J. Phys. Chem. 1995, 99, 7776-7781
7776
Fourier Transform Infrared Spectroscopic Studies of the Effect of Ca2+Binding on the States of Aspartic Acid Side Chains in Bacteriorhodopsin Satoshi Masuda, Masayuki Nara, and Mitsuo Tasumi* Department of Chemistry, School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
Mostafa A. El-Sayed* School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400
Janos K. Lanyi Department of Physiology, University of California, Imine, California 9271 7 Received: August 22, 1994; In Final Form: November 30, 1994@
Effects of Ca2+ binding on the states of aspartic acid residues near the retinal pocket and the secondary structure of bacteriorhodopsin (bR) have been studied by infrared spectroscopy in combination with the Fourier self-deconvolution technique. The band at 1754 cm-l of deionized bR is assigned to the C=O stretch of the COOH group of protonated Asp-85, and the band at 1405 cm-l of metal-ion-bound bR to the symmetric stretch of the COO- group of deprotonated Asp-85, on the basis of the infrared spectra of a bR mutant in which Asp-85 is replaced with Asn. The binding of Ca2+to the second high affinity site causes deprotonation of the Asp-85 COOH group and the blue to purple color change. The Ca2+ binding causes changes in the secondary structure of bR.
Introduction Bacteriorhodopsin (bR) is the chromophoric protein in the light-energy transducing purple membrane of Halobacterium salinarium (H. salinarium).1,2 It contains all-trans-retinal as the chromophore, which is covalently bound to the €-amino group of Lys-216 via a protonated Schiff-base linkage. Upon absorption of visible light, the chromophore is isomerized to the 13-cis form, and bR experiences a photochemical cycle with the following several intermediates distinguished mainly by visible ab~orption:~ BR K KL L M N 0 BR. In this process, bR transports one proton per one photon from the cytoplasm side of the purple membrane to the exterior side. The resulting proton gradient is then used by the bacteria for the synthesis of ATP.4 Well-washed purple membranes contain 3-4 mol of Mg2+ and 1 mol of CaZ+per 1 mol of bR.5 Removal of these cations or acidification causes a color transition from purple (A, = 568 nm) to blue (Am, = 605 nm).5-9 In the blue protein, the L to M transformation is inhibited and the proton pumping does not O C C U ~ . ~ Thus, .~ the metal cations are important for the proton-pumping activity and color regulation of bR. There are two possibilities for the binding of the metal cations to bR; one is the binding to some specific sites inside bR (e.g., the COO- groups of amino acid side and the other is nonspecific binding to the membrane surface.15 Recently, Zhang et al.l0 have obtained the binding constants of Ca2+ by measuring the concentration of free Ca2+ in the suspension of purple membranes regenerated with CaC12. They have shown that bR has two high affinity sites for Ca2+ (K1 = 3.5 x lo5 M-l, K2 = 6.3 x 104 M-l) and four to six low affinity sites (K3 = 3.5 x lo3 M-l). On Ca2+ binding to the lower of the two high affinity sites, bR experiences the color transition from blue to purple. It has been proposed" that the high affinity sites are not far from the retinal pocket, although ligands for
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@Abstract published in Advance ACS Abstracts, May 1, 1995.
