In the following, we examine specific structural features of wild-type bacteriorhodopsin and the D96A and T46V mutants. In such comparisons, if the differences in the atomic positions or the cavity sizes are real they must exceed the magnitude of positional error between different crystals of the same kind. For the wild-type, this variability was estimated from the averages and standard deviations in independent models refined from diffraction data from five crystals, with resolutions ranging from 1.43 to 1.60 Å. Two of these are published, alone or as part of determination of a photocycle intermediate (35
), but three are from newly collected data. For the T46V mutant the positional errors are calculated from models for four crystals, with resolutions between 1.84 and 1.92 Å. The crystallographic resolution for D96A is 2.0 Å (two crystals). There was no detectable twinning in the crystals of the two mutants, i.e. the quality of the data is more or less equivalent to the wild-type. The statistics for the data and the refinement of models for the D96A and T46V mutants are given in . The standard deviations for inter-atomic distances in the five wild type models will be given in the comparisons below, and are ± 0.06 – 0.20 Å depending on the location, in line with earlier attempts to determine crystal-to-crystal reproducibility in our bacteriorhodopsin crystals (37
). When we conclude that the structural feature is different it is only when the change of inter-atomic distance or cavity size is well above the corresponding standard deviation (or in the case of D96A the range from two independent crystals).
Table 1 X-ray data collection and refinement statistics for data sets from a non-illuminated and an illuminated crystal of the D96A bacteriorhodopsin mutant. The illumination at 295K with red laser, as described in Methods, converted bacteriorhodopsin in the (more ...)
Our intent was to compare also the structural changes in the M (last M substate, or M2
’) states produced by illumination of the D96A and D96N mutants under the same conditions, i.e., at ambient temperature. Although it would be more relevant to transport function to use the M state of the wild-type as reference, this is not possible. In the wild-type protein, M decays too rapidly at room temperature to accumulate in amounts more than a few percent under usual illumination conditions. To populate this state, in the two studies of wild-type M produced in cubic phase grown crystals at ambient temperature (10
), the illumination was continued for a short time (1 sec) as the crystals were rapidly cooled, and thus the M was produced at some undefined temperature below ambient. Such a regime produces either a claimed mixture (38
) of early and late M, or in our experience (10
) an earlier M state than M2
’. In both cases, the retinal assumed different configurations than in M of D96N. In two other studies, the illumination of the crystals was at 210 K (36
) and 230 K (40
), and the M produced was also earlier M, i.e., M1
, with lesser changes than the later M states. In yet another study of the wild-type M intermediate, with crystals of a different kind where the crystal contacts were suggested to slow the decay of M and allow its accumulation at ambient temperature (41
), the structural changes do not seem to be comparable to those in cubic phase crystals. In this case, there was virtually no change from the BR state near residue 96, where our attention is focused, but a sliding movement of helix G not seen in the other M structures was reported. The M state of the E204Q mutant (12
), on the other hand, shows similarities to the structure of the M intermediate of D96N. Although the M produced is an earlier sub-state because the E204Q mutation blocks proton release to the extracellular surface, i.e., the M2
’ reaction (6
), the illumination conditions are most similar to those used for the D96A and D96N mutants.
Because the illuminated crystals contained little of the non-illuminated state of the protein, both models for M, from D96A in this study and from D96N earlier (13
), are the results of refinements from data with full occupancy. The resolution of the data for M of D96N was 2.0 Å, and for D96A it is 2.08 Å (the latter from a crystal with merohedral twinning, see , as in the earlier report).
Structural Changes in the Non-illuminated State from Replacement of Asp-96
The crystal structure of the non-illuminated F219L mutant had revealed (10
) that the cavity created by the side-chain replacement is filled by two water molecules hydrogen-bonded to each other and to an existing water (wat501). Comparison of the 2Fobs
electron density maps of the immediate neighborhood of residue 96 in the D96A mutant and the wild type () shows, however, that this is not always the case. The cavity between Ala-96 and Thr-46 from the smaller Ala side-chain, with a volume of 23–25 Å3
, contains no density. As in the wild-type, the side-chain of Thr-46 donates a hydrogen-bond to the peptide C=O of Phe-42 (not shown in ), but the energy penalty of inserting a water molecule into this low-dielectric matrix is evidently not compensated by the hydrogen-bond this water would donate to OG1 of Thr-46. In contrast, replacement of the protonated carboxyl of residue 96 with an amide in D96N causes (13
) insertion of a water molecule to bridge the amide ND2 and OG1 of Thr-46, and creates no cavity.
FIGURE 1 Electron density (2Fobs − Fcalc) maps of the Asp-96 region in the non-illuminated state, in wild-type bacteriorhodopsin (a), the D96A mutant (b), and in T46V (c). Contour level at 1 σ. Wild-type model and map from 1C3W (32).
