Azide accelerates the decay of M-intermediate of pharaonis phoborhodopsin

Natronobacterium pharaonis has retinal proteins, one of which is pharaonis phoborhodopsin, abbreviated as ppR (or called pharaonis sensory rhodopsin II, psR-II). This pigment protein functions as a photoreceptor of the negative phototaxis of this bacterium. On photoexcitation ppR undergoes photocycling; the photoexcited state relaxes in the dark and returns to the original state via several intermediates. The photocycle of ppR resembles that of bR except in wavelengths and rate. The cycle of bR is completed in 10 ms while that of ppR takes seconds. The Arrhenius analysis of M-intermediate (ppR(M)) decay which is rate-limiting revealed that the slow decay is due to the large negative activation entropy of ppR. The addition of azide increases the decay rate 300-fold (at pH 7); Arrhenius analysis revealed decreases in the activation energy (activation enthalpy) and a further decrease in the activation entropy.     

Association of pharaonis phoborhodopsin with its cognate transducer decreases the photo-dependent reactivity by water-soluble reagents of azide and hydroxylamine.

pharaonis phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a receptor of the negative phototaxis of Natronobacterium pharaonis. In halobacterial membrane, ppR forms a complex with its transducer pHtrII, and this complex transmits the light signal to the sensory system in the cytoplasm. In the present work, the truncated transducer, t-Htr, was used which interacts with ppR [Sudo et al. (2001) Photochem. Photobiol. 74, 489-494]. Two water-soluble reagents, hydroxylamine and azide, reacted both with the transducer-free ppR and with the complex ppR/t-Htr (the complex between ppR and its truncated transducer). In the dark, the bleaching rates caused by hydroxylamine were not significantly changed between transducer-free ppR and ppR/t-Htr, or that of the free ppR was a little slower. Illumination accelerated the bleach rates, which is consistent with our previous conclusion that the reaction occurs selectively at the M-intermediate, but the rate of the complex was about 7.4-fold slower than that of the transducer-free ppR. Azide accelerated the M-decay, and its reaction rate of ppR/t-Htr was about 4.6-fold slower than free ppR. These findings suggest that the transducer binding decreases the water accessibility around the chromophore at the M-intermediate. Its implication is discussed.     

Interaction of Asn105 with the retinal chromophore during photoisomerization of pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. ppR has a blue-shifted absorption spectrum with a spectral shoulder, which is highly unique for the archaeal rhodopsin family. The primary reaction of ppR is a cis-trans photoisomerization of the retinal chromophore to form the K intermediate, like the well-studied proton pump bacteriorhodopsin (BR). Recent comparative FTIR spectroscopy of the K states in ppR and BR revealed that more extended structural changes take place in ppR than in BR with respect to chromophore distortion and protein structural changes [Kandori, H., Shimono, K., Sudo, Y., Iwamoto, M., Shichida, Y., and Kamo, N. (2001) Biochemistry 40, 9238-9246]. FTIR spectroscopy of the N105D mutant protein reported here assigns the vibrational bands at 1704 and 1700 cm(-1) as C=O stretches of Asn105 in ppR and ppR(K), respectively. A comparative investigation between ppR and BR further reveals that the structure at position 105 in ppR is similar to that of the corresponding position (Asp115) in BR; this observation is supported by the recent X-ray crystallographic structures of ppR [Luecke, H., Schobert, B., Lanyi, J. K., Spudich, E. N., and Spudich, J. L. (2001) Science 293, 1499-1503; Royant, A., Nollert, P., Edman, K., Neutze, R., Landau, E. M., Pebay-Peyroulla, E., and Navarro, J. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 10131-10136]. Nevertheless, structural changes upon photoisomerization at position 105 in ppR are greater than those at position 115 in BR. As a consequence of a unique chromophore-protein interaction in ppR, extended protein structural changes accompanying retinal photoisomerization occur, and these include Asn105 which is approximately 7 A from the retinal chromophore.     