Ca2+have not been specified yet. Most of the low affinity sites are on the cytoplasm surface near the C-terminus.12 The position of A, of deionized or acidified bR (-605 nm) is similar to that of a bR mutant in which Asp-85 is replaced with Asn (DUN). On this basis, it has been p r o p o ~ e d ~ 2that ' ~ J ~the Asp-85 side chain is protonated in deionized or acidified bR. A I3C NMR study18 has supported this view. Infrared spectroscopy is a powerful tool for detecting small structural changes in proteins. Infrared difference spectra between BR (the ground state of bR) and its photointermediates have been measured by trapping photointermediates at low temperatures under illumination of visible light, and structural changes occurring in the chromophore and apoprotein in photoreaction steps have been studied.19-21 The infrared bands arising from retinal have been assigned by using isotope substitution.22 The bands above 3400 cm-' have been assigned to the OH stretches of the COOH groups and water molecules embedded in the membrane and to the NH stretch of a tryptophan r e s i d ~ e . ~On ~ -the ~ ~basis of these results, interactions between Asp residues and water molecule have been d i s c ~ s s e d . *Assignments ~~~~ of the C=O stretching bands of the COOH groups observed in the 1770-1720-cm-' region have been discussed most extensively. There are four Asp residues in the membrane-embedded region of bR. The infrared spectra of the bR mutants in which different Asp residues are individually replaced by Asn residues have been measured, and the bands of BR and photointermediates in the 1770-1720-cm-l region have been assigned to the respective Asp residue^.^^.^^ The Schiff-base proton is considered to be released and transferred to Asp-85 upon the L to M transformation. Deionized bR has also been studied by infrared difference s p e c t r o s c ~ p y . Structural ~ ~ ~ ~ ~ changes accompanying the blue to purple transition initiated by exposing a film containing deionized bR to NH3 have been studied, and it has been proposed that water-exposed acidic side chains in deionized blue bR are p r o t ~ n a t e d .The ~ ~ difference spectrum of deionized bR
0022-365419512099-7776$09.00/0 0 1995 American Chemical Society
Infrared Studies of Ca2+ Binding to Bacteriorhodopsin between BR and L and that between dark-adapted BR and lightadapted BR have been measured.30 Infrared spectroscopy is also useful in studying the protein secondary ~ t r u c t u r e . ~ ' -The ~ ~ position of the amide-I band (amide-I being a mode mainly consisting of the peptide C=O stretch) is known to be sensitive to the secondary structure. To obtain information on the secondary structure, Fourier selfdeconvolution and curve-fitting techniques32have been applied to bR,36-38and it has been shown that bR has a high a-helix content (52-73%), which agrees with the results obtained by using electron cryomicroscopy. Polarized infrared spectra have also been measured for the oriented purple membranes, and the direction of the transmembrane a-helices and the accessibility of these helices to external water have been disc~ssed.~' Except for studies on the amide-I band, infrared studies of retinal proteins have been mostly performed by obtaining difference spectra. Difference spectroscopy is an excellent method for detecting small spectral changes arising from perturbations in local structures of a protein molecule. However, if the bands relating to structural changes are weak, broad, or overlapping with one another, application of resolution-enhancement techniques such as Fourier self-deconvolution (FSD)39is more effective in extracting useful information. The purpose of this paper is 2-fold. One is to demonstrate that usefulness of infrared spectroscopy as a method for studying interactions between metal ions and proteins. The other is to obtain new information on the interaction between Ca2+ and deionized bR by analyzing the infrared spectra of metal-ionbound, deionized and Ca2+-regenerated bR with varying Ca2+l deionized bR ratios (Ca2+ titrations).
Experimental Section 1. Materials. Halobacterium salinarium was grown from master slants of ET1001 strain provided by Professor R. Bogomolni (University of California, Santa Cruz). To obtain D U N , a clone containing the replacement of Asp-85 with Asn was constructed from the H. salinarium L-33 strain by transformation with a shuttle vector containing the modified bop gene, as described.40 bR was isolated by a combination of meth0ds.4~9~~ The pH of the sample containing wild or mutant bR in the metal-ion-bound state was 6.8 Deionized bR was prepared by passing a sample containing metal-ion-bound bR through a column of Bio-Rad AG 50W-X4 cation exchanger in the hydrogen form.g The pH of the sample containing deionized bR was 4.0. bR regenerated with Ca2+ was prepared by adding 1 mM CaC12 solution to a solution containing deionized bR (denoted as bR*) with various molar Ca2+/bR* ratios, and the regeneration was checked by visible absorption. No buffer was used during all sample preparation (including the preparation of metal-ion-bound bR) in order to avoid contamination of other metal cations. Samples for infrared measurements were prepared by centrifugation of membrane suspension at 16 000 rpm. 2. Measurements. Infrared spectra were obtained on a JEOL JIR- 100 Fourier transform infrared spectrophotometer equipped with an MCT detector (Judson) at 2-cm-' resolution. The sample was placed between two CaF2 plates with a 15-pm Teflon spacer. Dissolution of Ca2+ from the CaF2 plates into the sample on the time scale of infrared measurements was shown to be insignificant in a careful study by Trewhella et al.43 In fact, there was no indication of such possibility in the observed spectra to be discussed later. During spectral measurements, the sample was kept at 15 O C by use of a cooling unit (Yamato BL-61). Metal-ion-bound bR has two forms in the ground state; one is the light-adapted form (BR56g) having
J. Phys. Chem., Vol. 99, No. 19, 1995 7777
WAVENUMBER I cm-' Figure 1. Infrared spectra (1800-1350 cm-') of (a) metal-ion-bound bR and (b) deionized bR.
