12002
J. Phys. Chem. 1996, 100, 12002-12007
Effect of Binding of Lanthanide Ions on the Bacteriorhodopsin Hexagonal Structure: An X-ray Study Jennifer A. Griffiths and M. A. El-Sayed* School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332
Malcom Capel Biology Department, BrookhaVen National Laboratories, Upton, New York ReceiVed: March 11, 1996; In Final Form: May 17, 1996X
The effect of the binding of trivalent lanthanide metal cations (Eu3+, Ho3+, and Dy3+) on the hexagonal structure of bacteriorhodopsin (bR) is investigated at different pH using x-Ray diffraction to examine films made by slow evaporation of the corresponding regenerated bR. It is observed that the lanthanide-regenerated bR (at a ratio of 2:1 metal ion to bR) does not form a 2D structure isomorphous to that of native bR or Ca2+-regenerated samples at low sample pH. The native bR hexagonal structure is recovered by titration of the sample with sodium hydroxide. The pH at which the hexagonal structure is recovered depends on the charge density of the lanthanide ion used for the regeneration. The higher the charge density of the ion, the higher the pH at which an isomorphous lattice is formed. A model is proposed in which at normal or low pH a complex bidentate and monodentate type binding (which disrupts the lattice hexagonal structure) exists between a lanthanide ion, the O- of PO2- groups, and/or the amino acid residues. At high pH, complexation with OH- takes place, which converts this binding to a simple monodentate type complex that leads to the recovery of the lattice structure. An equation is derived for the pH at which this conversion takes place and is found to be proportional to the binding constant of the lanthanide ions to the O- of the PO2- groups or the amino acid residues and inversely proportional to the binding constant of the lanthanide ion to the OHgroups. This predicts an increase of conversion pH with the charge density of the lanthanide ion, as observed.
Introduction Bacteriorhodopsin (bR), which is the only protein found in the purple membrane (PM) of Halobacterium salinarium, is, like chlorophyll, a natural photosynthetic system.1-3 bR contains as a chromophore an all-trans retinal, which is covalently bound via a Schiff base linkage to the -amino group of Lys216.4 Upon absorbing a photon, the protein undergoes a photocycle in which multiple intermediates have been identified (for a review, see ref 5). The final step in the photocycle returns bR to its native structure, from which it can begin the cycle again.5 This photocycle results in the translocation of protons from the interior to the extracellular side of the purple membrane.5 The proton gradient created by this mechanism is used by the bacteria for metabolic processes such as ATP synthesis.6,7 bR has ∼2-4 mol of Ca2+ and Mg2+ bound per 1 mol of bR at pH ∼6 in the wild type protein.8-10 Removal of these metal cations changes the purple color of wild type bR (λmax ) 570 nm) to blue (λmax ) 604 nm).8,9 The consequence of the removal of these cations is that the blue membrane does not undergo a complete photocycle and thus does not pump protons.8,9 The efforts in the study of metal cations in bR11,12 have been directed toward answering two general questions: (1) what is the binding mechanism for the cations in the protein and how do they control the color of the membrane, and (2) how do the cations functionally influence the proton pumping mechanism of bR (that is, do the cations act to stabilize a structural conformation that is necessary for proton pumping or are they involved in the energy transduction pathway directly)? There are two distinct models for the location of the cationbinding sites and the mechanism through which they affect the X
Abstract published in AdVance ACS Abstracts, July 1, 1996.
