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PubMed2002

Structural basis for sensory rhodopsin function.

The crystal structure of sensory rhodopsin II from Natronobacterium pharaonis was recently solved at 2.1 A resolution from lipidic cubic phase-grown crystals. A critical analysis of previous structure-function studies is possible within the framework of the high-resolution structure of this photoreceptor. Based on the structure, a molecular understanding emerges of the efficiency and selectivity of the photoisomerization reaction, of the interaction of the sensory receptor and its cognate transducer protein HtrII, and of the mechanism of spectral tuning in photoreceptors. The architecture of the retinal binding pocket is compact, representing a major determinant for the selective binding of the chromophore, all-trans retinal to the apoprotein, opsin. Several chromophore-protein interactions revealed by the structure were not predicted by previous mutagenesis and spectroscopic analyses. The structure suggests likely mechanisms by which photoisomerization triggers the activation of sensory rhodopsin II, and highlights the possibility of a unified mechanism of signaling mediated by sensory receptors, including visual rhodopsins. Future investigations using time-resolved crystallography, structural dynamics, and computational studies will provide the basis to unveil the molecular mechanisms of sensory receptors-mediated transmembrane signaling.

Amino Acid Sequence↗
PubMed2000

Charge motions during the photocycle of pharaonis halorhodopsin.

Oriented gel samples were prepared from halorhodopsin-containing membranes from Natronobacterium pharaonis, and their photoelectric responses to laser flash excitation were measured at different chloride concentrations. The fast component of the current signal displayed a characteristic dependency on chloride concentration, and could be interpreted as a sum of two signals that correspond to the responses at high-chloride and no-chloride, but high-sulfate, concentration. The chloride concentration-dependent transition between the two signals followed the titration curve determined earlier from spectroscopic titration. The voltage signal was very similar to that reported by another group (Kalaidzidis, I. V., Y. L. Kalaidzidis, and A. D. Kaulen. 1998. FEBS Lett. 427:59-63). The absorption kinetics, measured at four wavelengths, fit the kinetic model we had proposed earlier. The calculated time-dependent concentrations of the intermediates were used to fit the voltage signal. Although no negative electric signal was observed at high chloride concentration, the calculated electrogenicity of the K intermediate was negative, and very similar to that of bacteriorhodopsin. The late photocycle intermediates (O, HR', and HR) had almost equal electrogenicities, explaining why no chloride-dependent time constant was identified earlier by Kalaidzidis et al. The calculated electrogenicities, and the spectroscopic information for the chloride release and uptake steps of the photocycle, suggest a mechanism for the chloride-translocation process in this pump.

Archaeal Proteins↗
PubMed2000

The M intermediate of Pharaonis phoborhodopsin is photoactive.

The retinal protein phoborhodopsin (pR) (also called sensory rhodopsin II) is a specialized photoreceptor pigment used for negative phototaxis in halobacteria. Upon absorption of light, the pigment is transformed into a short-wavelength intermediate, M, that most likely is the signaling state (or its precursor) that triggers the motility response of the cell. The M intermediate thermally decays into the initial pigment, completing the cycle of transformations. In this study we attempted to determine whether M can be converted into the initial state by light. The M intermediate was trapped by the illumination of a water glycerol suspension of phoborhodopsin from Natronobacterium pharaonis called pharaonis phoborhodopsin (ppR) with yellow light (>450 nm) at -50 degrees C. The M intermediate absorbing at 390 nm is stable in the dark at this temperature. We found, however, that M is converted into the initial (or spectrally similar) state with an absorption maximum at 501 nm upon illumination with 380-nm light at -60 degrees C. The reversible transformations ppR if M are accompanied by the perturbation of tryptophan(s) and probably tyrosine(s) residues, as reflected by changes in the UV absorption band. Illumination at lower temperature (-160 degrees C) reveals two intermediates in the photoconversion of M, which we termed M' (or M'(404)) and ppR' (or ppR'(496)). A third photoproduct, ppR'(504), is formed at -110 degrees C during thermal transformations of M'(404) and ppR'(496). The absorption spectrum of M'(404) (maximum at 404 nm) consists of distinct vibronic bands at 362, 382, 404, and 420 nm that are different from the vibronic bands of M at 348, 368, 390, and 415 nm. ppR'(496) has an absorption band that is shifted to shorter wavelengths by 5 nm compared to the initial ppR, whereas ppR'(504) is redshifted by at least 3 nm. As in bacteriorhodopsin, photoexcitation of the M intermediate of ppR and, presumably, photoisomerization of the chromophore during the M --> M' transition result in a dramatic increase in the proton affinity of the Schiff base, followed by its reprotonation during the M' --> ppR' transition. Because the latter reaction occurs at very low temperature, the proton is most likely taken from the counterion (Asp(75)) rather than from the bulk. The phototransformation of M reveals a certain heterogeneity of the pigment, which probably reflects different populations of M or its photoproduct M'. Photoconversion of the M intermediate provides a possible pathway for photoreception in halobacteria and a useful tool for studying the mechanisms of signal transduction by phoborhodopsin (sensory rhodopsin II).