The Asp to Ala residue replacement in D96A, and the consequent loss of the hydrogen-bond between Asp-96 and Thr-46 that connects helices B and C, will perturb this region. As shown in , where the model of D96A is overlaid on the wild type structure (latter in green), the cytoplasmic ends of helices B and C move apart. As a result, the distance between CA atoms of residue 96 and Thr-46 increases by nearly 1 Å. Another indication of perturbation is the increase of a small cavity in the protein, bounded by helices B, C, and G, that extends from the Thr-46 region toward the retinal (not shown). In the wild-type this cavity has a volume of 75 ± 4 Å3, but in D96A it nearly doubles in size, to 136 – 143 Å3. Otherwise, the main-chain and side-chain displacements from the D96A mutation are local, however, and do not extend significantly toward the center of the protein beyond Leu-48 on helix B and Leu-93 on helix C ().
FIGURE 2 Comparison of the Asp-96/Thr-46 region that connects helices B and C, in the non-illuminated states in D96A and the wild-type. The model for the mutant is shown with atomic colors, the wild-type model (32) in green.
Although the primary main-chain and side-chain perturbations from the D96A mutation in do not reach the retinal region, water molecules are affected as far away as the Schiff base and beyond, i.e. at a distance as far as 15 Å. Because these water molecules form hydrogen-bonds with functionally important residues and play prominent roles in the proton transport mechanism (42
), their displacements may be meaningful.
We had suggested earlier that the retinal region is connected to the Asp-96/Thr-46 region through a continuous chain of covalent and hydrogen-bonds that appear to have a functional role during the photocycle (12
). The structure of the D96A mutant (atomic color model in ) reveals that the retinal Schiff base region is influenced by the displacement of Thr-46 via the same chain. In the wild type, wat501 bridges helices G and F through hydrogen-bonds to the peptide O of Ala-215 and NH1 of Trp-182 (green model in ). The hydrogen-bond of wat501 to Trp-182 is essentially unchanged in the D96A mutant (its length is 2.91 – 3.05 Å vs. 2.80 ± 0.06 Å in the wild-type). However, the hydrogen-bond between wat501 and the O of Ala-215 is broken
as the inter-atomic distance increases from 2.97 ± 0.07 Å in the wild-type to 3.30–3.52 Å. This appears to be an indirect consequence of the displacement of helix B at Thr-46, as follows. Wat502 moves with the movement of Thr-46 described above because its hydrogen-bonds with the peptide O of Thr-46 (2.87 – 3.00 Å vs. 2.95 ± 0.09 Å in the wild type) and with the peptide O of Lys-216 also (2.94 – 2.95 Å vs. 3.02 ± 0.12 Å in the wild-type) are maintained (). Ala-215 is linked to the cytoplasmic region via the hydrogen-bond of the peptide O of Lys-216 to wat502. Displacement of wat502 therefore causes the main-chain of helix G to move at Lys-216 and Ala-215, and the peptide O of Ala-215 moves away from wat501, its hydrogen-bonding partner in the wild type protein.
FIGURE 3 Comparison of the retinal region of the non-illuminated states of the D96A (a) and T46V (b) mutants (shown with atomic colors) with the wild-type (32) (in green).
Unexpectedly, as shown in , water molecules are displaced in the D96A mutant in the extracellular region also. Wat402 is coordinated by the Schiff base nitrogen (NZ of Lys-216) and OD2 of two anionic residues, Asp-85 and Asp-212. Wat402 is displaced strongly toward Asp-85 (the length of its hydrogen-bond with OD2 of Asp-85 is 2.30–2.46 Å vs. 2.67 ± 0.13 Å in the wild type), while its distance to Asp-212 is nearly unaffected (2.99–3.30 Å vs. 3.04 ± 0.20 Å in the wild type). Surprisingly, in the D96A mutant, wat401, the link of Asp-85 to the extracellular aqueous network, moves close enough to wat402 to form a new hydrogen-bond (inter-water distance is decreased from 3.68 ± 0.17 Å in the wild type to 2.87 – 3.06 Å). The approach of wat401 and wat402 is partly from the displacement of wat402, but mostly from the movement of wat401 (). In spite of this movement, the hydrogen-bonds of wat401 to its partners in the wild-type (Asp-85 and wat406) are relatively unaffected (not shown in ). Likewise, although there is minor redistribution of side-chains in the extracellular region, no hydrogen-bonds are broken or formed as a result of the movement of wat401.
The observed movements of protein atoms, and particularly water far from the site of the D96A mutation, urge caution in interpreting the phenotypes of all mutations in terms of local effects, and in assigning O-H bands to specific water molecules solely on the basis of the location of mutations that affect them.
