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Originally published In Press as doi:10.1074/jbc.M300709200 on October 7, 2003

J. Biol. Chem., Vol. 279, Issue 3, 2147-2158, January 16, 2004
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Deformation of Helix C in the Low Temperature L-intermediate of Bacteriorhodopsin*

Karl Edman,a Antoine Royant,bc Gisela Larsson,d Frida Jacobson,a Tom Taylor,e David van der Spoel,f Ehud M. Landau,gh Eva Pebay-Peyroula,bi and Richard Neutzeaj

From the aDepartment of Chemistry and Bioscience, Chalmers University of Technology, Box 462, S-40530 Gothenburg, Sweden, the bInstitut de Biologie Structurale, UMR5075, Commissariat à l'Energie Atomique-CNRS-Université Joseph Fourier, 41 rue Jules Horowitz, F-38027 Grenoble Cedex 1, France, the cEuropean Synchrotron Radiation Facility, 6 rue Jules Horowitz, BP 220, F-38043 Grenoble Cedex, France, the dDepartment of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius vag 12, 106 91 Stockholm, Sweden, the eDepartment of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Centre, Box 590, 751 24 Uppsala, Sweden, the fDepartment of Cell and Molecular Biology, Uppsala University, Box 596, 751 24 Uppsala; Sweden, and the gMembrane Protein Laboratory, Sealy Center for Structural Biology, and Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, Texas 77555-0437

Received for publication, January 22, 2003 , and in revised form, October 7, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
X-ray and electron diffraction studies of specific reaction intermediates, or reaction intermediate analogues, have produced a consistent picture of the structural mechanism of light-driven proton pumping by bacteriorhodopsin. Of central importance within this picture is the structure of the L-intermediate, which follows the retinal all-trans to 13-cis photoisomerization step of the K-intermediate and sets the stage for the primary proton transfer event from the positively charged Schiff base to the negatively charged Asp-85. Here we report the structural changes in bacteriorhodopsin following red light illumination at 150 K. Single crystal microspectrophotometry showed that only the L-intermediate is populated in three-dimensional crystals under these conditions. The experimental difference Fourier electron density map and refined crystallographic structure were consistent with those previously presented (Royant, A., Edman, K., Ursby, T., Pebay-Peyroula, E., Landau, E. M., and Neutze, R. (2000) Nature 406, 645-648; Royant, A., Edman, K., Ursby, T., Pebay-Peyroula, E., Landau, E. M., and Neutze, R. (2001) Photochem. Photobiol. 74, 794-804). Based on the refined crystallographic structures, molecular dynamic simulations were used to examine the influence of the conformational change of the protein that is associated with the K-to-L transition on retinal dynamics. Implications regarding the structural mechanism for proton pumping by bacteriorhodopsin are discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Light-driven vectorial proton translocation is basic to the mechanism of energy transduction by photosynthetic systems. Bacteriorhodopsin (bR)1 is the simplest known light-driven proton pump and has long served as a model system for understanding how protons may be transported "up hill" against a transmembrane proton motive potential. bR contains seven transmembrane {alpha}-helices that surround a proton translocation channel lined with strategically placed charged residues (3). Depending upon their protonation states, which change in a well orchestrated cascade as a proton is transported across the cell membrane, these charged residues can serve as either proton donors or proton acceptors. Light activation of the chromophore, an all-trans-retinal molecule covalently attached to Lys-216 in helix G via a protonated Schiff base (the primary proton donor) results in the 13-cis-retinal configuration with two-thirds quantum efficiency. Steric conflicts and mechanical stress resulting from photoisomerization initiate a sequence of conformational changes that can be characterized spectroscopically and that perturb the local environment of several key residues, strongly affecting their pKa values and creating transient pathways for proton transfer.

The specific spectral intermediates of the bR photocycle have been well characterized, and a common reaction scheme is: bR570 -> K590 {leftrightarrow} L550 {leftrightarrow} M412 {leftrightarrow} N560 {leftrightarrow} O640 -> bR570 (sub-scripts denote the wavelengths of the respective absorption maxima). M412 is usually associated with two distinct intermediates (4), commonly referred to as the early and late M-states, whose spectrally silent transition correlates with a conformational change on the cytoplasmic half of the protein. In general, at low temperatures the thermal motions that aid the crossing of energy barriers associated with specific transitions can be reduced or even frozen out, and light activation of three-dimensional crystals can populate specific photocycle intermediates (5-9). Using this approach, a number of x-ray and electron diffraction studies on light-driven structural changes in bR (1, 10-16) and in bR mutants (17-19), as well as bR mutant intermediate state analogues (20, 21), have been presented recently. When taken together with an acceptance of the limitations of the techniques of kinetic crystallography, most structures combine to yield a remarkably consistent picture of the structural mechanism of outwardly directed proton pumping by bR (22).

As with the majority of studies of the low temperature L-intermediate (LLT) of bR (15, 23-29), our previous work on the structural changes associated with a build up of L was performed at 170 K. In that study three-dimensional crystals of bR were illuminated for 30 s with green light, thereby populating primarily K170K, followed by a further 40-s relaxation in the dark (1) in which L170K evolved. Subsequently the crystals were quenched in liquid nitrogen. As stated in that study, this trapping protocol produced predominantly the L-intermediate, although the existence of other species was detected and acknowledged (1) and was later quantified as a mixture of K170K, L170K, and M170K in the ratio 1:3:1 (2). Due to the mixture of spectral species the interpretation of our earlier structural result has been controversial (30-32). As such it is valuable to repeat the intermediate trapping experiment under conditions that produce pure L-intermediate in three-dimensional bR crystals.

In this work we present spectral and structural results following red light illumination of three dimensional bR crystals cooled to 150 K. The spectral analysis, difference Fourier map, and refined crystallographic structure demonstrate that the light-induced structural changes previously reported (1) were correctly interpreted as characterizing the build up of LLT. It follows that the mechanistic model of vectorial proton transport by bR should incorporate the fact that significant rearrangements of water molecules on the EC half of the protein, a reorientation of the guanidinium group of Arg-82 toward the EC medium, and a local flex of helix C toward the proton translocation channel centered near Asp-85 all occur after retinal photoisomerization yet prior to the primary proton transfer event from the Schiff base to Asp-85. Building upon the refined structural models, molecular dynamic simulations are presented that illustrate how the geometry of the retinal is perturbed by the conformational change in the protein associated with the K-to-L transition, providing structural insight into how the L-intermediate sets the stage for the primary proton transfer from the Schiff base to Asp-85 in the L-to-M transition.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Protein Crystallization—bR was crystallized using a lipidic cubic phase matrix as described previously (33). These crystals grow as hexagonal plates typically 70 µm across and less than 10 µm thick. Crystals were harvested from the viscous lipidic environment by a lipase treatment (34). For both microspectrophotometry and x-ray diffraction studies, crystals were light adapted (35) at room temperature by several minutes of exposure to bright white light immediately prior to being mounted on cryoloops and flash frozen in liquid nitrogen.

