©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of Water in Retinal Complexation to Bacterio-opsin (*)

Itay Rousso (1), Igor Brodsky (2), Aaron Lewis (2)(§), Mordechai Sheves (1)(§)

From the (1) Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel and the (2) Department of Applied Physics, Hebrew University of Jerusalem, Jerusalem 91904, Israel

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A system is described that allows for the delineation of the factors that effect the complexation of retinal to the apoprotein of bacteriorhodopsin. This complexation is investigated in various states of hydration, in HO and DO, at a variety of pH levels, with mutant membranes and labeled retinals. The complexation reaction was also investigated using absorption spectroscopy and vibrational spectra using difference Fourier transform infrared spectroscopy. The results demonstrate the crucial role of water in controlling the protein conformations that lead to protein/ligand binding reactions and begin to shed new light on the protein control of a reaction that normally cannot take place in an aqueous medium.


INTRODUCTION

The role of water in protein structure is an important question in both the theoretical and experimental understandings of the structure-function relationships that exist in proteins (1) . In this paper we use the binding of the retinal chromophore to bacterio-opsin in different environments of humidity in order to decipher the role played by water in this protein structure.

Bacteriorhodopsin is a membrane protein that is found in a specialized membrane called the purple membrane that is in the plasma membrane of the salt loving bacterium Halobacterium salinarium(2) (see Refs. 3-6 for recent reviews). Bacteriorhodopsin, which absorbs light at 568 nm in its light-adapted form (bR),() is the only protein found in this membrane, and it is organized in a hexagonal crystalline structure in the membrane. Its structure has been determined to approximately 3.5-Å resolution in a pioneering series of investigations, using electron diffraction and image reconstruction techniques (7) . The protein exhibits numerous light-induced structural transitions. Many of these transitions are associated with absorption changes in this chromoprotein that is composed of the retinal chromophore complexed covalently via a protonated Schiff base (8) to the amino group of lysine 216 in the protein. During these transitions the Schiff base is deprotonated yielding intermediate M (8) .

The binding of the chromophore to this amino group is an important question both in terms of the protein structural changes that are occurring during the complexation and in terms of understanding the chemical principles involved in the formation of the Schiff base between a carbonyl and an amino group under physiological pH despite the relatively high pK of uncomplexed amino groups. One of the first studies that defined a new approach to studying this problem was by Oesterhelt and colleagues (9, 10). In this approach a chromophore in the all-trans configuration was added to a suspension of bacterio-opsin at 0 °C and under such conditions it was possible to trap a complex that absorbed at 430/460 nm that eventually converted to bacteriorhodopsin. It was suggested (11) that the initial complex was composed of retinal rather than a Schiff base or protonated Schiff base. More recently, a similar complex was obtained when a mutant protein was analyzed in which lysine 216 was replaced by alanine (12) . This result further indicated that the blue absorbing complex was due to a retinal form of the chromophore. It was also shown that 9-cis- and 11-cis-retinal complexes with bacterio-opsin do not form bacteriorhodopsin until irradiation. These latter experiments form the basis for the studies reported in this paper.

The first studies to indicate the critical role of water in the function of the purple membrane was by Korenstein and Hess (13) . These workers demonstrated that the decay of the intermediate M that is produced when bR absorbs light is drastically affected by the amount of water present in the membrane. Varo and Keszthelyi (14) have demonstrated that the photoelectric signals of bR are altered as a function of amount of water. Ehrenberg et al.(15) and Doukas et al.(16) demonstrated the rapid (millisecond) exchange rate of the Schiff base proton in spite of the high pK of this group. The presence of water in the binding site and its role in stabilizing the protonated Schiff base was suggested by DuPuis et al.(17) . Later Hildebrand and Stockburger (18) noted on the basis of resonance Raman studies of dried membrane the importance of water in stabilizing the protonated Schiff base in bacteriorhodopsin.