Little of this cascade of displacements between Thr-46 and the retinal region is evident in the earlier determined structure (13
) of the more conservative mutant, D96N. The chain of hydrogen-bonds through O of Thr-46, wat502, and O of Lys-216 is unaffected by the smaller movement of helix C in this mutant, and the hydrogen-bond between O of Ala-215 and wat501 is not broken (not shown). The cytoplasmic cavity between Thr-46 and the retinal is only slightly increased, if at all, relative to the wild-type (to 82 Å3
vs. 75 ± 4 Å). The changes in the extracellular region are also less in D96N. Thus, although wat402 moves closer to OD2 of Asp-85 in D96N (2.30 Å vs. 2.67 ± 0.13 Å in the wild type) as in D96A, the inter-atomic distance between wat401 and wat402 is decreased (to 3.36 Å vs. 3.68 ± 0.17 Å in the wild type) but not sufficiently to form a hydrogen-bond.
Structural Changes in the Non-illuminated State from Replacement of Thr-46
The Thr to Val change for residue 46 breaks its hydrogen-bond with Asp-96 as does the D96A mutation, but there are two important differences. First, the side-chain is replaced without change of volume. Second, the energy penalty of burying the highly polar COOH of residue 96 without a hydrogen-bonding partner in T46V must be greater than burying the OH of the Thr in D96A. Indeed, the 2Fobs − Fcalc electron density map in this region is different in the T46V and the D96A mutants (compare ). There is no new cavity at residue 96 as in D96A, because a new water molecule, wat504, intercalates and forms a hydrogen-bond with OD2 of Asp-96 and O of Ile-45.
Consistent with the absence of a cavity at residue 96 and the formation of a new hydrogen-bond that retains the link between helices B and C, perturbation of the Asp-96 region in the T46V mutant is less than in D96A (). The distance between the CA atoms of Asp-96 and residue 46 is increased by less than 0.5 Å relative to the wild-type, and this change is from movement of both residues rather than mainly of Thr-46 as in D96A (). The cavity that extends toward retinal is increased in size, but less than in D96A, from 75 ± 4 Å3 in the wild-type to 94 ± 5 Å3. As in the D96A mutant, the perturbation is local, and little change is observed a few residues away on either helix B or C ().
FIGURE 4 Comparison of the retinal region of the non-illuminated states of the D96A (a) and T46V (b) mutants (shown with atomic colors) with the wild-type (32) (in green).
Consistent with the smaller perturbation at residue 46, the retinal region is less affected. As shown in , the hydrogen-bond of Ala-215 with wat501 is maintained (inter-atomic distance 2.97 ± 0.06 Å). Wat401 does not move significantly, and a new hydrogen-bond between wat402 and wat401 is not formed (inter-atomic distance 3.41 ± 0.09 Å).
In an earlier study of 2-dimensional crystals of the T46V mutant (17
), a density feature at helix C was observed in the non-illuminated T46V vs. non-illuminated wild type difference projection map. Its origin may well be the separation of helices B and C in .
Structural Changes in the M State from Replacement of Asp-96
Given the structural changes in the non-illuminated D96A mutant described above, it would be informative to know how they affect the structure of the M state. The decay of the M state is greatly slowed by replacement of Asp-96 with a non-protonatable residue, particularly at higher pH (31
), and this allows accumulation of the last M state (M2
’) in a photostationary state. Indeed, as with D96N crystals at neutral pH (13
), a full color-change from purple to yellow upon illumination of D96A crystals at pH 8.5 at ambient temperature indicated the virtually complete conversion to the M state. This state was then trapped by rapidly dropping the temperature to 100K, as before (13
electron density maps of the non-illuminated and M states of D96A, respectively. As expected, the trapped M state of this mutant bears considerable similarity to the earlier reported (13
) M of D96N: the retinal is 13-cis,15-anti, wat402 and wat406 are absent in the map (latter not shown), wat401 forms a hydrogen-bond with OD2 of Asp-85, and the Arg-82 side-chain is rotated toward the extracellular surface. An unexpected feature in M is a new water molecule, wat507, hydrogen-bonded to wat501. A water molecule had been reported (13
) at this location in the non-illuminated, although not in the M state, of D96N.
Electron density (2Fobs − Fcalc) maps of the retinal region in the non-illuminated state (a) and the trapped M state (b), in the D96A mutant. Contour level at 1 σ.
