were observed in the NR(K) minus NR difference spectra. This suggests a unique hydrogen-bonded network of the Schiff base region, which may be responsible for the lack of the reprotonation switch in NR.

Amino Acid Sequence↗

PubMedDate not supplied

Mechanisms of photoisomerization of polyenes in confined media: from organic glasses to protein binding cavities.

Photochemical reactivities of model organic systems (stilbene and diphenylbutadiene) in organic glasses were first examined and compared with those in solution and in organized media. These observations were in turn compared with reactivities of polyene chromophores in protein binding cavities (specifically PYP, rhodopsin and bacteriorhodopsin). The obvious conclusion is that the preference for the most volume-conserving Hula-twist mechanism isomerization in organic glasses is because of the close interaction between the guest and the host molecules. In organized media (zeolites, crystals and protein binding cavities), the residual empty space coupled with any specific guest-host interactions that are characteristic of a given system, could lead to involvement of the more volume-demanding one-bond-flip (i.e. torsional relaxation) or bicycle-pedal or an extended HT process in photoisomerization.

Bacterial Proteins↗

PubMed2001

Pharaonis phoborhodopsin binds to its cognate truncated transducer even in the presence of a detergent with a 1:1 stoichiometry.

Pharaonis phoborhodopsin (ppR) (also pharaonis sensory rhodopsin II) is a receptor of the negative phototaxis of Natronobacterium pharaonis. ppR forms a complex with its pharaonis halobacterial transducer (pHtrII), and this complex transmits the light signal to the sensory system in the cytoplasm. The expressed C-terminal-His tagged ppR and C-terminal-His tagged truncated pHtrII (t-Htr) in Escherichia coli (His means the 6x histidine tag) form a complex even in the presence of 0.1% of n-dodecyl-beta-D-maltoside, and the M-decay of the complex became about twice slower than that of ppR alone. The photocycling rates under varying concentration ratios of ppR to t-Htr in the presence of detergent were measured. The data were analyzed on the following assumptions: (1) the M-decay of both ppR alone and the complex followed a single exponential decay with different time constants; and (2) the M-decay under varying concentration ratios of ppR to t-Htr, therefore, followed a biexponential decay function which combined the decay of the free ppR and that of the complex as photoreactive species. From these analyses we estimated the dissociation constant (15.2 +/- 1.8 microM) and the number of binding sites (1.2 +/- 0.08).

Archaeal Proteins↗

PubMed2004

Role of charged residues of pharaonis phoborhodopsin (sensory rhodopsin II) in its interaction with the transducer protein.

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, NpSRII) is a receptor for negative phototaxis in Natronomonas (Natronobacterium) pharaonis. In membranes, it forms a 2:2 complex with its transducer protein, pHtrII, which transmits light signals into the cytoplasmic space through protein-protein interactions. We previously found that a specific deprotonated carboxyl of ppR or pHtrII strengthens their binding [Sudo, Y., et al. (2002) Biophys. J. 83, 427-432]. In this study we aim to identify this carboxyl group. Since the D75N mutant has only one photointermediate (ppR(O)(-)(like)) whose existence spans the millisecond time range, the analysis of its decay rate is simple. We prepared various D75N mutants such as D75N/D214N, D75N/K157Q/R162Q/R164Q (D75N/3Gln), D75N/D193N, and D75N/D193E, among which only D75N/D193N did not show pH dependence with regard to the ppR(O)(-)(like) decay rate and K(D) value for binding, implying that the carboxyl group in question is from Asp-193. The pK(a) of this group decreased to below 2 when a complex was formed. Therefore, we conclude that Asp-193(p)()(pR) is connected to the distant transducer-ppR binding surface via hydrogen bonds, thereby modulating its pK(a). In addition, we discuss the importance of Arg-162(p)()(pR) with respect to the binding activity.

Amino Acid Sequence↗

PubMed2004

Proton release and uptake of pharaonis phoborhodopsin (sensory rhodopsin II) reconstituted into phospholipids.

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a photo-receptor for negative phototaxis in Natronobacterium pharaonis. During the photoreaction cycle (photocycle), ppR exhibits intraprotein proton movements, resulting in proton pumping from the cytoplasmic to the extracellular side, although it is weak. In this study, light-induced proton uptake and release of ppR reconstituted with phospholipid were analyzed using a SnO(2) electrode. The reconstituted ppR exhibited properties in proton uptake and release that are different from those of dodecyl maltoside solubilized samples. It showed fast proton release before the decay of ppR(M) (M-photointermediate) followed by proton uptake, which was similar to that of bacteriorhodopsin (BR), a light-driven proton pump. Mutant analysis assigned Asp193 to one (major) of the members of the proton-releasing group (PRG). Fast proton release was observed only when the pH was approximately 5-8 in the presence of Cl(-). When Cl(-) was replaced with SO(4)(2-), the reconstituted ppR did not exhibit fast proton release at any pH, suggesting Cl(-) binding around PRG. PRG in BR consists of Glu204 (Asp193 in ppR) and Glu194 (Pro183 in ppR). Replacement of Pro183 by Glu/Asp, a negatively charged residue, led to Cl(-)-independent fast proton release. The transducer binding affected the properties of PRG in ppR in the ground state and in the ppR(M) state, suggesting that interaction with the transducer extends to the extracellular surface of ppR. Differences and similarities in the molecular mechanism of the proton movement between ppR and BR are discussed.

Amino Acid Substitution↗

PubMed1995

1.4 A 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.

Amino Acid Sequence↗

PubMed1987

Properties of a water-soluble, yellow protein isolated from a halophilic phototrophic bacterium that has photochemical activity analogous to sensory rhodopsin.

A water-soluble yellow protein, previously discovered in the purple photosynthetic bacterium Ectothiorhodospira halophila, contains a chromophore which has an absorbance maximum at 446 nm. The protein is now shown to be photoactive. A pulse of 445-nm laser light caused the 446-nm peak to be partially bleached and red-shifted in a time less than 1 microsecond. The intermediate thus formed was subsequently further bleached in the dark in a biphasic process occurring in approximately 20 ms. Finally, the absorbance of native protein was restored in a first-order process occurring over several seconds. These kinetic processes are remarkably similar to those of sensory rhodopsin from Halobacterium, and to a lesser extent bacteriorhodopsin and halorhodopsin; although these proteins are membrane-bound, they have absorbance maxima at about 570 nm, and they cycle more rapidly. In attempts to remove the chromophore for identification, it was found that a variety of methods of denaturation of the protein caused transient or permanent conversion to a form which has an absorbance maximum near 340 nm. Thus, by analogy to the rhodopsins, the absorption at 446 nm in the native protein appears to result from a 106-nm red shift of the chromophore induced by the protein. Acid denaturation followed by extraction with organic solvents established that the chromophore could be removed from the protein. It is not identical with all-trans-retinal and remains to be identified, although it could still be a related pigment. The E. halophila yellow protein has a circular dichroism spectrum which indicates little alpha-helical secondary structure (19%).(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