Single Crystal Microspectrophotometry—Single crystal microspectrophotometry (36, 37) was used to characterize both the ground state (Fig. 1a) and spectral intermediate (Fig. 1b) trapped within three-dimensional crystals of bR. An Oxford Cryosystems nitrogen gas stream held the crystal at a constant temperature of 150 K. The reaction was initiated using a 20-milliwatt, 635 nm laser diode coupled to a 225-µm-diameter optical fiber, which guided light to within ~300 µm of the crystal position incident at an angle ~30° relative to that of the crystal plate. The optical fiber was decoupled relative to the red laser, and the light flux was measured using a Newport 818-UV photometer with the same red light intensity being used for both x-ray diffraction and microspectrophotometry experiments (estimated to be of the order of 5 watts/cm2 at the crystal position). The ground state spectrum (Fig. 1a) and the difference spectrum between that recorded during red light illumination and the ground state spectrum (Fig. 1b, discontinuous line) were recorded using a continuous wave halogen lamp with a probe spot diameter of ~15 µm. The difference spectrum between the spectrum recorded following 30-s illumination and 40-s delay in the dark and the ground state spectrum (Fig. 1b, continuous line) was recorded using a 10-µs Xenon flash lamp. An Ocean Optics SD-2000 CCD spectrophotometer was used to record spectra enabling a broader wavelength domain to be sampled with significantly higher sensitivity than in previous work (2).



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  		FIG. 1.
Spectral analysis of three dimensional bR crystals cooled to 150 K. a, ground state absorption spectrum. b, the difference spectrum between that recorded while illuminating with red light and that without illumination (shows a discontinuity at 635 nm due to scatter of the red light) and the difference spectrum between that recorded after 30-s illumination with red light followed by a further delay of 40-s in the dark and that without illumination. The latter difference spectrum is reproduced with permission from Elsevier from Ref. 22 (previously scaled relative to the ground state absorption spectrum of Ref. 2).

 
X-ray Data Collection—Light-adapted (35) frozen bR crystals were illuminated at 150 K with red light ({lambda} = 635 nm) for 30 s followed by a further 40-s delay in the dark and were then quenched in liquid nitrogen. Approximately 20 min following off-line trapping, crystals were mounted on the x-ray goniometer of beamline ID14-EH2 of the European Synchrotron Radiation Facility. A first (coarse) alignment of the crystal on the goniometer head was made using red light, and the final adjustment (a few seconds) was made using dim white light. An x-ray wavelength of 0.93 Å was used for data collection with an ADSC Quantum4 CCD detector. 120 frames of 1°/frame were collected with 30-s x-ray exposure/frame. Data were processed using the HKL package (38) and the CCP4 suite (39). Crystallographic data and refinement statistics are summarized in Table I.


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  		TABLE I
Diffraction data and refinement statistics

 
Merohedral Twinning—The degree of merohedral twinning was determined using Britton plots and Yeates statistics (40) as described in Ref. 41 and yielded a value of 27% twinning. Crystallographic data were detwinned according to the formula (41),

(Eq. 1)

(Eq. 2)
prior to further crystallographic analysis where Idetwin is the intensity corresponding to diffraction from an untwinned crystal, Iobs is the (twinned) measured intensity, the two reflections h1 and h2 are related by the twin operation but not by crystallographic symmetry, and x is the degree of merohedral twinning. When following this procedure the detwinned data is independent of any structural model, and the number of independent observations is not reduced, but the errors associated with detwinning increase with increasing twinning, approaching infinity as x approaches 0.5 (41). Consequently when following this protocol the refined crystallographic R-factors are typically somewhat larger than might be expected. For perfectly twinned (or almost perfectly twinned) crystallographic data (from which several intermediate structures of bR have been reported (12-15, 17, 18)), x = 0.5, and it is not possible to retrieve the diffraction data corresponding to an untwinned crystal from the observations alone. Rather when calculating electron density maps the "detwinned" observations are recovered according to the formula (42),

(Eq. 3)

(Eq. 4)
or variations thereof, which depend explicitly upon intensities calculated from the structural model, Icalc(h) (the convention is that Idetwin and Icalc correspond to diffraction from only half of the crystal). In this case each observation Iobs(h1) and its associated twin Iobs(h2) cannot be measured independently (i.e. Iobs(h1) = Iobs(h2)), and the number of independent observations at any given resolution is reduced almost exactly by a half relative to data with low levels of twinning. Consequently refined crystallographic R-factors from perfectly twinned data are typically a factor of lower than for low (or un-)twinned data (40-42), and the explicit dependence of the detwinned data on the model itself (Equations 3 and 4) introduces additional model bias in electron density maps. Our work on the ground state (43) and KLT-state (10) of bR was free from merohedral twinning, and our previous work on LLT (x = 0.26 (1)) as well as that presented here (x = 0.27) had sufficiently low levels of twinning such that data were detwinned according to Equations 1 and 2 prior to further structural analysis.

Difference Fourier Analysis—Detwinned crystallographic data were scaled together with the ground state observations (Protein Data Bank entry 1QHJ [PDB] ) using the CCP4 suite (39). The difference Fourier electron density map was calculated by Fourier transform of (Fexc-Fgnd)·exp(i·{Phi}gnd) using the refined ground state model (Protein Data Bank entry 1QHJ [PDB] ) for phases (43). The structural changes described here were interpreted directly from the difference electron density map and were confirmed by partial occupancy refinement.