S0022-3654(96)00741-1 CCC: $12.00
color and function of the purple membrane. In the model of Szundi and Stoeckenius,13 the metal cations are bound to the membrane surface as free positive charges. The effect of the positive charges on the surface is to decrease the surface negative charge density. The high surface pH generated on the surface by the presence of the cations (the Gouy-Chapman effect) is sufficient to keep the counterion (aspartate) deprotonated and allow the amino acid to act as a proton acceptor during the photocycle. Thus removal of the metal cations decreases the surface pH, allowing the protonation of the aspartate counterion. This results in a change in the retinal color to produce blue bR. Conversely, several groups14-16 propose that there are specific binding sites within the protein between specific amino acid residues. In this model, the cation could be more directly involved in controlling the membrane color through interaction with the retinal and variation in the pKa of the protonated Schiff base. This latter binding mechanism, in which there is direct and specific association between the protein and the metal cation, has found significant support. Multiple spectroscopic studies17-20 have reached the conclusion that there are more than two classes of cation-binding sites in the deionized blue membrane, with different binding affinities and therefore different binding environments. Potentiometric titration studies20 of blue bR with Ca2+ concluded that there is a high-affinity Ca2+-binding site within the active site of the protein. It was suggested that Asp85, Asp212, Tyr185, and Arg82 might make the binding site for this Ca2+. The results of the potentiometric studies on mutants21 and on bR with the C-terminus cleaved22 suggest the presence of two metal cations within the protein whose binding constants are sensitive to charges on Asp85 and Asp212 as well as other low-affinity binding sites on the surface. The results of FTIR studies of Ca2+-regenerated bR and D85N mutant gave © 1996 American Chemical Society
Effect of Binding of Lanthanide Ions indication of the coupling between Ca2+ and the O- of Asp85.23 More recent theoretical simulations24 of the two-photon spectrum of the retinal in bR found good agreement with experimental results if the Ca2+ is bound to the O- of Asp85 and Asp212. The authors suggest24 that this cation is the second high-affinity cation observed in the potentiometric titrations. Several attempts have been made to locate the binding site of the metal cations through structural studies of 2D bR crystals using isomorphous replacement of the native cations. Stroud and co-workers25,26 have found up to four distinct binding sites located on the R helices of the protein using isomorphous replacement of the native cations with Pb2+. All of these studies are limited to information on the cation-binding site in the plane of the membrane (xy axis) due to the lack of order along the perpendicular (z) axis. Engelhardt et al.27 have utilized EXAFS on Fe3+-regenerated blue bR. The authors infer from the results that the metal cations are in an octahedral geometry coupled to 5.8 ( 1.5 carboxylate ligands, although the data cannot distinguish between nitrogen and oxygen ligands. Despite the wealth of research on metal cations in bR, there is still uncertainty in the answers to the questions posed earlier. To further understanding in these areas, we have used X-ray diffraction to monitor the effects of various lanthanide metal cations on the structure of bacteriorhodopsin when bound to the two high-affinity binding sites of bR. The trivalent lanthanides have been used extensively as replacements for the calcium in metalloproteins due to their similarity in size and binding to Ca2+ and Mg2+ as well as value in studies of fluorescence, EPR, NMR, etc.28,29 X-ray diffraction can be used as a direct probe of any changes in the hexagonal structure of purple membrane upon cation binding. The structure of the wild type bR in purple membrane has been investigated by X-ray diffraction,30 and there is a detailed investigation on the threedimensional structure by Henderson and co-workers with electron microscopy.31 The bR molecules interact to form a trimer structure, and in the purple membrane and in solid samples, they form a hexagonal structure of trimers with a unit cell length of 62.45 Å. The films made of regenerated bR sample solutions made by mixing 1:2 deionized bR and lanthanide metal cations at low pH are found to possess different structure from that of native bR. As the pH increases, the native bR structure is recovered at a pH that depends on the metal cation used. These results are in accordance with our previously observed FTIR results, which concluded that these metal cations bind strongly to the PO2- groups of the phospholipids of the membrane via bidentate type binding at low pH, which is changed to weaker monodentate type binding at high pH. Combining the X-ray and FTIR results, one concludes that complex multiple binding with metal cation lanthanides is the cause of the changes in the tertiary structure of the lanthanide-regenerated bR at low pH. Materials and Methods Purple membrane was prepared from the Halobacterium salinarium strain ET1001 as described previously.32 Blue membrane was prepared by passing the purple membrane over a cation exchange column (Bio-Rad). The final pH of the blue membrane was ∼4 with λmax ) 604 nm. All samples were suspended in Nanopure water (resistivity 10-18 MΩ cm) and kept in plastic tubes to avoid contamination from other cations. Concentration of the protein was determined using the following extinction coefficients: 570 ) 63 000 cm-1 M-1 and 608 ) 60 000 cm-1M-1. Calcium-regenerated samples were prepared by adding a ratio of 5:1 CaCl2 (10 mM) solution to deionized bR. These samples were equilibrated for at least 4 h. Lan-