all-trans-retinal, and the other is the dark-adapted form (BR548) having 13-cis-retinal. Without illumination of visible light, metal-ion-bound bR is a mixture of BR5.58 and BR548 in a ratio of 60:40,44 and BR548 is immediately converted into BR568 by light absorption. However, deionized bR in the ground state is a 1:l mixture of these two forms even under illumination of visible light.45 All infrared spectra were measured for darkadapted samples without illumination of visible light. In order to obtain the spectra of purple membranes without interference from water absorption, the spectrum of deioinized and distilled water was carefully subtracted from the observed spectra.& Because the observed bands were very broad and consisted of many partially overlapping bands, FSD39was used to enhance the spectral resolution. In practice, FSD was performed according to the method of Jones and S h i m ~ k o s h by i ~ ~using the parameters 20 = 19.5 cm-' and L = 0.195 cm, and the apodization function used was of the Bessel type.
Results and Discussion The infrared spectra of metal-ion-bound and deionized bR are shown in Figure 1. The two spectra are almost identical, except for a small shift in the peak positions of the amide-I bands, which are located at 1659 cm-' for metal-ion-bound bR and at 1661 cm-' for deionized bR. After applying FSD to the observed spectra, differences between metal-ion-bound and deionized bR are clearly seen, in the regions of 1770-1720 cm-' (C=O stretch of the COOH group), 1700-1600 cm-' (amide-I), and 1410- 1400 cm-' (COO- symmetric stretch). These differences (or bands) are very weak in intensity (note the varying absorbance scales in Figures 1-7), but such weakness is natural because they are caused by a local change in the large protein molecule; i.e., each of the bands in the 1770-1720-cm-' region arises from a single COOH group and the band in the 1410-1400-~m-~region from a single COOgroup in the protein consisting of 248 amino acid residues (vide post). In most papers dealing with the infrared spectra of retinal protein^,'^-*^ the difference-spectrum method is used to detect small spectral differences between photointermediates, and the observed spectral differences are enlarged lo3 to 10" times on the absorbance scale. In the original spectra, bands due to the retinal moiety are not readily recognizable on an ordinary
Masuda et al.
7778 J. Phys. Chem., Vol. 99, No. 19, 1995
W
0
z
2U %m a
1800
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WAVENUMBER / cm-'
1 30
1420
1410
1400
1390
WAVENUMBER cm-'
CaCl*/bR*,and (e) bR* (deionized bR).
Figure 3. Deconvoluted infrared spectra (1430-1390 cm-') of (a) metal-ion-bound bR, (b) 4:1,CaClz/bR*, (c) 2:l CaC12/bR*, (d) 1:l CaC12/bR*, and ( e ) bR* (deionized bR).
absorbance scale. This means that the band intensities relating to the single COOWCOO- group are of the same order of magnitude as those relating to the retinal moiety. Implications of these differences between the metal-ion-bound and deionized forms of bR will be discussed below. 1. States of Aspartic Side Chains. The deconvoluted spectra of metal-ion-bound and deionized bR in the regions of 1800-1700 and 1430-1390 cm-' are shown, respectively, in Figures 2 and 3, together with those for samples of bR regenerated with Ca2+, in which Ca2+ and deionized bR (bR*) are mixed with molar ratios of 4:1, 2:1, and 1:l Ca2+/bR*. In the deconvoluted spectrum of metal-ion-bound bR in Figure 2a, the band at 1741 cm-' and the shoulder at 1735 cm-' are assigned,28respectively, to the C=O stretch of the COOH groups of protonated Asp-96 and Asp-1 15 on the basis of the spectra obtained for the bR mutants in which different Asp residues are individually replaced by Asn residues. The spectrum of the 4:l Caz+/bR* sample (Figure 2b) is similar to that of metal-ion-bound bR (Figure 2a). With decreasing Ca2+/ bR* ratio, the Asp-115 band shifts to higher wavenumbers and a new band at 1754 cm-' begins to be observed. The 1754cm-' band is not so obvious in the spectrum of the 2:l Ca2+/ bR* sample (Figure 2c) as in that of the 1:l CaZf/bR* sample (Figure 2d), whereas the upshift of the Asp-115 band takes place in the spectrum of the 2:l Ca2+/bR* sample. This suggests that the two kinds of spectral changes are not strongly correlated, if not independent. The upshift of the Asp-115 band indicates that the microenvironment of the Asp-1 15 COOH group changes with decreasing Ca2+ content. The appearance of the 1754-cm-' band for deionized bR indicates that a COO- group in metal-ion-bound bR undergoes protonation in the deionization process. Then, one may expect to find, in the COO- symmetric stretching region of Figure 3, a band which loses its intensity with decreasing Ca2+ content.