Structural Refinement—Partial occupancy refinement was performed, using CNS (44), by refining one excited state conformation (labeled LLT or L150K, which was allowed to change conformation), while the ground state model (with complementary occupancy) was held fixed. After the models were aligned as rigid bodies, a simulated annealing refinement of LLT was performed to escape bias toward the initial model. The crystallographic occupancy was determined by first selecting 10 evenly spaced (from 0 to 100%) values for the crystallographic occupancy, refining the structure of LLT with this occupancy held fixed, and then refining the occupancy with the model for LLT held fixed. The crystallographic occupancy converged from above and from below to a value of 50% (see Ref. 2 for details of the method). As with previous work (1, 9, 10), planar constraints were placed on the geometry of the retinal during refinement of LLT since no paired electron density peaks indicating the nature and extent of distortion of the retinal geometry were visible at a high confidence level in the difference Fourier map.

Molecular Dynamic Simulations—Molecular dynamic simulations were used to investigate the influence of the K-to-L structural transition on retinal dynamics. Crystallographically resolved water molecules and additional water molecules predicted using Dowser (104) were included in these simulations. Dowser assigns non-crystallographic water molecules by constructing a molecular surface from the input structure file and finding positions where a solvent probe (with a default radius of 0.2 Å) touches three atoms simultaneously. Energies were computed for buried waters, and those with a stabilization energy of at least -2 kcal/mol were included in the model. Lipid molecules were removed from the model, which was instead inserted into a slab of low temperature argon atoms (45) with enhanced Lennard Jones repulsions between argon and water ({sigma} = 0.36 nm and {epsilon} = 0.85 kJ/mol) so as to mimic the hydrophobic membrane environment. Simulations of the intact membrane bilayer have been reported (46) but were not considered essential for this work. Time-dependent protein dynamic simulations of 500-ps duration were performed using the chemical simulation software package GROMACS (47) with a time step of 1 fs. The Gromos-96 force field was used throughout (48) and was modified to include parameters for the retinal and Schiff base where partial charges for simulations (49) were kindly provided by E. Tajkhorshid,2 and torsional displacements from planarity were subject to a harmonic energy penalty of ~320 kJ·mol-1·radian-2. All protein C{alpha} atoms were restrained to their crystal positions by a force constant of 100 kJ·mol·nm-1. Equilibration of this system for 20 ps resulted in models with well relaxed argon and water molecules, and the potential energy in any given simulation converged within 80 ps. The root mean square deviation of the non-hydrogen atoms of the average trajectory structures (from 100 to 300 ps) deviated from the crystallographic structures by ~1.0 Å.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Spectral Analysis—Fig. 1a shows the absorption spectrum of light-adapted bR (35) in the ground state recorded from a three-dimensional bR crystal cooled to 150 K using single crystal microspectrophotometry (36, 37). In Fig. 1b, two difference spectra (illuminated state spectrum minus the ground state spectrum) recorded at 150 K are presented: one recorded with simultaneous red light illumination contains a discontinuity at 635 nm due to scatter of the red laser light from the crystal and in agreement with difference spectra presented in Ref. 15 and one recorded after 30-s red light illumination followed by a further 40-s delay in the dark and in agreement with difference spectra presented in Ref. 16 (data are reproduced from Fig. 5c of Ref. 22; in bold the same spectrum is filtered and overlaid to reduce noise). It was necessary to reduce the red light intensity when recording the difference spectrum simultaneously with red light illumination to avoid overloading the CCD detector at 635 nm (due to scatter from the sample) and distorting the measurement. The other difference spectrum was recorded following the same illumination conditions (intensity and protocol) as used for x-ray diffraction studies. Although the two difference spectra differ to some extent, both show significant depletion of the optical density near 610 nm and a positive feature near 520 nm. These features characterize a build up of the low temperature L-intermediate (30). In contrast to the spectral results from three-dimensional crystals at 170 K (1, 2, 15, 16), there is no positive feature at 410 nm in either difference spectrum, which would indicate a contribution from the low temperature M-intermediate (MLT). In addition to lowering the temperature of the crystal by 20 K, the use of a more sensitive spectrophotometer (see "Materials and Methods") reduced considerably the white light flux through the crystal required to record a difference spectrum with a good signal-to-noise ratio, thus reducing the probability that the measurement itself created some MLT.



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  		FIG. 5.
Lipid molecules in the archaeal rhodopsins. a, the location of a purple membrane lipid molecule adjacent to helix C in bR. b, the location of an analogous lipid molecule in the structure of halorhodopsin. Helices B, C, and D are labeled.

 
From these spectral results it is apparent that this trapping protocol established a high population of L150K in three-dimensional bR crystals with negligible contamination by K150K (since it is depleted by the red light pump) and M150K (since its fingerprint is not visible in the difference spectrum). It should also be apparent that the two difference spectra in Fig. 1b, recorded from the same crystal either during or following red light illumination at 150 K, are qualitatively similar yet quantitatively distinct. This reinforces the fact that what is assigned as the spectral fingerprint of LLT can depend upon the details of the measurement itself. The problem of what constitutes an "authentic" fingerprint of LLT is apparent in the literature since the difference spectrum for LLT deriving from the work of Ebrey and co-workers (23) following red light illumination at 170 K is not the same as that recorded by Balashov et al. (50) following green light illumination at 93 K, heating to 173 K, cooling to 93 K, and finally red light illumination at 93 K. In addition to other differences, the change in extinction coefficient in converting bR to LLT found in Ref. 50 is 50% greater than that found in Ref. 23. Using the change in extinction coefficient deriving from the simpler trapping protocol (23) (i.e. that 100% conversion of bR to LLT decreases the absorption near 610 nm by 44% of the bR maximum at 570 nm), which is in good agreement with several time-resolved room temperature studies on L (4, 51, 52), we find a population estimate (from the continuous difference spectrum in Fig. 1b) of 40% of bR being converted to LLT under the conditions described here. Because the white light probe photoreverts LLT to bR during the measurement itself, the population of LLT trapped in three-dimensional crystals under these conditions may be underestimated. The discontinuous difference spectrum cannot be used for this population estimate since the red light intensity was lowered to avoid detector saturation near 635 nm.

