Cognate Transducer Protein Research Articles

Retinal Configuration of ppR Intermediates Revealed by Photoirradiation Solid-State NMR and DFT

Pharanois phoborhodopsin (ppR) from Natronomonas pharaonis is a transmembrane photoreceptor protein involved in negative phototaxis. Structural changes in ppR triggered by photoisomerization of the retinal chromophore are transmitted to its cognate transducer protein (pHtrII) through a cyclic photoreaction pathway involving several photointermediates. This pathway is called the photocycle. It is important to understand the detailed configurational changes of retinal during the photocycle. We previously observed one of the photointermediates (M-intermediates) by in situ photoirradiation solid-state NMR experiments. In this study, we further observed the 13C cross-polarization magic-angle-spinning NMR signals of late photointermediates such as O- and N′-intermediates by illumination with green light (520 nm). Under blue-light (365 nm) irradiation of the M-intermediates, 13C cross-polarization magic-angle-spinning NMR signals of 14- and 20-13C-labeled retinal in the O-intermediate appeared at 115.4 and 16.4 ppm and were assigned to the 13-trans, 15-syn configuration. The signals caused by the N′-intermediate appeared at 115.4 and 23.9 ppm and were assigned to the 13-cis configuration, and they were in an equilibrium state with the O-intermediate during thermal decay of the M-intermediates at −60°C. Thus, photoirradiation NMR studies revealed the photoreaction pathways from the M- to O-intermediates and the equilibrium state between the N′- and O-intermediate. Further, we evaluated the detailed retinal configurations in the O- and N′-intermediates by performing a density functional theory chemical shift calculation. The results showed that the N′-intermediate has a 63° twisted retinal state due to the 13-cis configuration. The retinal configurations of the O- and N′-intermediates were determined to be 13-trans, 15-syn, and 13-cis, respectively, based on the chemical shift values of [20-13C] and [14-13C] retinal obtained by photoirradiation solid-state NMR and density functional theory calculation.

Thermal and Spectroscopic Characterization of a Proton Pumping Rhodopsin from an Extreme Thermophile

So far retinylidene proteins (∼rhodopsin) have not been discovered in thermophilic organisms. In this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium Thermus thermophilus, which lives in a hot spring at around 75 °C. The gene for the retinylidene protein, named thermophilic rhodopsin (TR), was chemically synthesized with codon optimization. The codon optimized TR protein was functionally expressed in the cell membranes of Escherichia coli cells and showed active proton transport upon photoillumination. Spectroscopic measurements revealed that the purified TR bound only all-trans-retinal as a chromophore and showed an absorption maximum at 530 nm. In addition, TR exhibited both photocycle kinetics and pH-dependent absorption changes, which are characteristic of rhodopsins. Of note, time-dependent thermal denaturation experiments revealed that TR maintained its absorption even at 75 °C, and the denaturation rate constant of TR was much lower than those of other proton pumping rhodopsins such as archaerhodopsin-3 (200 ×), Haloquadratum walsbyi bacteriorhodopsin (by 10-times), and Gloeobacter rhodopsin (100 ×). Thus, these results suggest that microbial rhodopsins are also distributed among thermophilic organisms and have high stability. TR should allow the investigation of the molecular mechanisms of ion transport and protein folding.

Protein-Protein Interaction Changes in an Archaeal Light-Signal Transduction

Negative phototaxis in Natronomonas pharaonis is initiated by transient interaction changes between photoreceptor and transducer. pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) and the cognate transducer protein, pHtrII, form a tight 2 : 2 complex in the unphotolyzed state, and the interaction is somehow altered during the photocycle of ppR. We have studied the signal transduction mechanism in the ppR/pHtrII system by means of low-temperature Fourier-transform infrared (FTIR) spectroscopy. In the paper, spectral comparison in the absence and presence of pHtrII provided fruitful information in atomic details, where vibrational bands were identified by the use of isotope-labeling and site-directed mutagenesis. From these studies, we established the two pathways of light-signal conversion from the receptor to the transducer; (i) from Lys205 (retinal) of ppR to Asn74 of pHtrII through Thr204 and Tyr199, and (ii) from Lys205 of ppR to the cytoplasmic loop region of pHtrII that links Gly83.