Groot et al.(19) , on the basis of N nuclear magnetic resonance studies, suggested the presence of water in the binding site, and the presence of strongly bound water in the interior of the protein was suggested from neutron diffraction (20) . Recently, it was suggested that the water in the binding site plays a major role in controlling the pK of the protonated Schiff base and its counter ion. A specific orientation between the donor and the acceptor groups that allows water molecules to form a defined structure and to bridge between these groups was suggested. This particular structure could stabilize the ion pair and modify the pK values (21) . This role of water in modifying the pK was recently supported by theoretical calculations (22) . Furthermore, molecular dynamics simulations of the bR ground state indicates the feasibility of the presence of water in the binding site and the bridging role of this water between the Schiff base and aspartic 85 and 212 that are in relatively close proximity to this covalent linkage (23) . Interaction of Asp-85 with a water molecule and the protonated Schiff base in the L intermediate of the photocycle was monitored by Fourier transform infrared spectroscopy (FTIR) studies (24).

In this paper we have combined the use of isomers of retinal that complex but do not form directly bR at room temperature and the use of dried films. We have studied these films in environments of controlled humidity in order to test the controlling role of water in the structural changes that occur when all-trans-retinal is formed with light and the protonated Schiff base is finally generated. A variety of investigations have been completed on these films including detailed absorption studies and Fourier transform infrared investigations in order to characterize the molecular changes that are occurring during the complexation of retinal with apoprotein. Our data indicate that the association of water is a crucial component in the sequence of reactions that occur in these dried films with light and is critical to the formation of the covalent bond between the retinal and the protein in order to generate the protonated Schiff base complex.


MATERIALS AND METHODS

Sample Preparation

Wild type, D96N, and D115N apomembranes were prepared according to Oesterhelt and Stoeckenius (2) . The apomembrane suspension was incubated with 9-cis-retinal at room temperature for 1 h. All manipulations were performed in dim red light. Labeled 9-cis-retinal at 14-C and 15-C with C was prepared, based on a previously described method (25).

Absorption Spectroscopy

Films were prepared by drying water suspension of apomembrane combined with 9-cis-retinal on glass slides. The drying was performed at room temperature using mild vacuum (15 mm Hg). A variable degree of hydration of the films was obtained by equilibrating the samples with different relative air humidities produced by saturated salt solutions. The glass slide with the desired salt solution was inserted into a home-made sealed cuvette. The 0% relative humidity was defined as the limiting dryness obtained by 2 mm Hg vacuum. The light source for the irradiations was a KL1500 electronic halogen lamp. All absorption measurements were carried out at room temperature in the dark and were recorded on an Hewlett Packard 8452A diode array spectrophotometer.

FTIR Difference Spectroscopy

The apomembrane combined with 9-cis-retinal films were prepared for FTIR spectroscopy by isopotential spin drying on an AgCl windows (26) . Spectra were recorded on a Brucker IFS113V Fourier transform infrared spectrometer. The 570 nm species was produced by irradiation of the apomembrane/9-cis-retinal film at 298 K with a 380 nm cutoff filter, for 5 min under constant 80% humidity. The light source for the irradiation was a 200-watt mercury lamp. The high humidity was produced using a home-made FTIR cell. The cell was made of two AgCl windows with a specially designed 5-mm spacer containing two pathways. The cell was connected to a water source and to a weak vacuum pump (30 mm Hg), which produced a constant flow of water vapor through the cell. Spectra were measured before irradiation and 15 min following it.

Extraction of Chromophores

The chromophores were extracted from the film using a 1:1 ethanol-hexane mixture according to Scherrer et al.(27) . The hexane layer was separated and analyzed by high performance liquid chromatography using a silica 60 column and eluted with 5% ethyl acetate in hexane.

Kinetic Analysis

In order to find the best model, which describes the kinetics of the process initiated by irradiation, we constructed a first order series reaction.

The most general case of a system of n components A, A, . . . A in which first-order reactions of the following type may occur between any two components is shown in Equation 1.

On-line formulae not verified for accuracy

The rate equation for any component is given by Equation 2,

On-line formulae not verified for accuracy

where K= -k except when i = j, which gives Equation 3.

On-line formulae not verified for accuracy

A particular solution for the rate equation is of the form shown in Equation 4.

On-line formulae not verified for accuracy

Substituting this solution in the differential equation produce a set of simultaneous homogeneous linear equations, shown by Equation 5.