Difference Fourier Analysis—It has been 3 decades since Henderson and Moffat (53) showed, from mathematical considerations, that a difference Fourier electron density map provides an accurate, sensitive, and unbiased method for observing limited structural changes. Since a difference Fourier map is simply the Fourier transform of the measured changes in structure factor amplitudes (Fexc-Fgnd) using the known ground state model for phases, it represents the measured changes in the electron density without any assumptions as to the nature of those changes. Consequently, if a feature is visible at a reasonable level of significance within a difference Fourier map, then it is a genuine feature of the experimental data. Conversely, if conformational changes emerge during crystallographic refinement that are not consistent with the difference Fourier map, then they are likely to be artifacts arising from the assumptions made and constraints relaxed or imposed during structural refinement or result from a false minimum during refinement. For this reason the presentation of difference Fourier maps has become the crystallographic standard in the study of light-driven structural changes in macromolecular systems such as myoglobin-CO complexes (5, 8, 54-56), photoactive yellow protein (6, 7, 57, 58), sensory rhodopsin II (9), and bR (1, 10, 11, 16, 19, 20, 22, 59-63).

Fig. 2 compares long distance views of the (Fexc-Fgnd) difference Fourier maps calculated from three independent experiments: that resulting from x-ray diffraction data recorded during green light illumination at 110 K (Fig. 2a, difference electron density features correspond to the K110K intermediate; this difference density map was first presented in Ref. 10); that resulting from x-ray diffraction data recorded following 30-s red light illumination at 150 K and a further 40-s delay in the dark prior to quenching in liquid nitrogen (this work) whereupon the diffraction data were recorded at 110 K (Fig. 2b, difference electron density features correspond to the L150K intermediate, diffraction data are summarized in Table I; this difference density map was first presented in Ref. 22); and that resulting from x-ray diffraction data recorded following 30-s green light illumination at 170 K and a further 40-s delay in the dark prior to quenching in liquid nitrogen whereupon the diffraction data were recorded at 110 K (Fig. 2c, difference electron density features (1) correspond to a mixture of the K170K, L170K, and M170K intermediates in the ratio 1:3:1 (2); this difference density map was first presented in Ref. 1). Fgnd is the measured structure factor amplitude for the ground state (Protein Data Bank entry 1QHJ [PDB] ; this data is free from twinning (43)). When an overview of the difference electron density map is presented (1, 7, 9, 10, 58) all sources of noise within the crystallographic data (including errors arising from detwinning the data according to Equations 1 and 2 as well negative densities arising from radiation damage (16)) are visualized. In Fig. 2, regions removed from the proton translocation channel (where no mechanistically relevant conformational changes are expected) serve as an internal control of these sources of noise. Although the experimental difference density maps of Figs. 2 and 3 have been presented elsewhere (1, 10, 22), they are reproduced here in order that the reader may judge the experimental significance of the structural changes described below.



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  		FIG. 2.
Long distance overview of the Fexc-Fgnd difference Fourier electron density maps resulting from green light illumination at 110 K contoured at 4.0 {sigma} (a), red light illumination at 150 K contoured at 3.2 {sigma} (b), and green light illumination at 170 K contoured at 3.4 {sigma} (c). Positive electron density changes are colored blue, and negative electron density changes are colored yellow. More details of the trapping protocols are given in the text. Overviews of the difference density maps depicted in a and c can be found in stereo in Refs. 10 and 1, respectively.

 



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  		FIG. 3.
Light-driven structural changes in bR at low temperature. Close-up views of the Fexc-Fgnd difference Fourier electron density maps resulting from continuous green light illumination at 110 K (contoured at 4.0 {sigma}) (a), red light illumination at 150 K (contoured at 2.8 {sigma}) (b), and green light illumination at 170 K (contoured at 3.4 {sigma}) (c). d, overlays of the refined crystallographic models for the ground state (multicolored, Protein Data Bank entry 1QHJ [PDB] ), following red light illumination at 150 K (red), and following green light illumination at 170 K (green, Protein Data Bank entry 1EOP [PDB] ). The difference density maps depicted in a, b, and c were first presented in Refs. 10, 22, and 1, respectively, and data are reproduced with permission. Helices C and G are labeled in panel a.

 
In addition to the clusters of difference electron density in the vicinity of the Schiff base of the retinal, the C-20 methyl group, and the backbone of Lys-216 (seen in all difference Fourier maps), Fig. 2, b and c, shows two further regions where features arise well above the inherent noise levels of the map: one is toward the EC side of the proton translocation channel, primarily associated with helix C, and the other lies immediately toward the EC side of the {beta}-ionone ring of the retinal, primarily associated with helix F. A detailed description of the well ordered structural changes associated with helix C is given below. Electron density changes on the EC side of helix F, however, are reflected as a series of "stacked" negative density features characteristic of increased flexibility of the protein in this region. A probable cause for these perturbations is pressure exerted by the retinal on the residues packing immediately to the EC side of the binding pocket (1), and its consequences are felt (above 150 K, Fig. 2, b and c, but not at 110 K, Fig. 2a) as far as the putative proton release dyad of Glu-194 and Glu-204 (64, 65). A likely effect of this increased flexibility is to aid proton release. While it has been established that proton release is assisted by the light-driven reorientation of the guanidinium group of Arg-82 toward the EC side (1, 17, 22, 66), as noted by Luecke (67) the release dyad is occluded from the EC medium in the bR state, and additional conformational changes (or at least increased flexibility near Glu-194 and Glu-204) are necessary to complete proton release.

Fig. 3, a-c, shows close up stereoviews of the difference Fourier electron density maps presented in Fig. 2 focusing on the proton translocation channel in the region immediately adjacent to the retinal on the EC side. The experimental conditions are identical to those described above with Fig. 3a showing the electron density changes associated with a build up of K110K (10), Fig. 3b corresponding to a buildup of L150K, and Fig. 3c corresponding to a mixture of K170K, L170K, and M170K in the ratio 1:3:1 (1, 2). Inspection of Figs. 2 and 3 reveals that the observed changes in electron density at 150 K (Fig. 3b) are considerably more extensive than those observed at 110 K (Figs. 2a and 3a) yet are in excellent agreement with those observed at 170 K (Figs. 2c and 3c). As such the combination of results shown here unambiguously establishes that the conformational changes previously reported (1) occur prior to proton transfer. That the protein does not undergo large structural changes upon proton transfer at 170 K, which may have otherwise dominated the difference density of Figs. 2c and 3c, is in keeping with the result of Ormos (68) who found that certain protein motions specific to the M-state of bR were "frozen out" below 240 K. Similarly Zaccai and co-workers (69) observed a dynamical phase transition at 150 K, which correlates with the quantitative change in the nature of the light-induced movements observed at 110 K (Figs. 2a and 3a) when compared with those observed at 150 and 170 K (Figs. 2, b and c, and 3, b and c) and correlate with the need for additional thermal motions to cross the energy barrier from KLT to LLT.

