Rotational resonance NMR study of the active site structure in bacteriorhodopsin: conformation of the Schiff base linkage.

Rotational resonance, a new solid-state NMR technique for determining internuclear distances, is used to measure a distance in the active site of bacteriorhodopsin (bR) that changes in different states of the protein. The experiments are targeted to the active site of bR through 13C labeling of both the retinal chromophore and the Lys side chains of the protein. The time course of the rotor-driven magnetization exchange between a pair of 13C nuclei is then observed to determine the dipolar coupling and therefore the internuclear distance. Using this approach, we have measured the distance from [14-13C]retinal to [epsilon-13C]Lys216 in dark-adapted bR in order to examine the structure of the retinal-protein linkage and its role in coupling the isomerizations of retinal to unidirectional proton transfer. This distance depends on the configuration of the intervening C=N bond. The 3.0 +/- 0.2 A distance observed in bR555 demonstrates that the C=N bond is syn, and the 4.1 +/- 0.3 A distance observed in bR568 demonstrates that the C=N bond is anti. These direct distance determinations independently confirm the configurations previously deduced from solid-state NMR chemical shift and resonance Raman vibrational spectra. The spectral selectivity of rotational resonance allows these two distances to be measured independently in a sample containing both bR555 and bR568; the presence of both states and of 25% lipid in the sample demonstrates the use of rotational resonance to measure an active site distance in a membrane protein with an effective molecular mass of about 85 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Structural investigation of the active site in bacteriorhodopsin: geometric constraints on the roles of Asp-85 and Asp-212 in the proton-pumping mechanism from solid state NMR

Constraints on the proximity of the carboxyl carbons of the Asp-85 and Asp-212 side chains to the 14-carbon of the retinal chromophore have been established for the bR(555), bR(568), and M(412) states of bacteriorhodopsin (bR) using solid-state NMR spectroscopy. These distances were examined via (13)C-(13)C magnetization exchange, which was observed in two-dimensional RF-driven recoupling (RFDR) and spin diffusion experiments. A comparison of relative RFDR cross-peak intensities with simulations of the NMR experiments yields distance measurements of 4.4 +/- 0.6 and 4.8 +/- 1.0 A for the [4-(13)C]Asp-212 to [14-(13)C]retinal distances in bR(568) and M(412), respectively. The spin diffusion data are consistent with these results and indicate that the Asp-212 to 14-C-retinal distance increases by 16 +/- 10% upon conversion to the M-state. The absence of cross-peaks from [14-(13)C]retinal to [4-(13)C]Asp-85 in all states and between any [4-(13)C]Asp residue and [14-(13)C]retinal in bR(555) indicates that these distances exceed 6.0 A. For bR(568), the NMR distance constraints are in agreement with the results from recent diffraction studies on intact membranes, while for the M state the NMR results agree with theoretical simulations employing two bound waters in the region of the Asp-85 and Asp-212 residues. The structural information provided by NMR should prove useful for refining the current understanding of the role of aspartic acid residues in the proton-pumping mechanism of bR.     

Light- and dark-adapted bacteriorhodopsin, a time-resolved neutron diffraction study

Recently, neutron diffraction experiments have revealed well-resolved and reversible changes in the protein conformation of bacteriorhodopsin (BR) between the light-adapted ground state and the M-intermediate of the proton pumping photocycle (Dencher, Dresselhaus, Zaccai and Büldt (1989) Proc. Natl. Acad. Sci. USA 86, 7876-7879). These changes are triggered by the light-induced isomerization of the chromophore retinal from the all-trans to the 13-cis configuration. Dark-adapted purple membranes contain a mixture of two pigment species with either the all-trans- or 13-cis-retinal isomer as chromophore. Employing a time-resolved neutron diffraction technique, no changes in protein conformation in the resolution regime of up to 7 A are observed during the transition between the two ground-state species 13-cis-BR and all-trans-BR. This is in line with the fact that the conversion of all-trans BR to 13-cis-BR involves an additional isomerization about the C15 = N Schiff's base bond, which in contrast to M formation minimizes retinal displacement and keeps the Schiff's base in the original protein environment. Furthermore, there is no indication for large-scale redistribution of water molecules in the purple membrane during light-dark adaptation.     

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.     