On-line formulae not verified for accuracy

The general solution of the differential equation is a linear combination of the particular solutions, as shown in Equation 6,

On-line formulae not verified for accuracy

where are the eigen values of the linear equations, B is the rth component of the ith eigen vector, and are coefficients that are determined from the initial conditions.


RESULTS

In this section we describe the variety of photochemical and thermal reactions that the apo-bR/9-cis-retinal complex (AbR) system undergoes. The first sequence of reactions that will be described will be associated with absorption of a photon by AbR and the subsequent changes that ensue in this complex.

The AbR when investigated as a film with an absorption of 390 nm does not form the bacteriorhodopsin pigment (570 nm) following light absorption, but is transformed with light into a species that absorbs light at 470 nm. The time for this transition at room temperature has approximately a half-life of 5 min. Following irradiation it is possible to extract the retinal chromophore and to analyze the isomeric configuration using high performance liquid chromatography. This analysis clearly indicates that the transformation is initiated by the isomerization of the retinal from 9-cis to a mixture of all-trans, 13-cis, 9-cis, and probably 11-cis isomers in a ratio of 41:26:18:15.

In Fig. 1the appearance of the 470 nm absorption is shown as a function of time after the irradiation has instantly generated the retinal isomer mixture, which also absorbs at 390 nm. The functional dependence in this figure was analyzed using a kinetic scheme that was based on a model indicating at least a biphasic behavior to the transformation. The mathematical fit obtained for serial kinetic schemes strongly supported such schemes over the alternative version with parallel kinetics.


Figure 1: The 390` to 470 transformation following irradiation with 380 nm cutoff filter. Difference absorption spectra measured every 120 s (- - -) and every 10 min (-). Spectrum8 was taken 20 min after irradiation. Inset, absorption spectra immediately after isomerization (a) and after 3 h (b).



If this mixture is exposed to 100% water vapor, a two-step process occurs (Fig. 2). First, the 470 nm absorption is converted to a species with an maximum at 570 nm on a time scale of 4 min. Second, the 390 nm species is also converted to a 570 nm absorption but with a slower time constant (30 min). When the conversion of the 390 nm species to 470 nm was carried out with a film that was prepared from DO, the kinetic time constants decreased by a factor of 2 (see Fig. 3 ).


Figure 2: Difference absorption spectra of addition of water to the 390/470 mixture. Spectra 1-5 were taken every 2 min, spectrum6 after 16 min of water addition and then every 8 min (spectrum11 was taken after 56 min).




Figure 3: Comparison of the HO ( ) and DO () kinetics in the 390 to 470 transformation.



Difference spectra of the transformation of 390 to 470 nm following light absorption has a very crucial dependence on the level of the humidity. Increasing the humidity causes the formation of an additional species at 570 nm. The ratio of the production of 470 as compared to 570 nm depends on the humidity. This is seen in Fig. 4 , which displays difference spectra as a function of controlled humidity. From these spectra the fraction of molecules in the 570 nm state can be plotted as a function of humidity (Fig. 4). In Fig. 5is seen the effect of 100% humidity on this transformation using a mutant in which aspartic acid 96 is replaced by asparagine (D96N). These spectra show that the ratio of 470 to 570 nm in this mutant favors the 470 nm species relative to the wild type. The D115N mutant was studied as well and showed similar behavior to the wild type (data not shown). In addition, the humidity-dependent transition is also dependent on pH. This is seen in Fig. 6for the wild type species in which the pH of a film with 15% humidity was modified. The amount of 570 nm species increased as the pH was elevated. The transition is characterized by a pK of 8.9.


Figure 4: Absorption difference spectra of the 390 to 470 transformation as a function of humidity for wild type. a-f, 100, 75, 66, 57, 20, 0% of relative humidity. Inset, plot of the percentage of 570 nm species formed as a function of humidity.




Figure 5: Difference absorption spectra of the 390 to 470 and 570 nm species for the D96N mutant at 100% humidity. Spectra were taken every 2 min (- - -), and every 10 min (- -). Spectrum8 was taken 20 min after irradiation.




Figure 6: Formation of the 570 nm species as a function of pH for a dry film of the wild type. The percentage was calculated as a fraction from the 470 and 570 mixture assuming equal extinction coefficient for both species.