Elsewhere in the protein, however, the M states of D96A and D96N are different. show models of the retinal region in the M states (atomic colors) of D96N and D96A, respectively, each superimposed on the non-illuminated state of the same mutant (blue color). In D96N () the rotation of the retinal C15
=NZ-CE segment with the deprotonated Schiff base from the extracellular to the cytoplasmic direction and the accompanying changes of the geometry of the retinal polyene chain and the Lys-216 side-chain move the main-chain of helix G at Lys-216 and therefore at Ala-215 (for a detailed discussion of this, see refs. 12
). Displacement of O of Ala-215 breaks its hydrogen-bond with wat501, and there is no electron density for this water in M. The connection of helix G to helix F through this water and Trp-182 is thereby eliminated, and the Trp ring is free to tip upward from the approach of the 13-methyl group of the retinal (). Movement of Lys-216 moves the connected wat502 and the O of Thr-46, moving OG1 of Thr-46 away from Asn-96. Inasmuch as the same kinds of changes occur at these locations in the trapped M state of the E204Q mutant (12
), this structural shift may occur in the wild-type M as well. It should have the rationale to lower the pKa
of Asp-96 so it will become the proton donor to the Schiff base.
FIGURE 6 Comparison of the retinal region of the trapped M states of the D96N (13) (a) and D96A (b) mutants (shown with atomic colors) with their respective non-illuminated states (in blue).
The displacements of Ala-215, Lys-216, wat502 and wat401 (but not Trp-182, and obviously not the retinal) in the M of D96N () are similar to those in the non-illuminated state of the D96A mutant (), although they are greater in magnitude. Thus, in both models the O of Ala-215 moves away from wat501, wat502 is moved by displacement of Lys-216, and wat401 approaches the two aspartate carboxyls. In the M state the perturbation originates at the retinal and spreads to Asn-96 via Thr-46 (), while in D96A the perturbation originates at residue 96 and spreads to Thr-46 and then the retinal region (). It appears that the same “track” of covalent and hydrogen-bonds can be the means for propagating structural perturbation in both directions.
The changes in the position of Ala-215, wat502, and wat401 in M of D96N will have occurred partly in the non-illuminated D96A already. Consistent with perturbation of this region, while the movements of the retinal in the M state of D96A () are similar to those of the M state of D96N (), the ensuing displacements of Lys-216, Ala-215, Lys-216, and wat501 are less extensive. For example, the movement of O of Ala-215 in the M state of D96N relative to the non-illuminated state is 0.97 Å, but in the M state of D96A it is only 0.55 Å. Water 502, connected to O of Lys-216, moves by 1.00 Å in M of D96N but 0.51 Å in the M state of D96A. As a result, in the M state of D96A communication of the retinal region with the Thr-46 region will have been impaired and Thr-46 should be less displaced.
compare the region of Thr-46 in the non-illuminated and M states, in D96N and D96A, respectively. In M of D96N the displacement of wat502 toward O of Thr-46 allows movement of the peptide segment, and the ensuing torsion of the main-chain moves OG1 of Thr-46 away from wat504 (by 0.88 Å). The inter-atomic distance between these atoms increases from 3.29 Å to 4.02 Å and the weak hydrogen-bond breaks. Because the other hydrogen-bonds in this region are maintained, the bulky side-chain of Phe-42, a participant of the cytoplasmic hydrophobic barrier (10
) like Phe-219, moves, presumably in anticipation of the increased hydration that allows conducting a proton to the retinal Schiff base in the next step of the photocycle.
FIGURE 7 Comparison of the Asp-96/Thr-46 region in the trapped M states of the D96N (13) (a) and the D96A (b) mutants. The models for M are shown with atomic colors, the models for their respective non-illuminated states in blue.
In M of D96A the lesser, and more importantly lateral, displacement of wat502 relative to O of Thr-46 results in lesser changes at Thr-46 (). In D96A OG1 of Thr-46 undergoes virtually no movement in M (its displacement is only 0.14 Å).
In the earlier described trapped M state of D96N, the cytoplasmic ends of helices F and G, as well as part of the E-F interhelical loop, were too disordered to model (13
). The disorder along helix F begins at Val-177, which is displaced in a manner consistent with the outward tilt of the cytoplasmic end of helix F detected in many other studies (16
). Thus, it may be the result of the tilt that disrupts the crystal locally, and as such constitutes evidence for this large-scale conformational shift. However, we detect neither disorder nor observable tilt of helix F in the trapped M of D96A, consistent with the lack of structural changes at the Ala-96/Thr-46 pair (). In this respect also, the M states of D96A and D96N seem to differ. Such a difference was reported before. In the projection map of the M state of a 2-dimensional crystal of D96G mutant (17
), density changes were detected at helices F and G, but the difference map had considerably less magnitude at helix F than in the M state of the wild-type and D96N. The lesser tilt of helix F was attributed to an alteration of conformational flexibility in the D96G mutant, but not in the more conservative D96N mutant. This observation and its interpretation are consistent with the 3-dimensional structure we report here for the D96A mutant.