Determination of retinal chromophore structure in bacteriorhodopsin with resonance Raman spectroscopy

The analysis of the vibrational spectrum of the retinal chromophore in bacteriorhodopsin with isotopic derivatives provides a powerful "structural dictionary" for the translation of vibrational frequencies and intensities into structural information. Of importance for the proton-pumping mechanism is the unambiguous determination of the configuration about the C13=C14 and C=N bonds, and the protonation state of the Schiff base nitrogen. Vibrational studies have shown that in light-adapted BR568 the Schiff base nitrogen is protonated and both the C13=C14 and C=N bonds are in a trans geometry. The formation of K625 involves the photochemical isomerization about only the C13=C14 bond which displaces the Schiff base proton into a different protein environment. Subsequent Schiff base deprotonation produces the M412 intermediate. Thermal reisomerization of the C13=C14 bond and reprotonation of the Schiff base occur in the M412------O640 transition, resetting the proton-pumping mechanism. The vibrational spectra can also be used to examine the conformation about the C--C single bonds. The frequency of the C14--C15 stretching vibration in BR568, K625, L550 and O640 argues that the C14--C15 conformation in these intermediates is s-trans. Conformational distortions of the chromophore have been identified in K625 and O640 through the observation of intense hydrogen out-of-plane wagging vibrations in the Raman spectra (see Fig. 2). These two intermediates are the direct products of chromophore isomerization. Thus it appears that following isomerization in a tight protein binding pocket, the chromophore cannot easily relax to a planar geometry. The analogous observation of intense hydrogen out-of-plane modes in the primary photoproduct in vision (Eyring et al., 1982) suggests that this may be a general phenomenon in protein-bound isomerizations. Future resonance Raman studies should provide even more details on how bacterio-opsin and retinal act in concert to produce an efficient light-energy convertor. Important unresolved questions involve the mechanism by which the protein catalyzes deprotonation of the L550 intermediate and the mechanism of the thermal conversion of M412 back to BR568. Also, it has been shown that under conditions of high ionic strength and/or low light intensity two protons are pumped per photocycle (Kuschmitz & Hess, 1981). How might this be accomplished?(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural changes in the L photointermediate of bacteriorhodopsin

The L to M reaction of the bacteriorhodopsin photocycle includes the crucial proton transfer from the retinal Schiff base to Asp85. In spite of the importance of the L state in deciding central issues of the transport mechanism in this pump, the serious disagreements among the three published crystallographic structures of L have remained unresolved. Here, we report on the X-ray diffraction structure of the L state, to 1.53-1.73 A resolutions, from replicate data sets collected from six independent crystals. Unlike earlier studies, the partial occupancy refinement uses diffraction intensities from the same crystals before and after the illumination to produce the trapped L state. The high reproducibility of inter-atomic distances, and bond angles and torsions of the retinal, lends credibility to the structural model. The photoisomerized 13-cis retinal in L is twisted at the C(13)=C(14) and C(15)=NZ double-bonds, and the Schiff base does not lose its connection to Wat402 and, therefore, to the proton acceptor Asp85. The protonation of Asp85 by the Schiff base in the L-->M reaction is likely to occur, therefore, via Wat402. It is evident from the structure of the L state that various conformational changes involving hydrogen-bonding residues and bound water molecules begin to propagate from the retinal to the protein at this stage already, and in both extracellular and cytoplasmic directions. Their rationales in the transport can be deduced from the way their amplitudes increase in the intermediates that follow L in the reaction cycle, and from the proton transfer reactions with which they are associated.     

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.     

High-field EPR studies of the structure and conformational changes of site-directed spin labeled bacteriorhodopsin

Cw and pulsed high-field EPR (95 GHz, 3.4 T) are performed on site-directed spin labeled bacteriorhodopsin (BR) mutants. The enhanced Zeeman splitting leads to spectra with resolved g-tensor components of the nitroxide spin label. The g(xx) component shift determined for 10 spin labels located in the cytoplasmic loop region and in the protein interior along the BR proton channel reveals a maximum close to position 46 between the proton donor D96 and the retinal. A plot of g(xx) versus A(zz) of the nitrogen discloses grouping of 12 spin labeled sites in protic and aprotic sites. Spin labels at positions 46, 167 and 171 show the aprotic character of the cytoplasmic moiety of the proton channel whereas nitroxides at positions 53, 194 and 129 reveal the protic environment in the extracellular channel. The enhanced sensitivity of high-field EPR with respect to anisotropic reorientational motion of nitroxides allows the characterization of different motional modes for spin labels bound to positions 167 and 170. The motional restriction of the nitroxide at position 167 of the double mutant V167C/D96N is decreased in the M(N) photo-intermediate. An outward shift of the cytoplasmic moiety of helix F in the M(N) intermediate would account for the high-field EPR results and is in agreement with diffraction and recent X-band EPR data.     