It has recently been suggested that the difference density features observed in Figs. 2a and 3a arise primarily from the effects of x-ray-induced radiation damage (16). This argument derives from careful studies showing that, after four complete diffraction data sets were recorded from a single crystal, negative difference density peaks arose on Asp-85, Wat-402, and some other negatively charged residues. Radiation damage, however, cannot explain the positive difference density features of Fig. 3a (which are complementary to most negative features) nor the clear evolution of structural changes in bR as the temperature of intermediate trapping (but not the x-ray diffraction data collection) is raised. Since similar x-ray exposures were used for the ground and intermediate state data sets, radiation damage-induced features of the difference Fourier maps would be expected to arise randomly between different data sets, yet the features seen in Fig. 3, a-c, arose only when making comparisons between data collected from crystals that were illuminated against data collected from crystals without illumination. It is possible that not all features of the difference Fourier map shown in Figs. 2a and 3a (2.1-Å resolution, 35% occupancy (10)) are visible at 2.6 Å with 13% intermediate state occupancy (16).

An alternative series of structural models for K (13), L (15), and an early M (14) has also recently been presented. The experimental conditions used in those studies were similar to the trapping conditions described above, yet the models are inconsistent with the experimental electron density differences shown in Figs. 2 and 3. In those studies the crystallographic R-factors failed to distinguish between the intermediate state and ground state models (i.e. Rfree did not change as the occupancy of the intermediate state was varied (13, 14)), and the features of the only difference density map presented (calculated by subtracting two maps rather than through the Fourier transform of the experimental differences) were contoured at a confidence level of 1 {sigma} at which difference density peaks appear on virtually all residues since 1 {sigma} lies far below the threshold for meaningful differences described by Henderson and Moffat (53). Nevertheless a structural mechanism for proton pumping by bR was proposed (15) based upon the distortions to the bond angles of retinal which arose as bond angle constraints were removed during structural refinement. Two of the difficulties that emerge within this picture are: how does the pKa of Asp-85 increase from 2.2 (70) and that of the Schiff decrease from 13.5 (71) such that a proton may be exchanged without any change to the H-bond network prior to proton transfer (13, 15), and why does it require a time scale of ms for the Schiff base nitrogen of retinal to reorient away from Wat-402 and toward the cytoplasm when retinal photoisomerization occurs within a few ps (72, 73)? The enhanced model bias intrinsic to partial occupancy refinement against perfectly twinned crystallographic data and the failure to determine the occupancy of the intermediate state from the crystallographic data may explain why the structural models for these intermediate states (13-15) are so similar to the ground state structure.

Global Conformational Changes in L150K—Difference Fourier analysis presents the experimental electron density changes upon light activation of bR, yet it is necessary to quantify the extent of the indicated movements and model these changes through structural refinement. Structural refinement with partial occupancy was performed using CNS (see "Materials and Methods") against the crystallographic data, which were first detwinned according to Equations 1 and 2 and converged to Rcryst = 25.2% and Rfree = 29.8% (Table I). Partial occupancy refinement yielded 50% crystallographic occupancy for LLT (see "Materials and Methods" and Ref. 2 for more details on the refinement of crystallographic occupancy), a value higher than that estimated from the spectral analysis (40%). When one conformation (the ground state) is held fixed, this can overestimate somewhat the occupancy of the species that is allowed to vary in conformation. As there were no paired difference electron density peaks to indicate one dominant (distorted) retinal conformation (Fig. 3b), planar constraints were imposed on the retinal during crystallographic refinement. Fig. 3d overlays in stereo the refined structural models for the ground state (multicolored, Protein Data Bank entry 1QHJ [PDB] ), that refined against data recorded after red light illumination at 150 K (red), and that refined against data recorded after green light illumination at 170 K (green, Protein Data Bank entry 1EOP [PDB] ). As expected from the similarity of the two difference Fourier maps (Figs. 2, b and c, and 3, b and c), structural refinement resulted in very similar models for both the red light and green light studies of LLT (Fig. 3d). Minor differences in the crystallographic coordinates of individual atoms reflect the underlying level of noise within the crystallographic models.

An unanticipated result from our earlier study at 170 K was the observation of a local bend in helix C, which we interpreted as facilitating the primary proton transfer event (1). In Fig. 3, b and c, the angle of view is chosen to illustrate how the strongest cluster of positive electron density features in the map follows the backbone of helix C and is primarily associated with residues Arg-82 and Asp-85. Complementary negative density features are visible behind helix C, and additional difference density peaks are visible on residues on either side of this region. As such, these experimental electron density changes can be unambiguously interpreted as arising from a local "bend" of helix C toward the proton translocation channel, and, due to the spectral purity of our trapping protocol (Fig. 1), this conformational change is assigned to LLT.

In Fig. 4 the nature and extent of this motion is quantified through structural refinement of L150K. In Fig. 4a the EC region of the bR (multicolored (43)), L150K (red), and L170K (green (1)) models are overlaid in stereo. A distinct movement of helix C of ~1.0 Å toward the center of the proton translocation channel is visible in both intermediate state structures. The statistical significance of these backbone movements within the structural models for LLT is illustrated in Fig. 4, b and c, through an ESCET error scaled distance difference matrix (74). Within the illustrated range of statistical significance (from 2.5 to 4 {sigma}) three features stand out: (i) a movement of residues 82-87 of helix C toward residues 200-216 of helix G and (to a lesser extent) toward residues 8-13 of helix A, which corresponds to the local bend of helix C described above; (ii) a movement of the backbone of Lys-216, which is covalently bound to the retinal and responds immediately to photoisomerization (10, 75); and (iii) a (somewhat weaker) outward movement of helix F immediately to the cytoplasmic side of Trp-182 due to a steric clash with the C-20 methyl group of the retinal, and this anticipates a larger movement of this helix later in the photocycle (20). What is also statistically significant in the ESCET analysis of the refined structure for L170K (Fig. 4c), but is somewhat weaker in the structure of L150K (Fig. 4b), is a movement of the E-F loop centered near Glu-194, which correlates with the stacked region of negative density on the EC side of helix F illustrated in Fig. 2, b and c, and which aids proton release. That the strongest features in this statistical analysis correlate with the strongest difference electron density peaks in Fig. 2, b and c, illustrates how the refined structural model accurately represents the observed electron density changes.