An interesting observation is that associated with the 470 nm species illumination. When the 470 nm species is irradiated with 470 nm cutoff filter (under steady state conditions) a few minutes after the initial 390 nm irradiation, approximately 70% of the 470 nm absorbing species is converted to a species with a 390 nm absorption maximum (Fig. 7). Nonetheless, if the irradiation is carried out 4 h after the initial 390 nm illumination, only approximately 45% is converted to the 390 nm species. Once contrived by this procedure, the newly formed 390 nm absorption species can thermally generate a species that has an absorption at 470 nm. The kinetics of the latter process is similar to the direct initial irradiation of the 390 nm species (Fig. 7). This 470 nm species can generate the 570 nm absorption following addition of water.


Figure 7: Transformation of 470 to 390 with irradiation as a function of time. Absorption was monitored at 470 nm. Inset, difference absorption maximum of the 470 to 390 nm transformation.



In addition to the above, a lysine mutant was investigated in which the active site lysine 216 is replaced by a glycine (28) . Films of this species indicated that the 390 to 470 nm conversion occurred in a similar fashion (see Fig. 8) to that observed in wild type films. It should be noted that when all the lysines, except the active site lysine, were acetylated, an identical 470 nm species was generated by light absorption of 9-cis-retinal complexed to acetylated apo-bR (AbR).


Figure 8: Difference absorption spectra of the 390 to 470 transition for K216G mutant. Spectra shown (1-7) were taken at 10 min intervals, while 1 represents a different spectrum between immediately after and 1 h after irradiation states.



The transition from 390 to 570 nm at high humidity has been investigated using FTIR. The spectra are shown in Fig. 9. This figure shows a difference FTIR spectrum of this transition of the wild type protein in HO and DO, and a difference spectrum of the same transition with the retinal labeled with C in the 14-C and 15-C carbons. Furthermore, in Fig. 10the spectra of two mutants are displayed of D96N and D115N in which the aspartic acid is replaced by asparagine.


Figure 9: Difference FTIR spectra of 390 to 570 nm transition. A, HO; B, DO; C, 14,15-C retinal in HO.




Figure 10: Difference FTIR spectra of 390 to 570 nm transition of two mutants D96N and D115N.




DISCUSSION

The analysis of the data presented in Fig. 1indicates that, after light irradiation of the initial 390 nm species, two 470 nm forms are present in equilibrium with an isomerized form of 390 nm that absorbs at the same wavelength as the original species, and we label this species for convenience 390`. This is based on the biphasic behavior of the kinetic transition that is plotted in Fig. 3, which used the 470 nm absorption as a probe for the kinetics. The best fit with the kinetic results is obtained using Fig. SI.


Figure SI: Scheme I.



This scheme gave a good fit for the rate constants indicated in . The suggestion that the 470 nm intermediate consists of two species is also supported by the ratio of /, which changes during the course of the reaction. In a short time (30 min) the ratio was smaller than that observed after 4 h. The calculations based on this kinetic model indicate that the percentage of the 390` species in the mixture is approximately 70, and the species with a 470 nm absorption is 30%.

The results shown in Fig. 8with the pigment obtained with the lysine 216 mutant indicate that a species with a similar absorption at 470 nm is obtained when 9-cis-retinal is added to the purified membrane of this mutant. Upon addition of 9-cis-retinal, the mutant has an absorption at 390 nm. When this complex is dried and irradiated, the resulting species has an absorption at 470 nm despite the fact that Schiff base formation is impossible. This indicates that in all probability the 470 nm species consists of a chromophore characterized by an aldehyde, rather than a protonated or non-protonated Schiff base. As noted under ``Results,'' the possibility that Schiff base formation may occur at some site other than the active site has been ruled out by a similar absorption obtained for a protein acetylated at all lysine sites other than the active site.