FTIR study of the photoisomerization processes in the 13-cis and all-trans forms of Anabaena sensory rhodopsin at 77 K

Archaeal-type rhodopsins can accommodate either all-trans- or 13-cis,15-syn-retinal in their chromophore binding site in the dark, but only the former isomer is functionally important. In contrast, Anabaena sensory rhodopsin (ASR), an archaeal-type rhodopsin found in eubacteria, exhibits a photochromic interconversion of both forms, suggesting that ASR functions as a photosensor which interacts with its 14 kDa soluble transducer differently in the all-trans and 13-cis,15-syn forms. In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the 13-cis,15-syn form of ASR (13C-ASR) at 77 K and compared the local structure around the chromophore and its structural changes upon retinal photoisomerization with those of the all-trans form (AT-ASR) [Furutani, Y., Kawanabe, A., Jung, K. H., and Kandori, H. (2005) Biochemistry 44, 12287-12296]. By use of [zeta-15N]lysine-labeled ASR, we identified the N-D stretching vibrations of the Schiff base (in D2O) at 2165 cm(-1) for 13C-ASR and at 2163 and 2125 cm(-1) for AT-ASR. The frequencies indicate strong hydrogen bonds of the Schiff base with a water molecule for both 13C-ASR and AT-ASR. In contrast, the N-D stretching vibration appears at 2351 cm(-1) and at 2483 cm(-1) for the K states of 13C-ASR (13C-ASR(K)) and AT-ASR (AT-ASR(K)), respectively, indicating that the Schiff base still forms a hydrogen bond in 13C-ASR(K). Rotational motion of the Schiff base upon retinal isomerization is probably smaller for 13C-ASR than for AT-ASR, the latter altering hydrogen bonding of the Schiff base similar to bacteriorhodopsin (BR), a light-driven proton pump. Appearance of several hydrogen-out-of-plane vibrations and amide I vibrations in 13C-ASR(K), but not in AT-ASR(K), suggests that structural changes are distributed widely along the polyene chain for 13C-ASR. On the other hand, retinal photoisomerization in AT-ASR breaks the hydrogen bond of the Schiff base, and localized structural changes in the Schiff base region are induced.     

Spectroscopic evidence for the formation of an N intermediate during the photocycle of sensory rhodopsin II (phoborhodopsin) from Natronobacterium pharaonis

Sensory rhodopsin II is a seven transmembrane helical retinal protein and functions as a photoreceptor protein in negative phototaxis of halophilic archaea. Sensory rhodopsin II from Natronomonas pharaonis (NpSRII) is stable under various conditions and can be expressed functionally in Escherichia coli cell membranes. Rhodopsins from microorganisms, known as microbial rhodopsins, exhibit a photocycle, and light irradiation of these molecules leads to a high-energy intermediate, which relaxes thermally to the original pigment after passing through several intermediates. For bacteriorhodopsin (BR), a light-driven proton pump, the photocycle is established as BR → K → L → M → N → O → BR. The photocycle of NpSRII is similar to that of BR except for N, i.e., M thermally decays into the O, and N has not been well characterized in the photocycle. Thus we here examined the second half of the photocycle in NpSRII, and in the present transient absorption study we found the formation of a new photointermediate whose absorption maximum is ～500 nm. This intermediate becomes pronounced in the presence of azide, which accelerates the decay of M. Transient resonance Raman spectroscopy was further applied to demonstrate that this intermediate contains a 13-cis retinal protonated Schiff base. However, detailed analysis of the transient absorption data indicated that M-decay does not directly produce N but rather produces O that is in equilibrium with N. These observations allowed us to propose a structural model for a photocycle that involves N.     

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.     