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  		FIG. 4.
Assignment of a local bend of helix C from the crystallographic models of LLT. a, stereoview of the backbone movement of helix C on the EC side of the retinal for L150K (red) and L170K (green). Helices A to G are labeled. b, ESCET error scaled distance difference matrix analysis (74) of the movements of C{alpha} atoms for L150K. c, an identical analysis for L170K.

 
A consequence of the observed flex of helix C is that Asp-85 (the primary proton acceptor) approaches the Schiff base (the primary proton donor) in LLT relative to KLT as was previously observed in solid state NMR studies of LLT at 170 K (27, 76) and was anticipated from the associated blue spectral shift (77). As this local bend of helix C was not observed in any of the x-ray structures of M reported to date (11, 12, 14, 17, 18) we have proposed that it may be transient, driven (in part) by the mutual electrostatic attraction between the positively charged Schiff base and the negatively charged Asp-85, which ceases following proton transfer in the L-to-M transition. In this context it is noteworthy that Pro-91, which is well conserved within the archaeal rhodopsin family (78), lies immediately to the cytoplasmic side of where the local bend of helix C is most pronounced and may provide additional flexibility. Furthermore, as illustrated in Fig. 5a, a well defined purple membrane lipid molecule is found immediately behind helix C of bR at this position. The "greasy" interactions between helix C and this lipid molecule, and perhaps to some extent the pressure exerted by the packing of lipids within the bR trimer (43), may also be ingredients that help to explain the apparent elasticity of this secondary structural element. Negative electron density, located behind this lipid (not shown) also supports this hypothesis. It is interesting to note that a lipid molecule is also found at a similar position in the x-ray structure of halorhodopsin (79) (Fig. 5b). It has recently been suggested that a local change in the conformation of helix C at this position may play a role in light-driven Cl- transport by halorhodopsin (80).

Conformational Changes near the Active Site in LLT—An important issue for the early intermediates is the degree of distortion from planarity of the retinal anticipated from spectral observations (72, 81, 82). It is, however, an inherent weakness of kinetic crystallography that subtle changes in bond angles in regions where the ground state and intermediate state models overlap cannot easily be resolved. The problem is compounded by the fact that the early intermediates can only be trapped with partial occupancy by the effects of x-ray-induced radiation damage to the retinal (16) and by the larger errors associated with twinned crystallographic data (see "Materials and Methods"). In our work this problem is highlighted by the fact that the three difference Fourier maps of Fig. 3 are not identical in the immediate vicinity of the C-13=C-14 isomerized bond. For these reasons we have always applied planar constraints to the retinal during structural refinement but acknowledge that this cannot yield the complete picture.

Despite these limitations, several features of our refined crystallographic models for KLT and LLT are consistent with a broad range of spectral observations. Retinal isomerization exerts a mechanical pull on the side chain of Lys-216, and an early movement of this side chain was anticipated on the basis of Fourier transform infrared spectroscopy studies on KLT (83) as have Fourier transform infrared spectroscopy studies on M also identified a movement of the backbone of Lys-216 (75). Movements of the side chain and backbone of Lys-216 are suggested in all difference Fourier maps of Fig. 3, and the nature and extent of these movements for L150K are illustrated in Fig. 3d. Furthermore, in the structures of KLT and LLT, the H-bond of the Schiff base nitrogen to Wat-402 (43, 84) is lost as a result of retinal isomerization. This result is in agreement with low temperature Fourier transform infrared studies of KLT since the Schiff base C=N stretch in bR (assigned at 1641 cm-1 in H2O) is shifted 13 cm-1 (to 1628 cm-1) in D2O, yet the corresponding shift (from 1608 to 1606 cm-1) is only 2 cm-1 in KLT (85). These results are usually taken to indicate the lack of a Schiff base H-bond in KLT (82, 85). Low temperature resonance Raman studies have led to the same conclusions based upon the lowered frequencies of the in-plane and out-of-plane hydrogen bending motions (86). It is also noteworthy that a water molecule located near the active site of a myoglobin-carbon monoxide complex was shown, by time resolved x-ray diffraction, to be dislocated within 100 ps of photoexcitation by a fs light pulse (56).

Our refined structures of KLT and LLT (Fig. 3d) show that C{epsilon} of Lys-216 moves to ~2.5 Å from the position originally occupied by Wat-402 in the ground state model (43, 84). Since C{epsilon} of Lys-216 cannot act as a H-bond donor to this water molecule, a steric clash results, and Wat-402 is displaced, visualized as negative electron density on this water molecule in all difference Fourier maps of Fig. 3. In recent molecular dynamic simulations of the K-intermediate, two-thirds of a series of 80-ps trajectories showed that Wat-402 is displaced by a steric conflict with C{epsilon} of Lys-216 (87) when a fluctuating charge model was used to simulate all water molecules within the protein, and the red shift in the calculated spectrum was in good agreement with that of KLT. When the H-bond interactions between Wat-402 and the Schiff base were enhanced by adding additional features, the same study suggested an additional early and highly distorted retinal conformation, which may also lie along the reaction coordinate. In this context it is noteworthy that a mechanism in which C{epsilon} displaces Wat-402 following retinal photoisomerization can also accommodate the intriguing observation that a covalent bond to Lys-216 is not strictly required for proton pumping activity in bR (88, 89). In those studies the K216G and K216A bR mutants, when reconstituted with a number of retinal alkylamine Schiff bases, preserved some proton pumping activity. Since an atom analogous to C{epsilon} of Lys-216 in bR was preserved in these studies (albeit it covalently bound to retinal and not to the protein), photoisomerization of the retinal could still displace Wat-402 through the structural mechanism described above.

Arguably the most important new idea to emerge from the high resolution x-ray diffraction studies of bR and its reaction intermediates was the central mechanistic role played by water molecules in vectorial proton transport (22). In the ground state structure of bR (43, 90) Wat-402 is a key water molecule since it has hydrogen bonds to both the primary proton donor (the Schiff base) and the primary proton acceptor (Asp-85, Fig. 6a), helping to stabilize their widely separated pKa values of 13.5 (71) and 2.2 (70), respectively, thus ensuring that the probability for proton transfer to occur in the resting state is negligible. Indeed theoretical studies have shown that the presence of Wat-402 stabilizes the protonated Schiff base, whereas a proton will spontaneously detach from the Schiff base only when Wat-402 is absent (91).