Archaeal Proteins↗
PubMed2001

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.

Archaeal Proteins↗
PubMed1997

Functional expression of pharaonis phoborhodopsin in Escherichia coli.

Pharaonis phoborhodopsin, the photoreceptor of the negative phototaxis of archaebacterial Natronobacterium pharaonis, was functionally expressed in the heterologous system of Escherichia coli. Flash-photolysis on a millisecond time scale indicated that the photochemical properties of ppR expressed in E. coli were the same as those of the native ppR in N. pharaonis. We concluded that the integral membrane protein ppR is correctly folded in vivo in the eubacterial E. coli membrane.

Archaeal Proteins↗
PubMed2000

Involvement of two groups in reversal of the bathochromic shift of pharaonis phoborhodopsin by chloride at low pH.

Pharaonis phoborhodopsin (ppR; or pharaonis sensory rhodopsin II, psRII) is a photophobic receptor of the halobacterium Natronobacterium pharaonis. Its lambdamax is at 496 nm, but upon acidification in the absence of chloride, lambdamax shifted to 522 nm. This bathochromic shift is thought to be caused by the protonation of Asp75, which corresponds to Asp85 of bacteriorhodopsin (bR). The D75N mutant, in which Asp75 was replaced by Asn, had its lambdamax at approximately 520 nm, supporting this mechanism for the bathochromic shift. A titration of the shift yielded a pKa of 3.5 for Asp75. In the presence of chloride, the spectral shifts were different: with a decrease in pH, a bathochromic shift was first observed, followed by a hypsochromic shift on further acidification. This was interpreted as: the disappearance of a negative charge by the protonation of Asp75 was compensated by the binding of chloride, but it is worthy to note that the binding requires the protonation of another proton-associable group other than Asp75. This is supported by the observation that in the presence of chloride, upon acidification, the lambdamax of D75N even showed a blue shift, showing that the protonation of a proton-associable group (pKa = 1.2) leads to the chloride binding that gives rise to a blue shift.

Archaeal Proteins↗
PubMed1999

Positioning proton-donating residues to the Schiff-base accelerates the M-decay of pharaonis phoborhodopsin expressed in Escherichia coli.

Phoborhodopsin (also called sensory rhodopsin II, sR-II) is a receptor for the negative phototaxis of Halobacterium salinarum (pR), and pharaonis phoborhodopsin (ppR) is the corresponding receptor of Natronobacterium pharaonis. pR and ppR are retinoid proteins and have a photocycle similar to that of bacteriorhodopsin (bR). A major difference between the photocycle of the ion pump bR and the sensor pR or ppR is found in their turnover rates which are much faster for bR. A reason for this difference might be found in the lack of a proton-donating residue to the Schiff base which is formed between the lysine of the opsin and retinal. To reconstruct a bR-like photochemical behavior, we expressed ppR mutants in Escherichia coli in which proton-donating groups have been reintroduced into the cytoplasmic proton channel. In measurement of the photocycle it could be shown that the F86D mutant of ppR (Phe86 was substituted by Asp) showed a faster decay of M-intermediate than the wild-type, which was even accelerated in the F86D/L40T double mutant.

Archaeal Proteins↗
PubMed2002

Early structural rearrangements in the photocycle of an integral membrane sensory receptor.

Sensory rhodopsins are the primary receptors of vision in animals and phototaxis in microorganisms. Light triggers the rapid isomerization of a buried retinal chromophore, which the protein both accommodates and amplifies into the larger structural rearrangements required for signaling. We trapped an early intermediate of the photocycle of sensory rhodopsin II from Natronobacterium pharaonis (pSRII) in 3D crystals and determined its X-ray structure to 2.3 A resolution. The observed structural rearrangements were localized near the retinal chromophore, with a key water molecule becoming disordered and the retinal's beta-ionone ring undergoing a prominent movement. Comparison with the early structural rearrangements of bacteriorhodopsin illustrates how modifications in the retinal binding pocket of pSRII allow subtle differences in the early relaxation of photoisomerized retinal.

Archaeal Proteins↗
PubMed2001

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.

Archaeal Proteins↗
PubMed2001

Molecular mechanism of spectral tuning in sensory rhodopsin II.