Conformational heterogeneity of transmembrane residues after the Schiff base reprotonation of bacteriorhodopsin: 15N CPMAS NMR of D85N/T170C membranes

bR, N-like and O-like intermediate states of [15N]methionine-labelled wild type and D85N/T170C bacteriorhodopsin were accumulated in native membranes by controlling the pH of the preparations. 15N cross polarization and magic angle sample spinning (CPMAS) NMR spectroscopy allowed resolution of seven out of nine resonances in the bR-state. It was possible to assign some of the observed resonances by using 13C/15N rotational echo double resonance (REDOR) NMR and Mn2+ quenching as well as D2O exchange, which helps to identify conformational changes after the bacteriorhodopsin Schiff base reprotonation. The significant differences in chemical shifts and linewidths detected for some of the resonances in N- and O-like samples indicate changes in conformation, structural heterogeneity or altered molecular dynamics in parts of the protein.     

Structure of the retinal chromophore in sensory rhodopsin I from resonance Raman spectroscopy

Sensory rhodopsin I (SR-I) is a retinal-containing pigment which functions as a phototaxis receptor in Halobacterium halobium. We have obtained resonance Raman vibrational spectra of the native membrane-bound form of SR587 and used these data to determine the structure of its retinal prosthetic group. The similar frequencies and intensities of the skeletal fingerprint modes in SR587, bacteriorhodopsin (BR568), and halorhodopsin (HR578) as well as the position of the dideuterio rocking mode when SR-I is regenerated with 12,14-D2 retinal (915 cm-1) demonstrate that the retinal chromophore has an all-trans configuration. The shift of the C = N stretching mode from 1628 cm-1 in H2O to 1620 cm-1 in D2O demonstrates that the chromophore in SR587 is bound to the protein by a protonated Schiff base linkage. The small shift of the 1195 cm-1 C14-C15 stretching mode in D2O establishes that the protonated Schiff base bond has an anti configuration. The low value of the Schiff base stretching frequency together with its small 8 cm-1 shift in D2O indicates that the Schiff base proton is weakly hydrogen bonded to its protein counterion. This suggests that the red shift in the absorption maximum of SR-I (587 nm) compared with HR (578 nm) and BR (568 nm) is due to a reduction of the electrostatic interaction between the protonated Schiff base group and its protein counterion.     

Low-temperature solid-state 13C NMR studies of the retinal chromophore in rhodopsin

Magic angle sample spinning (MASS) 13C NMR spectra have been obtained of bovine rhodopsin regenerated with retinal prosthetic groups isotopically enriched with 13C at C-5 and C-14. In order to observe the 13C retinal chromophore resonances, it was necessary to employ low temperatures (-15-----35 degrees C) to restrict rotational diffusion of the protein. The isotropic chemical shift and principal values of the chemical shift tensor of the 13C-5 label indicate that the retinal chromophore is in the twisted 6-s-cis conformation in rhodopsin, in contrast to the planar 6-s-trans conformation found in bacteriorhodopsin. The 13C-14 isotropic shift and shift tensor principal values show that the Schiff base C = N bond is anti. Furthermore, the 13C-14 chemical shift (121.2 ppm) is within the range of values (120-123 ppm) exhibited by protonated (C = N anti) Schiff base model compounds, indicating that the C = N linkage is protonated. Our results are discussed with regard to the mechanism of wavelength regulation in rhodopsin.     

Coupling photoisomerization of retinal to directional transport in bacteriorhodopsin

In order to understand how isomerization of the retinal drives unidirectional transmembrane ion transport in bacteriorhodopsin, we determined the atomic structures of the BR state and M photointermediate of the E204Q mutant, to 1.7 and 1.8 A resolution, respectively. Comparison of this M, in which proton release to the extracellular surface is blocked, with the previously determined M in the D96N mutant indicates that the changes in the extracellular region are initiated by changes in the electrostatic interactions of the retinal Schiff base with Asp85 and Asp212, but those on the cytoplasmic side originate from steric conflict of the 13-methyl retinal group with Trp182 and distortion of the pi-bulge of helix G. The structural changes suggest that protonation of Asp85 initiates a cascade of atomic displacements in the extracellular region that cause release of a proton to the surface. The progressive relaxation of the strained 13-cis retinal chain with deprotonated Schiff base, in turn, initiates atomic displacements in the cytoplasmic region that cause the intercalation of a hydrogen-bonded water molecule between Thr46 and Asp96. This accounts for the lowering of the pK(a) of Asp96, which then reprotonates the Schiff base via a newly formed chain of water molecules that is extending toward the Schiff base.     

Structure of an early intermediate in the M-state phase of the bacteriorhodopsin photocycle.