FTIR spectroscopy of the M photointermediate in pharaonis rhoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. During the photocycle of ppR, the Schiff base of the retinal chromophore is deprotonated upon formation of the M intermediate (ppR(M)). The present FTIR spectroscopy of ppR(M) revealed that the Schiff base proton is transferred to Asp-75, which corresponds to Asp-85 in a light-driven proton-pump bacteriorhodopsin (BR). In addition, the C==O stretching vibrations of Asn-105 were assigned for ppR and ppR(M). The common hydrogen-bonding alterations in Asn-105 of ppR and Asp-115 of BR were found in the process from photoisomerization (K intermediate) to the primary proton transfer (M intermediate). These results implicate similar protein structural changes between ppR and BR. However, BR(M) decays to BR(N) accompanying a proton transfer from Asp-96 to the Schiff base and largely changed protein structure. In the D96N mutant protein of BR that lacks a proton donor to the Schiff base, the N-like protein structure was observed with the deprotonated Schiff base (called M(N)) at alkaline pH. In ppR, such an N-like (M(N)-like) structure was not observed at alkaline pH, suggesting that the protein structure of the M state activates its transducer protein.     

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.     

Photo-induced proton transport of pharaonis phoborhodopsin (sensory rhodopsin II) is ceased by association with the transducer

Phoborhodopsin (pR; also sensory rhodopsin II, sRII) is a retinoid protein in Halobacterium salinarum and works as a receptor of negative phototaxis. Pharaonis phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. In bacterial membrane, ppR forms a complex with its transducer pHtrII, and this complex transmits the light signal to the sensory system in the cytoplasm. We expressed pHtrII-free ppR or ppR-pHtrII complex in H. salinarum Pho81/wr(-) cells. Flash-photolysis experiments showed no essential changes between pHtrII-free ppR and the complex. Using SnO2 electrode, which works as a sensitive pH electrode, and envelope membrane vesicles, we showed the photo-induced outward proton transport. This membranous proton transport was also shown using membrane vesicles from Escherichia coli in which ppR was functionally expressed. On the other hand, the proton transport was ceased when ppR formed a complex with pHtrII. Using membrane sheet, it was shown that the complex undergoes first proton uptake and then release during the photocycle, the same as pHtrII-free ppR, although the net proton transport ceases. Taking into consideration that the complex of sRII (pR) and its transducer undergoes extracellular proton circulation (J. Sasaki and J. L., Biophys. J. 77:2145-2152), we inferred that association with pHtrII closes a cytoplasmic channel of ppR, which lead to the extracellular proton circulation.     

Internal water molecules of pharaonis phoborhodopsin studied by low-temperature infrared spectroscopy.

In the Schiff base region of bacteriorhodopsin (BR), a light-driven proton-pump protein, three internal water molecules are involved in a pentagonal cluster structure. These water molecules constitute a hydrogen-bonding network consisting of two positively charged groups, the Schiff base and Arg82, and two negatively charged groups, Asp85 and Asp212. Previous infrared spectroscopy of BR revealed stretching vibrations of such water molecules under strong hydrogen-bonding conditions using spectral differences in D2O and D2(18O) [Kandori and Shichida (2000) J. Am. Chem. Soc. 122, 11745-11746]. The present study extends the infrared analysis to another archaeal rhodopsin, pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin-II, psR-II), involved in the negative phototaxis of Natronobacterium pharaonis. Despite functional differences between ppR and BR, similar spectral features of water bands were observed before and after photoisomerization of the retinal chromophore at 77 K. This implies that the structure and the structural changes of internal water molecules are similar between ppR and BR. Higher stretching frequencies of the bridged water in ppR suggest that the water-containing pentagonal cluster structure is considerably distorted in ppR. These observations are consistent with the crystallographic structures of ppR and BR. The water structure and structural changes upon photoisomerization of ppR are discussed here on the basis of their infrared spectra.     