J. Phys. Chem., Vol. 100, No. 29, 1996 12003 thanide-regenerated samples were prepared by adding solutions of EuCl3 (10 mM), DyCl3 (10 mM), and HoCl3 (10mM) (Sigma, 99.99% purity) to deionized bR at a stoichiometric ratio of 2:1 lanthanide cation to bR. pH titrations of the regenerated samples were done using NaOH (10 mM). All samples were washed with Nanopure water after regeneration and prior to film preparation to remove any residual, unbound cations. X-ray samples were prepared by drying at a relative humidity of 86% small drops (2-3 mm) of concentrated protein on 12 µm Mylar film. Samples were prepared for each metal cation with a range of pH values. The pH of each sample was titrated with NaOH after addition of the metal cation to the bR solution. Reported sample pH values were measured prior to drying the sample and, as such, do not necessarily reflect the pH of the dried sample. After drying, the sample was mounted in a sealed holder in which the relative humidity was kept at 100% prior to and during the experiment. The sample holder was mounted with the purple membrane (PM) films perpendicular to the X-ray beam. X-ray diffraction data were acquired at NSLS beamline X-12B using a two-dimensional multiwire gas proportional imaging detector.33 The X12B beamline provides an intense (order 1011 photons/s) monochromatic beam with a focus spot approximately 350 × 350 µm (full width at half-maximum) placed at the detector position.34 This system has an active area 10 × 10 cm, with a nominal resolution (pixel size) of 200 × 200 µm when using argon as the working medium. Heliumfilled flight paths were employed to minimize the contribution of air scatter to the background. Fluorescence XAFS spectra were measured for all specimens to confirm binding of the replacement lanthanide ions. Individual diffraction patterns were integrated for 300 s, corrected for detector response nonuniformity, and radially averaged using bin widths equal to the detector pixel dimension. Background intensity profiles were collected for an empty sample cell, corrected for changes in incident flux, and subtracted from the bR sample data. Data was collected for 5 min on each sample. In a damage control study, no shift in the position of the Bragg reflections was observed for 5 h radiation exposure, although significant destruction of the lattice and sample bleaching were observed for longer exposure times. Results Figures 1 and 2 show the radially averaged X-ray diffraction patterns from 2D films of deionized PM that are reconstituted at a lanthanide to bR ratio of 2:1 with either Ho3+ or Dy3+ (data for Eu3+ regeneration not shown). The intensities are plotted in reciprocal space for intensity vs Q (A-1). Diffraction patterns were obtained at multiple pH’s, as indicated in the figures. It can be observed that the PM lattice diffracts to higher resolution with increasing pH, reaching a final pH at which the radial average is identical to that of wild type bR films. In addition, the Bragg peaks shift continuously, with the higher pH corresponding to samples with a hexagonal lattice that is similar to wild type bR. It should be noted that the final pH at which the Bragg peak positions are the same as that observed for wild type bR depends on the lanthanide cation. In general, the final pH necessary increases as Eu3+ < Dy3+ < Ho3+, with the Eu3+-regenerated samples gaining a structure isomorphous to wild type bR at pH 7. Analysis of the experimentally determined radial profile of the wild type regenerated sample gives a hexagonal lattice unit cell length of 63.1 Å, which is in reasonable agreement with that of 62.45 Å observed by Henderson and co-workers with electron microscopy31 for the wild type bR. The retention of the basic topological features of the radial distribution in all
12004 J. Phys. Chem., Vol. 100, No. 29, 1996
Figure 1. Intensity profiles obtained by radially averaging deionized bR samples regenerated with Ho3+ at a ratio of 2:1 ion to bR. Three different samples are shown in which the pH was varied from 4.6 to 10 by titration with NaOH prior to film formation. It is observed that the purple membrane lattice diffracts to higher resolution with increasing pH. In addition, the diffraction peaks shift continuously with increasing pH. At pH 10 the Ho3+-regenerated sample has a hexagonal structure isomorphous to wild type bR. The intensity profile of wild type bR is shown for comparison.