Characterization of a Signaling Complex Composed of Sensory Rhodopsin I and Its Cognate Transducer Protein from the EubacteriumSalinibacter ruber

Sensory rhodopsin I (SRI) exists in the cell membranes of microorganisms such as the archaeon Halobacterium salinarum and is a photosensor responsible for positive and negative phototaxis. SRI forms a signaling complex with its cognate transducer protein, HtrI, in the membrane. That complex transmits light signals to the flagellar motor through changes in protein-protein interactions with the kinase CheA and the adaptor protein CheW, which controls the direction of the rotation of the flagellar motor. Recently, we cloned and characterized Salinibacter sensory rhodopsin I (SrSRI), which is the first SRI-like protein identified in eubacteria [Kitajima-Ihara, T., et al. (2008) J. Biol. Chem. 283, 23533-23541]. Here we cloned and expressed SrSRI with its full-length transducer protein, SrHtrI, as a fusion construct. We succeeded in producing the complex in Escherichia coli as a recombinant protein with high quality having all-trans-retinal as a chromophore for SRI, although the expression level was low (0.10 mg/L of culture). In addition, we report here the photochemical properties of the SrSRI-SrHtrI complex using time-resolved laser flash spectroscopy and other spectroscopic techniques and compare them to SrSRI without SrHtrI.

Functional Expression of the Signaling Complex Sensory Rhodopsin II/Transducer II from Halobacterium salinarum in Escherichia coli†

Sensory rhodopsin II, a photoreceptor from Halobacterium salinarum (HsSRII), in complex with its cognate transducer protein (HsHtrII) triggers the photophobic response via a cytoplasmic two-component signaling cascade. HsHtrII possess in addition to the HsSRII binding and the cytoplasmic domains an extracellular serine-receptor domain. Here we describe the properties of HsSRII and HsHtrII and those of various shortened transducer analogs, heterologously expressed in Escherichia coli. HsSRII displays the photocycle typical of archaeal photosensors with prolonged kinetics. Using an isothermal titration calorimetric analysis for this complex a dissociation constant of 1.1 microm was obtained similar to that of the corresponding transducer/receptor pair from Natronobacterium pharaonis. A shortened transducer lacking the extracellular and cytoplasmic domain is also sufficient to bind the receptor with a slightly lower affinity. The dissociation constant of serine binding to the extracellular domain was determined to be about 5 microm. This result is in line with the proposal that the extracellular domain indeed is a serine receptor.

3P-214 細菌の光センサーSRIとトランスデューサーHtrlの複合体の性質(光生物-視覚・光受容,第47回日本生物物理学会年会)

3P-214 細菌の光センサーSRIとトランスデューサーHtrlの複合体の性質(光生物-視覚・光受容,第47回日本生物物理学会年会)

Protein–Protein Interaction of a Pharaonis Halorhodopsin Mutant Forming a Complex with Pharaonis Halobacterial Transducer Protein II Detected by Fourier‐Transform Infrared Spectroscopy†

Pharaonis halorhodopsin (pHR) functions as a light-driven inward chloride ion pump in Natoronomonas pharaonis, while pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, pSRII), is a light sensor for negative phototaxis. ppR forms a 2:2 complex with its cognate transducer protein (pHtrII) through intramembranous hydrogen bonds: Tyr199(ppR)-Asn74(pHtrII) and Thr189(ppR)-Glu43 (pHtrII), Ser62(pHtrII). It was reported that a pHR mutant (P240T/F250Y), which possesses the hydrogen-bonding sites, impairs its pumping activity upon complexation with pHtrII. In this study, effect of the complexation with pHtrII on the structural changes upon formation of the K, L(1) and L(2) intermediates of pHR was investigated by use of Fourier-transform infrared spectroscopy. The vibrational changes of Tyr250(pHR) and Asn74(pHtrII) were detected for the L(1) and L(2) intermediates, supporting that Tyr250(pHR) forms a hydrogen bond with Asn74(pHtrII) as similarly to Tyr199(ppR). The conformational changes of the retinal chromophore were never affected by complexation with pHtrII, but amide-I vibrations were clearly different in the absence and presence of pHtrII. The molecular environment around Asp156(pHR) in helix D is also slightly affected. These additional structural changes are probably related to blocking of translocation of a chloride ion from the extracellular to the cytoplasmic side during the photocycle.