FTIR spectroscopy of the O photointermediate in pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor protein for negative phototaxis in Natronobacterium pharaonis. During the photocycle of ppR, the retinal chromophore is thermally isomerized from the 13-cis to all-trans form. We employed FTIR spectroscopy of ppR at 260 K and pH 5 to reveal that this isomerization occurs upon formation of the O intermediate (ppR(O)) by using ppR samples reconstituted with 12,14-D(2)-labeled retinal. In ppR(O), C=O stretching vibrations of protonated carboxylates newly appear at 1757 (+)/1722 (-) cm(-1) in H(2)O and at 1747 (+)/1718 (-) cm(-1) in D(2)O in addition to the 1765 (+) cm(-1) band of Asp75. Amide I vibrations are basically similar between ppR(M) and ppR(O), whereas unique bands of ppR(O) are also observed such as the negative 1656 cm(-1) band in D(2)O and intense bands at 1686 (-)/1674 (+) cm(-1). In addition, O-D stretching vibrations of water molecules in the entire mid-infrared region are assigned for ppR(M) and ppR(O), the latter being unique for ppR, since it can be detected at low temperature (260 K). The ppR(M) minus ppR difference spectra lack the lowest frequency water band (2215 cm(-1)) observed in the ppR(K) minus ppR spectra, which is probably associated with water that interacts with the negative charges in the Schiff base region. It is likely that the proton transfer from the Schiff base to Asp75 in ppR(M) can be explained by a hydration switch of a water from Asp75 to Asp201, as was proposed for the light-driven proton-pump bacteriorhodopsin (hydration switch model) [Tanimoto, T., Furutani, Y., and Kandori, H. (2003) Biochemistry 42, 2300-2306]. In the transition from ppR(M) to ppR(O), a hydrogen-bonding alteration takes place for another water molecule that forms a strong hydrogen bond.     

Vibrational modes of the protonated Schiff base in pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. Recent X-ray crystallographic structures showed that ppR and bacteriorhodopsin (BR), a light-driven proton pump, possess similar molecular environments of the retinal Schiff base. Nevertheless, absorption spectra are different by 70 nm between ppR and BR, suggesting the different chromophore-protein interactions involving the Schiff base region. In this article, we identify frequencies of the Schiff base vibrations in the ppR(K) minus ppR difference spectra by means of low-temperature FTIR spectroscopy of [zeta-(15)N]lysine-labeled ppR. The N-D stretch in D(2)O was found at 2140 and 2091 cm(-1) for ppR, which are shifted to a lower frequency by 32-33 cm(-1) compared to those for BR. This observation indicates the stronger hydrogen bond of the Schiff base in ppR than in BR. The N-D stretch of the Schiff base and O-D stretch of water molecules are located at the different frequencies in ppR, while they appear in the same frequency region in BR [Kandori, H., Belenky, M., and Herzfeld, J. (2002) Biochemistry 41, 6026-6031]. These differences could be correlated with the distorted pentagonal cluster structure in ppR. In contrast, the N-D stretch of ppR(K) was found at 2474 cm(-1), which is close in frequency to that of BR(K). The O-D stretch of Thr79 was also assigned at 2512 and 2474 cm(-1) for ppR and ppR(K), respectively. These frequencies are close to those of BR, suggesting the interaction of Thr79 and Asp75 in ppR is similar to that of Thr89 and Asp85 in BR.     

Photocycle of phoborhodopsin from haloalkaliphilic bacterium (Natronobacterium pharaonis) studied by low-temperature spectrophotometry.