the low-pH lanthanide-regenerated samples indicates that the hexagonal structure of bR is retained. However, the unit cell dimension is smaller at low pH, as evidenced by the shift of the Bragg peaks. The unit cell length is found to vary from 57 Å at low pH (this value varies depending on the initial pH of the sample and the metal ion used for regeneration) to 63.1 Å at high pH; that is, as the hydroxyl concentration is increased, the hexagonal unit cell dimensions increase. A second observation is that the full width at half-maximum (fwhm) of the diffraction peaks is larger at low pH and narrows as the pH is increased, reaching a final fwhm that is comparable to the Ca2+regenerated sample. In general, the width of a Bragg peak is related to any combination of the following effects: (1) lattice distortion, (2) small crystallite sizes, or (3) structural factors. To check that the change with pH was not a general effect observed in any metal cation regenerated bR sample, Figure 3 shows the pH dependence of the Ca2+-regenerated sample at a ratio of 5:1 cation to bR from pH 4 to 10. There is no observed shift in the peak positions in the radial profile under these conditions. The presence of the lanthanide cations at high pH in the bR films was confirmed by EXAFS on the same samples used for diffraction. Figure 4 shows a typical EXAFS scan for a Ho3+regenerated sample at pH 9.0. The presence of the EXAFS signal implies that titration of the sample with NaOH did not simply replace the lanthanide cations with Na+ cations. In addition, as the samples are washed after regeneration and prior to film preparation it is unlikely that the lanthanide cations are located interstitially between the membrane planes. Discussion There are four interesting results of this experiment to address: (1) the change of the hexagonal structure observed
Griffiths et al.
Figure 2. Intensity profiles obtained by radially averaging deionized bR samples regenerated with Dy3+ at a ratio of 2:1 ion to bR. The pH of the samples was varied from 6.5 to 9. As for Ho3+, the purple membrane lattice diffracts to higher resolution with increasing pH, and the peaks shift continuously with increasing pH. However, Dy3+ regains the hexagonal structure of wild type bR at a pH of 9, lower than observed for Ho3+. The intensity profile of wild type bR is shown for comparison.
Figure 3. Radially integrated intensity profiles for Ca2+-regenerated bR (6:1 ion to bR) at pH 7, 8.5, 9.5, and 10.5. For comparison of different samples, the four radial profiles were scaled to the average column sum and are plotted overlapping each other. In contrast to the pH dependence of the lanthanide-regenerated samples, there is no pH dependence of the Ca2+-regenerated samples; that is, all spectra are identical.
upon regenerating bR at low pH with lanthanide cations but not observed with Ca2+, (2) the recovery of the hexagonal unit cell with increasing bulk pH, (3) the change in the hexagonal unit cell dimensions with increasing bulk pH, and (4) the dependence of the pH value (at which the hexagonal structure isomorphous with wild type bR is recovered) on the charge density of the lanthanide ion used for regeneration.
Effect of Binding of Lanthanide Ions
J. Phys. Chem., Vol. 100, No. 29, 1996 12005 this perturbation. This is not the case in the simple nondirectional ionic type binding of Ca2+ ions. The complex strong interhelices binding of the lanthanide ions could be changed if the bidentate PO2- lanthanide amino acid complex is changed into a simple monodentate complex with OH-. This changes the helix-helix flexibility and could lead to structural changes in the tertiary structure. In this case, the experimentally observed pH dependence at which the conversion of the protein-protein or protein-PO2- complex changes into a simple monodentate complex depends on the equilibrium constant, K, of the reaction O2
O2 O1
Figure 4. Fluorescence XAFS spectra of Ho3+-regenerated bR at pH 9. The presence of the XAFS signal indicates that Ho3+ is bound to bR and not displaced by Na+ with increasing pH.