Steric Constraint in the Primary Photoproduct of Sensory Rhodopsin II Is a Prerequisite for Light-Signal Transfer to HtrII

Sensory rhodopsin II (SRII, also called pharaonis phoborhodopsin, ppR) is responsible for negative phototaxis in Natronomonas pharaonis. Photoisomerization of the retinal chromophore from all- trans to 13- cis initiates conformational changes in the protein, leading to activation of the cognate transducer protein (HtrII). We previously observed enhancement of the C 14-D stretching vibration of the retinal chromophore at 2244 cm (-1) upon formation of the K state and interpreted that a steric constraint occurs at the C 14D group in SRII K. Here, we identify the counterpart of the C 14D group as Thr204, because the C 14-D stretching signal disappeared in T204A, T204S, and T204C mutants as well as a C 14-HOOP (hydrogen out-of-plane) vibration at 864 cm (-1). Although the K state of the wild-type bacteriorhodopsin (BR), a light-driven proton pump, possesses neither 2244 nor 864 cm (-1) bands, both signals appeared for the K state of a triple mutant of BR that functions as a light sensor (P200T/V210Y/A215T). We found a positive correlation between these vibrational amplitudes of the C 14 atom at 77 K and the physiological phototaxis response. These observations strongly suggest that the steric constraint between the C 14 group of retinal and Thr204 of the protein is a prerequisite for light-signal transduction by SRII.

S11I5 膜蛋白質間相互作用による光情報伝達を考える(膜蛋白質を介したエネルギー・情報変換機構を若手研究者が深<考える,シンポジウム,第45回日本生物物理学会年会)

S11I5 膜蛋白質間相互作用による光情報伝達を考える(膜蛋白質を介したエネルギー・情報変換機構を若手研究者が深<考える,シンポジウム,第45回日本生物物理学会年会)

Assignment of the Hydrogen-Out-Of-Plane and -in-Plane Vibrations of the Retinal Chromophore in the K Intermediate of pharaonis Phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor protein for negative phototaxis in Natronomonas pharaonis. Photoisomerization of the retinal chromophore from all-trans to 13-cis initiates conformational changes of the protein leading to activation of the cognate transducer protein (pHtrII). Elucidation of the initial photoreaction, formation of the K intermediate of ppR, is important for understanding the mechanism of storage of photon energy. We have reported the K minus ppR Fourier transform infrared (FTIR) spectra, including several vibrational bands of the retinal, the protein, and internal water molecules. It is interesting that more vibrational bands were observed in the hydrogen-out-of-plane (HOOP) region than for the light-driven proton pump, bacteriorhodopsin. This result implied that the steric constraints on the retinal chromophore in the binding pocket of ppR are distributed more widely upon formation of the initial intermediate. In this study, we assigned the HOOP and hydrogen-in-plane vibrations by means of low-temperature FTIR spectroscopy applied to ppR reconstituted with retinal deuterated at C7, C8, C10-C12, C14, and C15. As a result, the 966 (+)/971 (-) and 958 (+)/961 (-) cm(-1) bands were assigned to the C7=C8 and C11=C12 Au HOOP modes, respectively, suggesting that the structural changes spread to the middle part of the retinal. The positive bands at 1001, 994, 987, and 979 cm(-1) were assigned to the C15-HOOP vibrations of the K intermediate, whose frequencies are similar to those of the K(L) intermediate of bacteriorhodopsin trapped at 135 K. Another positive band at 864 cm(-1) was assigned to the C14-HOOP vibration. Relatively many positive bands of hydrogen-in-plane vibrations supported the wide distribution of structural changes of the retinal as well. These results imply that the light energy was stored mainly in the distortions around the Schiff base region while some part of the energy was transferred to the distal part of the retinal.

Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phosphotransfer in vitro.