This deduction is most interesting in view of the fact that retinals normally absorb in solution at around 380 nm. This means that the red-shift induced upon complexation is indeed most significant (5000 cm). One suggestion for this observation is that a strong hydrogen bonding may exist with the aldehyde moiety (probably in addition to ring-chain planarization enforced by the protein; Refs. 9 and 10). This possibility was also proposed by Schweiger et al.(12) for the 430/460 nm complex formed by binding of all trans retinal to bacterio-opsin in solution to form bacteriorhodopsin. It has been shown (29) that a red-shift in a retinal Schiff base can be induced by hydrogen bonding with a carboxyl group or bound water. Similar red-shifts could conceivably be induced in a retinal chromophore. One effect of such hydrogen bonding could be to increase the positive charge at the carbonyl, which would enhance nucleophilic attack of the amino group that is needed for the retinal-lysine complexation.

An interesting observation seen in the results presented in Fig. 3and is the observation of a deuterium effect in the kinetics on the formation of the 470 nm species. Relative to the kinetics in HO there was an increase in the time of formation of 470 in DO by a factor of 2. This might indicate the involvement of a proton movement in this transformation, and this could be related to the suggestion above of the possible effect of strong hydrogen bonding and possibly even partial protonation of the aldehyde end group that could result in a red-shifted aldehyde absorption. Nonetheless, we cannot rule out, at the present time, other factors that may result in this red-shift including the effect of protein groups surrounding the retinal. Interestingly, as described above, the retinal complexed with the dried membrane of mutant K216G also has an absorption at 470 nm. This indicates that the amino group of the active site lysine does not appear to be affecting the absorption of the retinal chromophore.

It is interesting to note that absorption of light by the 470 nm species in the dried film produces an absorption at 390 nm, i.e. losing the observed red-shift on the retinal. It is presumed that this effect of the absorption of light is to produce isomeric forms of retinal. Such isomerization is accompanied by the loss of the specific chromophore interactions with its environment.

In these dried films a species absorbing close to 570 nm cannot be formed. However, if the films with an initial 390 nm absorption are irradiated in high humidity, a 570 nm species is generated. This absorption maximum is consistent with the formation of a protonated Schiff base, and the result demonstrates that water is a crucial factor in order to accomplish a reaction between the retinal chromophore and the amino group of lysine 216. The amino group would have to be deprotonated in order for the nucleophilic attack of the nitrogen on the aldehyde carbon to proceed. Nonetheless a clarification of the protonation state of the amino group will have to be obtained before further deductions can be made on the mechanism. By comparison, in rhodopsin it has been determined (30) that the pK of the active site lysine is close to 10 resembling the normal pK for such a group. If in the 470 nm species the lysine has a similar pK, then the amino group should be protonated rather than unprotonated; thus, the complexation would be difficult to explain at pH 7 based on generally accepted principles.

The effect of water on the formation of 570 nm species exhibits a cooperative behavior, and this is seen in Fig. 4. From the data in Fig. 4, a Hill coefficient can be derived, and this coefficient has been deduced to be 3 ± 0.2. Thus, the data seem to indicate that three binding sites have to be filled with water molecules in order for the protein structure to be in a state that allows the formation of the 570 nm species. As shown in Fig. 5 and Fig. 6, this water-dependent formation of the purple species is affected by the pH of the medium and also when mutant D96N is analyzed (see Fig. 6).

A possible scheme that incorporates all of our data is shown by Fig. SII.


Figure SII: Scheme II Solid arrows represent where the data strongly suggest the equilibria shown, while dashed arrows represent hypothetical equilibria between species.



The data indicate that the 470d (dry) species is formed by subsequent conformational transitions that result from the photochemistry and then the hydration induced structural changes lead to the 470w (wet) species that transforms to 570. Future studies will be aimed at understanding the relative importance of the various pathways shown above.

In Fig. 6 we note that increasing the pH of the film increases the percentage of the 570 species that is produced at low humidity. In essence a low humidity film (15% humidity) that should in fact produce very little 570 nm species can be made to generate up to 35% of the species absorbing at 570 nm. The effect of high pH could be related to the deprotonation of a group in the protein, which alters the protein conformation and enhances the Schiff base formation. One possible way in which such a high pK protein group could effect the formation of 570 would be by enhancing, through a protein structural change, a critical water pool required for complexation of the retinal and the lysine.