The experimental results suggest two mechanisms for the pH dependent interaction of the lanthanide ion with the membrane protein. One possibility is that the trivalent charge on the lanthanide ion repels or attracts neighboring amino acids or acidic lipid head groups. As a result, the protein and the membrane undergo slight conformational changes that preclude the formation of the hexagonal lattice of wild type bR. In this case, the increase in pH could simply increase the number of OH- ions bound to the lanthanide ion and thus decrease the positive charge of the trivalent cation felt by the protein and/or lipids. Eventually a point is reached where the net charge that these groups feel from the trivalent cation is similar to that for the divalent cation, and the purple membrane assumes a conformation similar to that of wild type or Ca2+-regenerated bR. The known basicities of the lanthanides suggest that this mechanism does not occur in our system. In general, the basicity of the lanthanide decreases35 as Eu3+ > Dy3+ g Ho3+. The key issue to note is that, on the basis of this trend, Ho3+ binds OH- more strongly than does Eu3+ or Dy3+. If the observed pH effect is to simply shield the trivalent charge from neighboring amino acids or phospholipids, then the effect should be seen at lower pH for the Ho3+ with a trend of increasing pH effect of Ho3+ < Dy3+ < Eu3+. However, the lanthanide ions that bind OH- more strongly in solution are observed in this experiment to require more OH- (higher pH) to regenerate the wild type bR lattice structure. A more likely possibility is that the lanthanide ion is chelated with more than one amino acid or lipid in the membrane. In general, lanthanide metal cations can have a coordination number of 6-9 in aqueous complexes.28,29 Oxygen is a favored ligand for these metal cations. If the lanthanide, with its greater coordination ability than the divalent alkaline metal cations, were to complex simultaneously with a greater number of amino acids in the protein (especially if they are on different helices) with a combination of binding to the amino acids and an O- from the PO2- of the lipid head group, it could alter the relative position of the helices. Only slight perturbations of the helices are necessary to significantly influence the packing of the bR trimer in a hexagonal lattice and thus affect the X-ray diffraction pattern. The known covalent coordination ability of the lanthanide ions that gives strong directional type bonding could lead to
L3+(R)4 + OH–
L3+(R)4 + O1
OH–
(1)
in which O1 and O2 stand for the O- of an amino acid residue on different helices or one on a helix and the other on the PO2head group, the R groups could be H2O or other amino acids, and L is either Ho, Dy, or Eu. The pH at which conversion is observed is that at which half of the multiple complex binding is converted into a monodentate type binding. This can be shown to be
pH ) log Kw/K
(2)
where Kw is the ion product of water. It is clear that the larger the equilibrium constant K for the above reaction, the lower the pH at which the conversion is expected to occur. For the above reaction, K can be obtained from the product of equilibrium constants of the following two reactions O2
O2–L3+(R)4 + OH–
OH–
L3+(R)4
KB(OH)
(3)
and O2
O2 O1
L3+(R)4
L3+(R)4 + O1
Kd(O 1 ) = 1/KB(O1 )
(4)
where KB(OH) is the binding constant for OH- and KB(O1) is the binding constant for amino acid number 1-L3+ or the PO2L3+ complex. Thus eq 2 above becomes
pH ) KwKB(O1)/KB(OH)
(5)
KB(O1) increases with the charge density of L. It is also known that KB(OH) decreases with the charge density.35 Equation 5 thus predicts an increase in the pH of the conversion from the protein binding to OH- binding as the charge density of the lanthanide ion increases, as obserVed. The above proposal is in agreement with the current available data on the binding configuration of lanthanides with amino acids in which the lanthanide can have either monodentate or bidentate binding with carboxylate groups. For example, for the crystal structure of rabbit muscle parvalbumin in which Yb3+ is substituted for Ca2+ it is proposed that the Yb3+ has three bidentate and five monodentate carboxylic ligands.36 Transferrin, an iron metal cation binding protein, has been shown37 to have an iron-binding site in which the iron is ligated by two tyrosines, one histidine, one monodentate aspartic acid, and one bidentate carbonate. Vibronic sideband spectroscopy38 of Yb3+substituted transferrin suggests that the monodentate binding of iron to the carboxylic group of aspartic acid in the native protein is replaced by a bidentate binding of the lanthanide. A similar effect was observed for the binding of Yb3+ to the