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  		FIG. 6.
Light-induced changes in the EC H-bond network. a, the location of water molecules in bR. b, the location of water molecules in LLT. Figs. 2, 3, 4, 5, 6 were drawn using the Swiss PDB Viewer (102).

 
Another key functional role for Wat-402 is suggested by crystallographic studies of the early intermediates (1, 10). As is apparent in the difference Fourier maps of Fig. 3, not only is there negative density on Wat-402, but negative density also appears on Wat-400 and Wat-401 in LLT. This suggests a domino-type effect in the EC direction such that the physical displacement of Wat-402 by a light-induced movement of the side chain of Lys-216 (Fig. 3d) induces correlated movements of Wat-400 and Wat-401. A new location for a water molecule is visible as a positive feature between the triad of negative density peaks on water molecules in the difference Fourier map of Fig. 3c and was labeled Wat-451 in Ref. 1. Positive electron density is also seen at this position in Fig. 3a, although this was not previously modeled as a water molecule in our KLT structure (10). While a positive feature at this position is not visible at the contour level of Fig. 3b, a water molecule could be placed at this position, and it refined with a reasonable B-factor (26.9 Å2) and was visible at a contour level of 1.8 {sigma} in the Fobs-Fcalc omit electron density map. A water molecule was also placed at this position within both the K110K and L150K models by Dowser (see "Materials and Methods") when using these crystallographic structures as starting points for identifying unassigned waters, and molecular dynamic simulations of the early structural rearrangements in bR (87), as well as those starting from the KLT and LLT crystallographic models (see below), show a stable water molecule at this position.

The rearrangement of water molecules along the proton translocation channel disrupts the H-bonds to the guanidinium group of Arg-82 (Fig. 6a), which moves in response (Figs. 3d and 6b, also visualized as paired negative and positive difference electron density peaks in Fig. 3, b and c, but not in a). The conformation of the side chain of Arg-82 in the bR ground state (43, 90) toward the active site (Fig. 6a) must be delicately balanced since, in the otherwise very similar ground state structure of pSRII (sensory rhodopsin II from Natronobacterium pharaonis) (92, 93), this conserved arginine is orientated toward the EC medium. A putative domino-like mechanism, whereby the displacement of Wat-402 by the side chain of Lys-216 can destabilize the conformation of Arg-82, is strongly supported by recent molecular dynamic studies in which this sequence of events was simulated (87). Due to this major rearrangement of H-bonds along the extracellular side of the proton translocation channel (Fig. 6b), helix C is destabilized and consequently flexes in toward the proton translocation channel as quantified through the crystallographic refinement above.

Simulations of Retinal Dynamics—An issue central to vectorial proton pumping by bR is the extent to which conformational changes in the protein can influence the accessibility of the Schiff base of retinal to either the EC or cytoplasmic proton translocation channels. This idea is central to long-standing frameworks such as the Isomerization-Switch-Transfer model for proton pumping by bR (94). To investigate the influence of the conformational change of the protein from K to L on the dynamics of retinal we performed a set of simulations using the refined crystallographic structures of KLT and LLT as starting points (see "Materials and Methods" for details). Since both the KLT and LLT simulations used equivalent parameters for the description of the retinal and protein, the observed differences in the dynamics of retinal stem only from subtle differences in the electrostatic environment of the retinal due to the conformational change from K-to-L.

Fig. 7 presents trajectories deriving from molecular dynamic simulations of the K- and L-intermediates. In both cases a stable water molecule was observed between Asp-85 and Asp-212 in the position of Wat-451 (Fig. 6b) as was also observed in recent molecular dynamic simulations of the early intermediates by Hayashi et al. (87). In Fig. 7c two structures averaged over a 200-ps trajectory (after energy minimization, see "Materials and Methods") for a simulation of K (blue) and L (red) are overlaid. It is apparent from these simulations that the retinal shows significant deviation from planarity in both K and L with the perturbations to torsional angles being most pronounced for the isomerized C-13=C-14 bond, although the maximum torsion angle for any one bond was not more than 17° in these average structures. Strikingly the prevalent conformation of the Schiff base of the retinal undergoes a distinct change in orientation as a consequence of the K-to-L structural transition. Whereas in K the retinal is distorted with the Schiff base N-H dipole being inclined toward helix G, in L this dipole reorients ~180° toward helix C. A similar switch in the orientation of the Schiff base nitrogen was observed in molecular dynamic simulations by Hayashi et al. (87), although in that work it was assigned as being associated with the K-to-KL transition.



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  		FIG. 7.
Molecular dynamic simulations of the retinal geometry. a, trajectory distances from the Schiff base nitrogen to O{delta}1 of Asp-85 (red) and to O{gamma} of Thr-89 (blue) and from O{gamma} of Thr-89 to O{delta}1 of Asp-85 (orange) arising from molecular dynamic simulations of K. b, an equivalent representation arising from molecular dynamic simulations of L. c, overlay of the average geometry of the retinal and surrounding residues recovered through molecular dynamic simulations of the K-intermediate (blue) and the L-intermediate (red). The Schiff base hydrogen atom is colored white. d, examples of extreme conformations of retinal geometry that arise during simulations of the L-intermediate suggesting proton transfer pathways either directly from the Schiff base to Asp-85 (gold) or via Thr-89 (red). Hydrogen atoms are colored white. This figure was drawn using MOLSCRIPT (103).