Let us now consider the role of water in Schiff base formation. It is logical to assume that around the Schiff base there are water molecules that are difficult to remove even in the dehydrated environments being considered in this paper. Nonetheless, our experimental results indicate that the Schiff base formation, a reaction that usually cannot take place in bulk water, is enhanced by the addition of water to the dehydrated protein. Therefore, it is a logical suggestion that the reaction enhancing water binds to a site removed from the Schiff base. This would suggest that this added water's effect on the Schiff base formation indicates a conformational change in the protein. In view of the inability of Schiff base formation to occur in bulk water environments, the water in the active site must be in very definite structural states in order to avoid hydrolyzing the Schiff base after its formation. Our present results do not clearly define at which stage in the steps toward complexation the network of structured water is achieved in the active site. However, such defined structures of water must be generated at least immediately preceding the reaction. Therefore, the protein structural transformation might play a role in altering the nature of water interactions around the Schiff base. In addition to the water structure being appropriate, other geometrical factors should play an important role in the Schiff base formation. Specifically, the geometry of the retinal carbonyl and the amino group of the lysine must be in the correct orientation and in close contact in order for a reaction to take place. In order to reach the appropriate complex of interactions essential for Schiff base formation, it is certainly important to consider how the pK of the lysine amino group could be tuned to allow the reaction to proceed. One way to tune this group for such a reaction would be to partially deprotonate the amino group by the presence of another appropriately positioned group in its vicinity. In this respect, we note that the reaction proceeds very slowly in the mutant D85N or at very low pH.() Based on this observation, we can suggest that aspartic 85 plays a major role in the reaction, which in turn proceeds via the partial protonation of Asp-85 by the amino group of the lysine. The presence of structured water could be integrated with this need to tune the amino group pK by an alteration in the geometry of water molecules that are thought to bridge the region between the amino group and aspartic 85 (21) . In summary, the protein conformational change induced by water could have multiple effects of altering the amino group/retinal geometry, changing the nature of the structured water and tuning the pK of the groups that must interact in order to form a Schiff base.

In order to obtain supporting evidence for some of the above conclusions, we have obtained difference Fourier transform infrared spectra of the transition between the 390 nm species in the wet state and its transformation into the state absorbing at 570 nm. The positive peaks in the difference spectrum in HO have a number of similarities to bacteriorhodopsin peaks that are seen in difference spectra between bR and its photochemically induced intermediates. For example, notice the characteristic bR bands in the fingerprint region between 1150 and 1200 cm and the C=C stretching vibration at 1530 cm.

Interesting behavior of the C=C stretching vibration was observed when the retinal was replaced by a retinal chromophore in which the 14-C and 15-C carbons were labeled with C. In this spectrum a split is seen in the C=C stretching vibration and two vibrations at 1530 cm and 1518 cm appear. Resonance Raman measurements (31) of bacteriorhodopsin with labeled retinal with C at 14-C and 15-C, as well as bR/M FTIR difference spectrum (data not shown) indicate only one C=C stretching frequency at 1518 cm and does not show the 1530 cm band. This observation might indicate that the 1530 cm vibration in the AbR/570 nm difference spectrum consists of another vibration besides the C=C stretching mode. The fingerprint region of the 570 nm species exhibits alteration due to 14-C and 15-C labeling similar to that observed in FTIR spectrum of bR. This suggests that the retinal conformation in the 570 nm species obtained by our experiments and bR are indeed similar. Nonetheless, because of the difference noted above in the C=C stretching mode, we cannot definitely conclude that our 570 nm species and bR are identical.