12006 J. Phys. Chem., Vol. 100, No. 29, 1996 calcium containing protein parvalbumin,39 in which the calcium has a monodentate binding with Asp92 and yet the lanthanide has a bidentate binding. Thus there is precedence for a change in ligation of the metal cation when the native divalent cations are replaced with trivalent lanthanide metal cations from monodentate binding to multiple complex binding. It has also been observed that trivalent lanthanide cations do not interact with the purple membrane in a mechanism that is completely analogous to calcium. Drachev et al.40,41 found that La3+ has an inhibitory effect on the decay of the M intermediate in the bR photocycle. As the pH is increased from 5.5 to 8.0, this inhibitory effect is increased until at pH 8.0 the M decay is about 103 times slower than in the wild type bR. Chemical modification42 of the carboxyl groups in which a positive charge is attached eliminates this inhibitory effect, suggesting that the La3+ may be bound in part to carboxyl groups. Mercier and Dupuis43 have suggested that the La3+ induces conformational changes in the protein that are different from either mono- or divalent cation perturbations. Recent results on lanthanide cation binding to the O- of the PO2- group of phospholipids provides additional support for the proposed mechanism of lanthanide binding in regenerated bR. Recently it was found44 that increasing the pH of the lanthanide-regenerated bR changes the broad band shape of the FTIR spectrum in such a manner as to indicate that the separation between these two vibrational frequencies increases. This leads to the conclusion that a bidentate type binding with the PO2- group in the lanthanide-regenerated bR is present, which changes to monodentate binding as OH- is added; that is, one of the lanthanide-O bonds in the bidentate complex is broken to give the monodentate type complex. The shift in these frequencies with increasing pH for regenerated bR with lanthanide ions strongly supports the proposal that these ions bind the PO2- groups via a multiple complex binding mechanism that is broken to monodentate type binding at higher pH. At the high pH, the separation is found to be similar to that observed for a Ca2+-regenerated (or wild type) bR sample, whose frequencies show no change with pH. The binding constant of the bidentate complex binding site of the lanthanides must be very high and might be even higher than that for the high-affinity sites found for Ca2+ in regenerated bR. This is concluded from the previous44 FTIR data that shows that the complex binding of lanthanides to PO2- was observed at low ratios of metal ion to bR. The monodentate binding might be much weaker. The question arises as to whether Ca2+ and Eu3+ have the same high affinity binding site or if different metal cations could have different binding sites of comparable affinity constants. Our previous study on the pH dependence of the FTIR spectrum of Ca2+ and lanthanides in the PO2-sMn+ binding has shown that at low pH the lanthanides form bidentate binding to PO2groups of the phospholipids which is not observed for Ca2+ binding.44 More recently, using the emission for Yb3+, it was found that the binding site of the unquenched lanthanide was involved in bidentate binding to the PO2- groups and binding to the protein residues as well.45 EXAFS studies on Mn2+ has recently shown46 that its binding seems to differ from the results of EXAFS studies27 on Fe3+. It was suggested46 that Fe3+ seems to bind to the phospholipid groups, while the Mn2+ seems to bind to the protein. For Nd3+ binding, the FTIR spectra at normal pH did not show effects of bidentate binding. This is consistent with eq 5. The 31P NMR results47 have shown that the NMR signal did not broaden until more than 2:1 Nd3+/bR was added. Combining this with the Ca2+ binding constant results of Zhang
Griffiths et al. et al.,20 it was previously concluded44 that Nd3+ binds to the PO2- groups in the weak surface type binding sites suggested by the Ca2+ potentiometric studies. From eq 5 it seems that the pH at which the conversion of bidentate complex to Nd3+ monodentate takes place is below the pH at which the deionized bR is eluted, i.e., below about pH 4. This would explain the absence of detecting bidentate binding in the FTIR spectra of Nd3+-regenerated bR at neutral pH.44 It thus appears that at least one strong binding site for lanthanide metal ions (and transition metal ions with 3+ charge) involves the bidentate complexation with the PO2- groups of the phospholipids (and some protein residues) and thus surfacelike sites. Since lanthanides are found to radiate strongly, this binding site might not involve water molecules that are known to quench the lanthanide emission. There are two observations that might suggest, however, that the second high-affinity site involves binding to both a number of water molecules and amino acid residues (Asp85 and Asp212). First is the conclusion of the presence of a high-affinity site near the retinal active site (near Asp85 and Asp212).21-24 Second is the fact that the Ca2+ and lanthanide ion binding to deionized bR regenerates the purple color and proton-pumping function, thus having this site involve water molecules within the proton release channel. The above considerations could also explain the previous results in the fluorescence of Eu3+-regenerated bR.15 Two emitting sites were observed, with very short lifetime, whose intensity and lifetime increased with retinal removal or deuteration of the aqueous medium. The Fo¨rster dipolar energy transfer mechanism of Eu3+ fluorescence quenching predicts48 a short distance (