A chimeric fusion protein consisting of Natronomonas pharaonis sensory rhodopsin II (SRII), fused by a flexible linker to the two transmembrane helices of its cognate transducer protein, HtrII, followed by the HtrII membrane-proximal cytoplasmic fragment joined to the cytoplasmic domains of the Escherichia coli chemotaxis receptor Tsr, was expressed in E. coli. Purified fusion chimera protein reconstituted in liposomes binds to E. coli CheA kinase in the presence of the coupling protein CheW, and activates CheA autophosphorylation activity. CheA kinase activity is stimulated by photoexcitation of the SRII domain of the fusion protein, as shown by the wavelength-dependence of photostimulated phosphotransfer to the E. coli flagellar motor response regulator CheY in the purified in vitro liposomal system. Further confirming the fidelity of the in vitro system, increased and decreased levels of CheA activation in vitro result from overmethylated and undermethylated fusion protein purified from methylesterase and methyltransferase-deficient E. coli, respectively. Photoexcitation of the undermethylated fusion protein resulted in a 3-fold increase in phosphotransfer over that of the dark state. The results directly demonstrate the coupling of SRII photoactivated states to histidine kinase activity, previously predicted on the basis of sequence homologies of the haloarchaeal phototaxis system components to those of E. coli chemotaxis. The fusion chimera provides the first tool for in vitro measurement of photosignaling activity of SRII-HtrII molecular complexes.

Conformational Changes Detected in a Sensory Rhodopsin II-Transducer Complex

Sensory rhodopsins (SRs) are light receptors that belong to the growing family of microbial rhodopsins. SRs have now been found in all three major domains of life including archaea, bacteria, and eukaryotes. One of the most extensively studied sensory rhodopsins is SRII, which controls a blue light avoidance motility response in the halophilic archaeon Natronobacterium pharaonis. This seven-helix integral membrane protein forms a tight intermolecular complex with its cognate transducer protein, HtrII. In this work, the structural changes occurring in a fusion complex consisting of SRII and the two transmembrane helices (TM1 and TM2) of HtrII were investigated by time-resolved Fourier transform infrared difference spectroscopy. Although most of the structural changes observed in SRII are conserved in the fusion complex, several distinct changes are found. A reduction in the intensity of a prominent amide I band observed for SRII indicates that its structural changes are altered in the fusion complex, possibly because of the close interaction of TM2 with the F helix, which interferes with the F helix outward tilt. Deprotonation of at least one Asp/Glu residue is detected in the transducer-free receptor with a pKa near 7 that is abolished or altered in the fusion complex. Changes are also detected in spectral regions characteristic of Asn and Tyr vibrations. At high hydration levels, transducer-fusion interactions lead to a stabilization of an M-like intermediate that most likely corresponds to an active signaling form of the transducer. These findings are discussed in the context of a recently elucidated x-ray structure of the fusion complex.

FTIR spectroscopy of the complex between pharaonis phoborhodopsin and its transducer protein.

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psRII) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. ppR activates the cognate transducer protein, pHtrII, upon absorption of light. ppR and pHtrII form a tight 2:2 complex in the unphotolyzed state, and the interaction is somehow altered during the photocycle of ppR. In this paper, we studied the influence of pHtrII on the structural changes occurring upon retinal photoisomerization in ppR by means of low-temperature FTIR spectroscopy. We trapped the K intermediate at 77 K and compared the ppR(K) minus ppR spectra in the absence and presence of pHtrII. There are no differences in the X-D stretching vibrations (2700-1900 cm(-1)) caused by presence of pHtrII. This result indicates that the hydrogen-bonding network in the Schiff base region is not altered by interaction with pHtrII, which is consistent with the same absorption spectrum of ppR with or without pHtrII. In contrast, the ppR(K) minus ppR infrared difference spectra are clearly influenced by the presence of pHtrII in amide-I (1680-1640 cm(-1)) and amide-A (3350-3250 cm(-1)) vibrations. The identical spectra for the complex of the unlabeled ppR and (13)C- or (15)N-labeled pHtrII indicate that the observed structural changes for the peptide backbone originate from ppR only and are altered by retinal photoisomerization. The changes do not come from pHtrII, implying that the light signal is not transmitted to pHtrII in ppR(K). In addition, we observed D(2)O-insensitive bands at 3479 (-)/3369 (+) cm(-1) only in the presence of pHtrII, which presumably originate from an X-H stretch of an amino acid side chain inside the protein.