In the region of carboxyl group vibrations, spectra have been obtained with native pigment and labeled retinal. In both spectra two peaks are observed a negative peak at 1743 cm and a positive peak at 1737 cm (Fig. 9). Both of these peaks disappear in the mutant in which aspartic 115 is replaced by asparagine (Fig. 10). This indicates that aspartic 115 undergoes a transition to a decreased hydrophobic environment in going from the wet 390 nm species to the 570 nm state. Despite this, there is a residual contribution in this region even in the 115 mutant, which would indicate that other carboxyl groups are also undergoing environmental changes in this transition. Similar difference spectra of the mutant with aspartic 96 replaced by asparagine show no significant change in this region of the spectrum, and this supports the role of an environmental change in aspartic 115 as discussed above. We note that environmental changes in aspartic 115 have also been reported by Rüdiger et al.(32) in a study of what appears to be an AbR/bR transformation. An additional point of interest is associated with the shifts experienced by this carboxyl transformation on resuspension of the native membrane in DO. As noted above, this carboxylic acid group is surrounded by a different environment in the 390 and 570 nm states. The spectra seen in Fig. 9indicate that the proton-deuterium exchange of this group in these two states is different. Notice that upon DO suspension, the positive band at 1737 cm down-shifts by 11 cm, as would be expected for such a group undergoing exchange. Nonetheless, all the data indicate that the band at 1743 cm is not down-shifted by this exchange. There is evidence in the spectra of an increase in intensity at 1748 cm, but this can be interpreted as an additional peak to the one at 1743 cm. Therefore, we conclude, that as a result of the 390 to 570 nm transformation, aspartic 115 alters its environment from a region in which H-D exchange is not possible to a region where such exchange occurs.

One interesting region that shows significant sensitivity to DO is the region around 1456 cm. This region could possibly have some contribution that is associated with the lysine vibrational modes and these modes could gain intensity by coupling to the dipolar C = N - H vibrations. This could explain the sensitivity of this region to DO.

The FTIR spectra indicate a major change in the negative peak at 1653 cm, which is in the region of the amide I vibrations, and this suggests an alteration in the backbone conformation in going from the 390 nm apo species to the 570 nm state. The lack of movement of this band when the retinal is labeled with C at 14-C and 15-C (Fig. 9, A and B) rules out the possible assignment of this band to the stretching vibration of the retinal carbonyl. Bands are also seen in the amide II region, which generally occurs between 1550 cm and 1600 cm. Of the two bands that are seen in this frequency regime, the one that appears at 1572 cm can be assigned to the C=C stretching vibration of the 390 nm state. We can make this assignment based on the effect of the isotopic substitutions in the retinal on the frequency of this vibrational mode. This then suggests that the remaining band at 1557 cm, which is unaffected by the retinal isotopic substitution, is associated with amide II frequencies, which reflect changes in the backbone conformation. In DO, however, the frequency of the band that is due to the C=C stretching mode of the retinal is not altered, while the band that appears to be associated with the amide II vibrations is split and shifts in frequency. This is consistent with the accepted analysis of the amide II vibrations containing significant N-H character. On the other hand, the amide I vibration that seems to appear at 1652 cm, which contains little N-H character, is not affected by DO. An additional interesting observation that can be deduced from this sensitivity of the amide II to DO is that the regions of the protein backbone undergoing changes in the 390 to 570 transition are exposed to water in the 390 nm species.


CONCLUSION

The results of this paper have highlighted the crucial role that water plays in the conformational states of bR that control the covalent binding of the retinal ligand. As can be seen in our results, dry apomembranes can bind retinal, but they do not have the ability to couple the retinal via a Schiff base to the amino group of the lysine. This affect of water is modified by protein groups such as aspartic acid 96 and pH. The binding of water appears to be a well defined process with a switching of the protein structure into a state that allows progress of the reaction to a 570 nm species after the binding of a criticial number of water molecules. In essence the system we have investigated here offers the possibility to study in a quantitative fashion the interesting reactions control the binding of retinal to lysine. It has always been recognized that in addition to the primary sequence of amino acids that water plays a crucial role in protein structure. Our results highlight the significance of this assumption.

  
Table: Calculated rate constants for formation of the 470 nm species



FOOTNOTES

*
This work was supported by the United States-Israel Binational Science Foundation, by the Human Frontier for the Promotion of Science and by the Israeli Fund for Basic Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dedicated to Prof. Koji Nakamishi on his 70th birthday.

§
To whom correspondence should be addressed.

The abbreviations used are: bR, bacteriorhodopsin; AbR, apo-bR/9-cis-retinal complex; FTIR, Fourier transform infrared.

I. Rousso, I. Brodsky, A. Lewis, and M. Sheves, unpublished results.


ACKNOWLEDGEMENTS

We thank Profs. R. Needleman and J. Lanyi for their generous gift of bR mutants.


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