Phoborhodopsin (pR) is the fourth retinal pigment of Halobacterium halobium and works as a photoreceptor for the negative phototactic response. A similar pigment was previously found in haloalkaliphilic bacterium (Natronbacterium pharaonis) and also works as the receptor of the negative phototactic response; this pigment is called pharaonis phoborhodopsin (ppR). In this paper, the photocycle of ppR was investigated by means of low-temperature spectrophotometry. The absorption maximum of ppR is located at 498 nm, while that of pR is at 487 nm. The absorption spectra of the two have similar vibrational structures. Irradiation of ppR below -100 degrees C produced a K-like intermediate (ppRK) which was a composite of two components. The original ppR and ppRK were perfectly photoreversible. On warming, ppRK was directly converted to an M-like intermediate without formation of the L-like intermediate. The M-like intermediate was converted to the O-like intermediate at pH 7.2, but the O-like intermediate was not detected at pH 9.0. The O-like intermediate then reverted to the original pigment. On the basis of these findings, the photocycle and the primary photochemical process of ppR are presented.     

Temperature-dependent interactions between photoactivated pharaonis phoborhodopsin and its transducer

Pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronomonas pharaonis. In membranes, it forms a 2:2 complex with its transducer protein pHtrII, and the association is weakened by 2 orders of magnitude in the M intermediate (ppR(M)). Such a change is believed to correspond to the transfer of the light signal to pHtrII. A previous Fourier transform infrared (FTIR) study observed hydrogen-bonding alteration of Asn74 in pHtrII in the M state, suggesting a light-signaling pathway from the receptor to the transducer [Furutani, Y., Kamada, K., Sudo, Y., Shimono, K., Kamo, N., and Kandori, H. (2005) Biochemistry 44, 2909-2915]. In this paper, we measure temperature dependence of the ppR(M) minus ppR spectra in the absence and presence of pHtrII at 250-293 K. Significant temperature dependence was observed for the amide-I vibrations of helices only for the ppR/pHtrII complex, where the amplitude of amide-I vibrations was reduced at room temperature. (13)C-Labeling of ppR or pHtrII revealed that such spectral changes of helices originate from ppR and not pHtrII. The hydrogen-bonding alteration of Asn74 in pHtrII was temperature-independent, implying that the observed helical structural perturbation in ppR takes place in different region. On the other hand, temperature-dependent structural changes of helices were diminished for the complex of ppR with the G83C and G83F mutants of pHtrII. Gly83 is believed to connect the transmembrane helix and cytosolic linker region in a flexible kink near the membrane surface of pHtrII, and its replacement by Cys or Phe abolishes the photosensory function. The present study provides direct experimental evidence that Gly83 plays an important structural role in the activation processes of the ppR/pHtrII complex. A molecular mechanism of protein structural changes in the ppR/pHtrII complex is discussed on the basis of the present FTIR results.     

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.     

Structural changes of pharaonis phoborhodopsin upon photoisomerization of the retinal chromophore: infrared spectral comparison with bacteriorhodopsin

Archaeal rhodopsins possess a retinal molecule as their chromophores, and their light energy and light signal conversions are triggered by all-trans to 13-cis isomerization of the retinal chromophore. Relaxation through structural changes of the protein then leads to functional processes, proton pump in bacteriorhodopsin and transducer activation in sensory rhodopsins. In the present paper, low-temperature Fourier transform infrared spectroscopy is applied to phoborhodopsin from Natronobacterium pharaonis (ppR), a photoreceptor for the negative phototaxis of the bacteria, and infrared spectral changes before and after photoisomerization are compared with those of bacteriorhodopsin (BR) at 77 K. Spectral comparison of the C--C stretching vibrations of the retinal chromophore shows that chromophore conformation of the polyene chain is similar between ppR and BR. This fact implies that the unique chromophore-protein interaction in ppR, such as the blue-shifted absorption spectrum with vibrational fine structure, originates from both ends, the beta-ionone ring and the Schiff base regions. In fact, less planer ring structure and stronger hydrogen bond of the Schiff base were suggested for ppR. Similar frequency changes upon photoisomerization are observed for the C==N stretch of the retinal Schiff base and the stretch of the neighboring threonine side chain (Thr79 in ppR and Thr89 in BR), suggesting that photoisomerization in ppR is driven by the motion of the Schiff base like BR. Nevertheless, the structure of the K state after photoisomerization is different between ppR and BR. In BR, chromophore distortion is localized in the Schiff base region, as shown in its hydrogen out-of-plane vibrations. In contrast, more extended structural changes take place in ppR in view of chromophore distortion and protein structural changes. Such structure of the K intermediate of ppR is probably correlated with its high thermal stability. In fact, almost identical infrared spectra are obtained between 77 and 170 K in ppR. Unique chromophore-protein interaction and photoisomerization processes in ppR are discussed on the basis of the present infrared spectral comparison with BR.     