 
Fig. 7, a and b, shows the distances from the Schiff base nitrogen to O{delta}1 of Asp-85 (red), from the Schiff base nitrogen to O{gamma} of Thr-89 (blue), and from O{gamma} of Thr-89 to O{delta}1 of Asp-85 (orange) for K and L over the same 200-ps trajectory. Interestingly a ground state H-bond from O{gamma} of Thr-89 to O{delta}2 of Asp-85 is replaced by a H-bond to O{delta}1 of Asp-85 in these intermediate state simulations due to a rotational movement of the Asp-85 carboxylate as already seen in the x-ray structures (1, 10) (Fig. 3d). The atomic distances illustrated in Fig. 7, a and b, fluctuate considerably, but on average, the Schiff base nitrogen is ~1.2 Å closer to Asp-85 in L than in K. From mechanistic grounds this would be expected since a proton must be transferred from the Schiff base to Asp-85 in the L-to-M transition, and hence a pathway must transiently form. In Fig. 7d two extreme retinal configurations for L are selected for which the N-H-O angle is favorable for H-bond formation, indicating the nature of temporary H-bonds that may link the primary proton donor to either Thr-89 (red) or Asp-85 (gold). These conformations strongly suggest the formation of transient low energy pathways for proton transfer away from the Schiff base to Asp-85 either through direct proton transfer (22) (Fig. 7d, gold) or via a coordinated proton transfer from the Schiff base to Thr-89 (Fig. 7d, red) simultaneous with a proton transfer from Thr-89 to Asp-85 (20). From these results it would appear that direct proton transfer may be slightly favored since the illustrated transient H-bond from the Schiff base nitrogen to O{delta}1 of Asp-85 is 3.1 Å (Fig. 7c, gold), whereas the distance from the Schiff base nitrogen to O{gamma} of Thr-89 is 3.6 Å (Fig. 7c, red; that from O{gamma} of Thr-89 to O{delta}1 of Asp-85 is 2.7 Å). However, more detailed calculations including the determination of the height of energy barriers to proton transfer through quantum mechanical methods are required to conclusively distinguish between these two putative pathways. It is also plausible that multiple pathways are possible for the primary proton transfer event.

It is highly significant that two pathways for proton transfer arise in simulations of L but do not arise in simulations of K. This illustrates how the reorientation of the guanidinium group of Arg-82 toward the EC medium and the local flex of helix C toward the proton translocation channel are necessary to create an electrostatic environment for efficient proton transfer. Recent criticism by Lanyi and Schobert (15) that our L-state structure could not support proton transfer because of the distance and angle of the Schiff base relative to Asp-85 appears to have overlooked the importance of dynamical fluctuations about the crystallographic structure.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Since every residue, with the exception of Asp-85, can be replaced without fundamentally altering the vectorial nature of the proton pump bR (95), then the details of the primary proton transfer event must be central to understanding the overall mechanism. For a proton to move from the Schiff base (pKa 13.5 (71)) to Asp-85 (pKa 2.2 (70)) orchestrated structural changes must occur that reverse the proton donor/acceptor relationship between these oppositely charged groups. From the refined structure of L150K determined here (Figs. 3d and 6b), the manner in which the protein utilizes conformational changes to significantly alter the pKa values of both the primary proton donor and acceptor in advance of proton transfer can be understood. Upon photoisomerization and dislocation of Wat-402, the loss of a stabilizing H-bond to the Schiff base (96), as well as the reorientation of the positively charged Schiff base into a hydrophobic region (67), lowers the pKa of the Schiff base by as much as 5 pKa units in L (97, 98). Conversely the loss of a H-bond of Asp-85 to a water molecule and change to its H-bond to Thr-89, the reorientation of Arg-82 away from the active site, and the local flex of helix C that enhances the interaction between the two negatively charged aspartic acid residues in LLT (1) all serve to increase the pKa of Asp-85 by perhaps as much as 8 pKa units (99). Thus the effective reversal of the original donor/acceptor relationship makes it possible for Asp-85 to accept the Schiff base proton. In addition to changing the pKa values of the key groups involved, a low barrier proton transfer pathway from the Schiff base to Asp-85 must (at least transiently) be created. Molecular dynamic simulations presented herein are highly suggestive as to the nature of this pathway. Associated with the K-to-L structural transition, the conformation of the retinal is perturbed to the extent that H-bonds from the Schiff base to either Thr-89 or Asp-85 can transiently form. These distorted retinal conformations (Fig. 7d) imply transient pathways for proton transfer from the Schiff base to Asp-85 either by direct proton transfer or via Thr-89.

Within this picture the observed local bend of helix C toward the proton translocation channel (Figs. 3 and 4) is unique to L and plays a central role in setting the stage for proton transfer since the new electrostatic environment of the retinal in L profoundly influences the conformational dynamics of retinal (Fig. 7). That a global rearrangement of a secondary structural element is associated with L explains why a time scale of µs is needed for L to build up at room temperature and incorporates the observation that Asp-85 approaches the Schiff base in L (27, 76). A testable consequence of this hypothesis is that for bR mutants that decrease (or increase) the primary proton donor/acceptor separation, the rate of Schiff base deprotonation should show a corresponding increase (or decrease). Indeed proton transfer in the D85E bR mutant (which lengthens the side chain and presumably decreases the Schiff base nitrogen-carboxylate group distance) is 10-fold faster than that for wild type bR (100). Conversely studies on the D96G/F171C/F219L triple bR mutant, for which the Schiff base-Asp-85 distance is increased by ~1 Å (20), show that the primary proton transfer event is slowed 20-fold (101). Following proton transfer the electrostatic attraction between Asp-85 and the Schiff base is cancelled, allowing the two groups to relax apart, relieving electrostatic strain on both the retinal (which could relax toward the cytoplasmic side) and helix C. Should the primary proton transfer step proceed from the Schiff base directly to Asp-85, then only a slight separation of these groups following proton transfer is needed to reinstate a large energy barrier, thus preventing the reverse proton transfer and imprinting directionality on the overall mechanism.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1R3P) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by the Swiss National Science Foundation, the European Union-BIOTECH, the Howard Hughes Medical Institute, The Swedish Science Research Council (VR), SWEGENE, the Swedish Strategic Research Foundation (SSF), and the French Ministry of Education and Research (MENR). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

h To whom correspondence may be addressed. E-mail: emlandau{at}utmb.edu. i To whom correspondence may be addressed. E-mail: pebay{at}godot.ibs.fr. j To whom correspondence may be addressed. E-mail: neutze{at}molbiotech.chalmers.se.

1 The abbreviations used are: bR, bacteriorhodopsin; LT, low temperature; EC, extracellular; CCD, charge-coupled device; Wat, water; {sigma}, root mean square electron density of the map. Back

2 E. Tajkhorshid, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank D. Bourgeois for access to the European Synchrotron Radiation Facility cryobench facilities and the staff of beamline ID14 for experimental support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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