Sensory rhodopsin II (SRII) is unique among the archaeal rhodopsins in having an absorption maximum near 500 nm, blue shifted roughly 70 nm from the other pigments. In addition, SRII displays vibronic structure in the lambda(max) absorption band, whereas the other pigments display fully broadened band maxima. The molecular origins responsible for both photophysical properties are examined here with reference to the 2.4 A crystal structure of sensory rhodopsin II (NpSRII) from Natronobacterium pharaonis. We use semiempirical molecular orbital theory (MOZYME) to optimize the chromophore within the chromophore binding site, and MNDO-PSDCI molecular orbital theory to calculate the spectroscopic properties. The entire first shell of the chromophore binding site is included in the MNDO-PSDCI SCF calculation, and full single and double configuration interaction is included for the chromophore pi-system. Through a comparison of corresponding calculations on the 1.55 A crystal structure of bacteriorhodopsin (bR), we identify the principal molecular mechanisms, and residues, responsible for the spectral blue shift in NpSRII. We conclude that the major source of the blue shift is associated with the significantly different positions of Arg-72 (Arg-82 in bR) in the two proteins. In NpSRII, this side chain has moved away from the chromophore Schiff base nitrogen and closer to the beta-ionylidene ring. This shift in position transfers this positively charged residue from a region of chromophore destabilization in bR to a region of chromophore stabilization in NpSRII, and is responsible for roughly half of the blue shift. Other important contributors include Asp-201, Thr-204, Tyr-174, Trp-76, and W402, the water molecule hydrogen bonded to the Schiff base proton. The W402 contribution, however, is a secondary effect that can be traced to the transposition of Arg-72. Indeed, secondary interactions among the residues contribute significantly to the properties of the binding site. We attribute the increased vibronic structure in NpSRII to the loss of Arg-72 dynamic inhomogeneity, and an increase in the intensity of the second excited (1)A(g)(-) -like state, which now appears as a separate feature within the lambda(max) band profile. The strongly allowed (1)B(u)(+)-like state and the higher-energy (1)A(g)(-) -like state are highly mixed in NpSRII, and the latter state borrows intensity from the former to achieve an observable oscillator strength.

Amino Acid Substitution↗
PubMed2003

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.

Amino Acid Substitution↗
PubMed2003

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.

Archaeal Proteins↗
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↗
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↗
PubMed2004

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.

Amides↗
PubMed2005

Role of putative anion-binding sites in cytoplasmic and extracellular channels of Natronomonas pharaonis halorhodopsin.

Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is an inward light-driven Cl(-) ion pump. For efficient Cl(-) transport, the existence of Cl(-)-binding or -interacting sites in both extracellular (EC) and cytoplasmic (CP) channels is postulated. Candidates include Arg123 and Thr126 in EC channels and Lys215 and Thr218 in CP channels. The roles played by these amino acid residues in anion binding and in the photocycle have been investigated by mutation of the amino acid residues at these positions. Anion binding was assayed by changes in circular dichroism and the shift in the absorption maximum upon addition of Cl(-) to anion-free NpHR. The binding affinity was affected in mutants in which certain EC residues had been replaced; this finding revealed the importance of Arg123. On the other hand, mutants in which certain residues in the CP channel were replaced (CP mutants) did not show changes in their dissociation constants. The photocycles of these mutants were also examined, and in the case of the EC mutants, the transition to the last step was greatly delayed; on the other hand, in the CP mutants, L2-photointermediate decay was significantly prolonged, except in the case of K215Q, which lacked the O-photointermediate. The importance of Thr218 for binding of Cl(-) to the CP channel was indicated by these results. On the basis of these observations, the possible anion transport mechanism of NpHR was discussed.

Anions↗
PubMed2005

Structural changes of the complex between pharaonis phoborhodopsin and its cognate transducer upon formation of the M photointermediate.

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 the association is weakened by 2 orders of magnitude in the M intermediate. Such change is believed to correspond to the transfer of the light signal to pHtrII. In this paper, we applied Fourier transform infrared (FTIR) spectroscopy to the active M intermediate in the absence and presence of pHtrII. The obtained difference FTIR spectra were surprisingly similar, notwithstanding the presence of pHtrII. This result strongly suggests that the transducer activation in the ppR-pHtrII system does not induce secondary structure alterations of the pHtrII itself. On the other hand, we found that the hydrogen bond of the OH group of Thr204 is altered in the primary K intermediate, but restored in the M intermediate. The hydrogen bond of Asn74 in pHtrII is strengthened in M, presumably because of the change in interaction with Tyr199 of ppR. These facts provided a light signaling pathway from Lys205 (retinal) of the receptor to Asn74 of the transducer through Thr204 and Tyr199. Transducer activation is likely to involve a relaxation of Thr204 in the receptor and hydrogen bonding alteration of Asn74 in the transducer, during which the helices of the transducer perform rigid-body motion without changing their secondary structures.

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↗