A pharaonis phoborhodopsin mutant with the same retinal binding site residues as in bacteriorhodopsin

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. ppR has a blue-shifted absorption maximum (500 nm) relative those of other archaeal rhodopsins such as the proton-pump bacteriorhodopsin (BR; 570 nm). Among the 25 amino acids that are within 5 A of the retinal chromophore, 10 are different in BR and ppR, and they are presumed to be crucial in determining the color of their chromophores. However, the spectral red shift in a multiple mutant of ppR, in which the retinal binding site was made similar to that of BR (BR/ppR), was smaller than 40% (lambda(max) = 524 nm) than expected. In the paper presented here, we report on low-temperature Fourier transform infrared (FTIR) spectroscopy of BR/ppR, and compare the infrared spectral changes before and after photoisomerization with those for ppR and BR. The C[bond]C stretch and hydrogen out-of-plane (HOOP) vibrations of BR/ppR were similar to those of BR, suggesting that the surrounding protein moiety of BR/ppR becomes like BR. However, BR/ppR exhibited a unique IR band regarding the hydrogen bond of the protonated Schiff base. It has been known that ppR has a stronger hydrogen bond for the Schiff base than BR as judged from the frequency difference between their C[double bond]NH and C[double bond]ND stretches. We now find that replacement of the 10 amino acids of BR with ppR (BR/ppR) does not weaken the hydrogen bond of the Schiff base. Rather, the hydrogen bond in BR/ppR is stronger than that in the native ppR. We conclude that the principal factor of the smaller than expected opsin shift in BR/ppR is the strong association of the Schiff base with the surrounding counterion complex.     

Proton transfer reactions in the F86D and F86E mutants of pharaonis phoborhodopsin (sensory rhodopsin II)

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII), a negative phototaxis receptor of Natronobacterium pharaonis, can use light to pump a proton in the absence of its transducer protein. However, the pump activity is much lower than that of the light-driven proton-pump bacteriorhodopsin (BR). ppR's pump activity is known to be increased in a mutant protein, in which Phe86 is replaced with Asp (F86D). Phe86 is the amino acid residue corresponding to Asp96 in BR, and we expect that Asp86 plays an important role in the proton transfer at the highly hydrophobic cytoplasmic domain of the F86D mutant ppR. In this article, we studied protein structural changes and proton transfer reactions during the photocycles of the F86D and F86E mutants in ppR by means of Fourier transform infrared (FTIR) spectroscopy and photoelectrochemical measurements using a tin oxide (SnO2) electrode. FTIR spectra of the unphotolyzed state and the K and M intermediates are very similar among F86D, F86E, and the wild type. Asp86 or Glu86 is protonated in F86D or F86E, respectively, and the pK(a) > 9. During the photocycle, the pK(a) is lowered and deprotonation of Asp86 or Glu86 is observed. Detection of both deprotonation of Asp86 or Glu86 and concomitant reprotonation of the 13-cis chromophore implies the presence of a proton channel between position 86 and the Schiff base. However, the photoelectrochemical measurements revealed proton release presumably from Asp86 or Glu86 to the cytoplasmic aqueous phase in the M state. This indicates that the ppR mutants do not have the BR-like mechanism that conducts a proton uniquely from Asp86 or Glu86 (Asp96 in BR) to the Schiff base, which is possible in BR by stepwise protein structural changes at the cytoplasmic side. In ppR, there is a single open structure at the cytoplasmic side (the M-like structure), which is shown by the lack of the N-like protein structure even in F86D and F86E at alkaline pH. Therefore, it is likely that a proton can be conducted in either direction, the Schiff base or the bulk, in the open M-like structure of F86D and F86E.     