The structure of an early M-intermediate of the wild-type bacteriorhodopsin photocycle formed by actinic illumination at 230 K has been determined by x-ray crystallography to a resolution of 2.0 A. Three-dimensional crystals were trapped by illuminating with actinic light at 230 K, followed by quenching in liquid nitrogen. Amide I, amide II, and other infrared absorption bands, recorded from single bacteriorhodopsin crystals, confirm that the M-substate formed represents a structure that occurs early after deprotonation of the Schiff base. Rotation about the retinal C13-C14 double bond appears to be complete, but a relatively large torsion angle of 26 degrees is still seen for the C14-C15 bond. The intramolecular stress associated with the isomerization of retinal and the subsequent deprotonation of the Schiff base generates numerous small but experimentally measurable structural changes within the protein. Many of the residues that are displaced during the formation of the late M (M(N)) substate formed by three-dimensional crystals of the D96N mutant (Luecke et al., 1999b) are positioned, in early M, between their resting-state locations and the ones which they will adopt at the end of the M phase. The relatively small magnitude of atomic displacements observed in this intermediate, and the well-defined positions adopted by nearly all of the atoms in the structure, may make the formation of this structure favorable to model (simulate) by molecular dynamics.     

Crystal structure of the 13-cis isomer of bacteriorhodopsin in the dark-adapted state

The atomic structure of the trans isomer of bacteriorhodopsin was determined previously by using a 3D crystal belonging to the space group P622. Here, a structure is reported for another isomer with the 13-cis, 15-syn retinal in a dark-adapted crystal. Structural comparison of the two isomers indicates that retinal isomerization around the C13[double bond]C14 and the C15[double bond]N bonds is accompanied by noticeable displacements of a few residues in the vicinity of the retinal Schiff base and small re-arrangement of the hydrogen-bonding network in the proton release channel. On the other hand, aromatic residues surrounding the retinal polyene chain were found to scarcely move during the dark/light adaptation. This result suggests that variation in the structural rigidity within the retinal-binding pocket is one of the important factors ensuring the stereospecific isomerization of retinal.     

Hydration switch model for the proton transfer in the Schiff base region of bacteriorhodopsin

In a light-driven proton-pump protein, bacteriorhodopsin (BR), protonated Schiff base of the retinal chromophore and Asp85 form ion-pair state, which is stabilized by a bridged water molecule. After light absorption, all-trans to 13-cis photoisomerization takes place, followed by the primary proton transfer from the Schiff base to Asp85 that triggers sequential proton transfer reactions for the pump. Fourier transform infrared (FTIR) spectroscopy first observed O-H stretching vibrations of water during the photocycle of BR, and accurate spectral acquisition has extended the water stretching frequencies into the entire stretching frequency region in D(2)O. This enabled to capture the water molecules hydrating with negative charges, and we have identified the water O-D stretch at 2171 cm(-1) as the bridged water interacting with Asp85. We found that retinal isomerization weakens the hydrogen bond in the K intermediate, but not in the later intermediates such as L, M, and N. On the basis of the observation particularly on the M intermediate, we proposed a model for the mechanism of proton transfer from the Schiff base to Asp85. In the "hydration switch model", hydration of a water molecule is switched in the M intermediate from Asp85 to Asp212. This will have raised the pK(a) of the proton acceptor, and the proton transfer is from the Schiff base to Asp85.     

Detergent delipidation and solubilization strategies for high-resolution NMR of the membrane protein bacteriorhodopsin

High-resolution NMR studies of bacteriorhodopsin require the availability of the detergent-solubilized protein with both high concentration and small rotational correlation time. A procedure is described for the optimized preparation of such samples. Bacteriorhodopsin was first delipidated by detergent treatment of purple membrane under nonsolubilizing conditions for the protein. The delipidated aggregated protein could then be solubilized into monomers at concentration close to millimolar by selected detergents. The solubilizing detergent had an important effect on the rotational correlation time of the protein as shown by measuring in each case the temperature-dependent stability of the protein, the size of the detergent-protein complex, and the detergent viscosity. Consistently, a strong influence of the detergent was also found on spectral resolution in 13C NMR spectra of solubilized bacteriorhodopsin labeled with [1-13C]phenylalanine. Best resolution was obtained using n-dodecylmaltoside as detergent, with which relatively narrow well resolved 13C NMR resonances were observed at 50 degrees C. It is suggested that high-resolution NMR studies performed with this detergent may contribute to the structural resolution of bacteriorhodopsin.