Linker region of a halobacterial transducer protein interacts directly with its sensor retinal protein

pHtrII, a pharaonis halobacterial transducer protein, possesses two transmembrane helices and forms a signaling complex with pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, NpSRII) within the halobacterial membrane. This complex transmits a light signal to the sensory system located in the cytoplasm. It has been suggested that the linker region connecting the transmembrane region and the methylation region of pHtrII is important for binding to ppR and subsequent photosignal transduction. In this study, we present evidence to suggest that the linker region itself interacts directly with ppR in addition to the interaction in the membrane region. An in vitro pull-down assay revealed that the linker region bound to ppR, and its dissociation constant (K(D)) was estimated to be approximately 10 microM using isothermal titration calorimetry (ITC). Solution NMR analyses showed that ppR interacted with the linker region of pHtrII (pHtrII(G83)(-)(Q149)) and resulted in the broadening of many peaks, indicating structural changes within this region. These results suggest that the pHtrII linker region interacts directly with ppR. There was no demonstrable interaction between the C-terminal region of ppR (ppR(Gly224)(-)(His247)) and either the linker region (pHtrII(G83)(-)(Q149)) or the transmembrane region (pHtrII(M1)(-)(E114)) of pHtrII. On the basis of the NMR, CD, and photochemical data, we discuss the structural changes and role of the linker region of pHtrII in relation to photosignal transduction.     

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.     

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.     

Did protein kinase regulatory mechanisms evolve through elaboration of a simple structural component?

Statistical analysis of the functional constraints acting on eukaryotic protein kinases (EPKs) and on distantly related kinases suggests that EPK regulatory mechanisms evolved around an ancient structural component whose most distinctive features include the HxD-motif adjoining the catalytic loop, the F-helix, an F-helix aspartate, and the DFG-motif adjoined to the activation loop. The HxD-histidine constitutes a convergence point for signal integration, as conserved interactions link it to key catalytic residues, to the F-helix aspartate, and to both ends of the DFG-motif. These and other conserved features appear to be associated with DFG conformational changes and with coordinated movements possibly associated with phosphate transfer and ADP release. The EPKs have acquired structural features that link this core component to likely substrate-interacting regions at either end of the F-helix (most notably involving an F-helix tryptophan) and to three regions undergoing conformational changes upon kinase activation: the activation segment, the C-helix, and the nucleotide-binding pocket.     

Chromophore configuration of pharaonis phoborhodopsin and its isomerization on photon absorption.

The configuration of the retinylidene chromophore in pharaonis phoborhodopsin (ppR) and its changes during the photoreaction cycle were investigated by means of a chromophore extraction method followed by HPLC analysis. The ppR has an all-trans chromophore, and unlike bacteriorhodopsin, it exhibits no dark isomerization of the chromophore. Irradiation of a ppR sample in the presence of 10 mM hydroxylamine, at which concentration a negligible amount of ppR was bleached, caused the formation of 90% 13-cis- and 10% all-trans-retinal oximes. Because the ppR sample under the continuous irradiation was a mixture containing original ppR, ppRM, and a small amount of ppRO, the above results showed that the chromophores of ppRM and ppRO are in a 13-cis form and an all-trans form, respectively. Therefore, the all-trans chromophore of ppR is isomerized to the 13-cis form on photon absorption, and it is thermally reisomerized to the all-trans form on the conversion process from ppRM to ppRO. The extracted retinal oximes from ppR and ppRO were mainly the 15-syn form, while that from ppRM was mainly the 15-anti form. This fact indicated that the attack of hydroxylamine on the chromophore is stereoselective owing to the unique structure of the chromophore binding site near the Schiff base region of the chromophore.