In fact, the band at 1405 cm-', which is very weak but definitely observed in the spectrum of metal-ion-bound bR (Figure 3a), disappears in the spectra of the 1:l Ca2+/bR* sample and deionized bR (Figure 3d,e). The fact that the 1405-cm-' band is not observed in the spectrum of deionized bR justifies the premise that dissolution of CaZ+from CaF2 cell windows into the sample is insignificant. It has long been known that the COO- symmetric stretch gives rise to a band around 1400 cm-1.48 The frequencies of the symmetric stretch of the metal-ion-bound acetate ion in various solid-state complexes have been studied e ~ t e n s i v e l y . ~ ~ Bands observed in this region of the infrared difference spectra of bR have also been assigned to the COO- symmetric s t r e t ~ h . We ~ ~ have , ~ ~ observed the infrared spectra of mixtures of sodium aspartate and CaC12 in aqueous solution at various mixing ratios. In the absence of Ca2+, the symmetric stretch of the COO- group of the aspartate side chain gives rise to a band at 1393 cm-'. This assignment is supported by the observation that aspartylphenylalanine methyl ester also has a band at the same wavenumber. At the Ca2+/aspartate ratio of 5, the corresponding band is found at 1399 cm-', and it shifts to 1420 cm-' or higher wavenumbers at very high Ca2+/ aspartate ratios. The frequencies of the symmetric stretch of the metal-ion-bound COO- group seem to be determined by two factors, namely, the type of coordination (unidentate, bidentate, or others) and the binding c ~ n s t a n t . ~Although ~,~~ the relationship between the COO- symmetric stretching frequency and these factors has not been fully understood, it is certain that the Ca2+-bound COO- group of Asp gives rise to a band at about 1400 cm-' or higher wavenumbers. Infrared bands due to the COO- antisymmetric stretch are also expected to give useful information on the metal-ion binding to the COOgroup.49,51,52 However, complete deuteration of the NH groups of the protein main chain is necessary to observe the COO-
Figure 2. Deconvoluted infrared spectra (1800-1700 cm-l) of (a) metal-ion-bound bR, (b) 4:l CaClz/bR*, (c) 2:l CaC12/bR*, (d) 1:l
Infrared Studies of Ca2+ Binding to Bacteriorhodopsin
1sbo
1750
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WAVENUMBER cm-' Figure 4. Deconvoluted infrared spectra (1800-1700 cm-l) of (a) metal-ion-bound D85N and (b) deionized D85N.
1430
1420
1410
1400
1390
WAVENUMBER cm-' Figure 5. Deconvoluted infrared spectra (1430-1390 cm-I) of (a) metal-ion-bound bR, (b) metal-ion-bound DUN, and (c) deionized
D85N.
antisymmetric stretching bands normally existing in the region of 1620-1550 cm-', where the amide-11 modes (the NH bend mixed with the CN stretch) also give rise to strong infrared bands. Since complete deuteration of the NH groups in bR is difficult, we have not been able to obtain information on the metal-ion binding to the Asp COO- groups in bR from the analysis of bands in the region of 1620-1550 cm-'. In order to identify the amino acid residue whose side-chain COOWCOO- group is responsible for the bands at 1754 and 1405 cm-', the infrared spectra of the metal-ion-bound and deionized forms of D85N have been measured, and the results in the regions of 1800-1700 and 1430-1390 cm-' are shown, respectively, in Figures 4 and 5. No band is observed at 1754 cm-' in the spectrum of deionized D85N (Figure 4b), and no band is observed at 1405 cm-' in the spectrum of metal-ionbound D85N (Figure 5b). [Although it seems as if there is a very weak band at about 1401-1400 cm-' in the latter spectrum, this is an artifact due to FSD giving rise to a negative peak at
J. Phys. Chem., Vol. 99, No. 19, 1995 1119 about 1397 cm-' weakly appearing in the tail of the band at 1392 cm-'. The same artifact is also seen in the deconvoluted spectra in Figure 3.1 The above observations convincingly indicate that the 1754-cm-' band in Figure 2e is attributable to the C=O stretch of the COOH group of the protonated Asp-85 and the 1405-cm-' band in Figure 3a to the symmetric stretch of the COO- group of the deprotonated Asp-85. This is consistent with the accepted notion that the Asp-85 side chain is protonated in deionized bR, which is supported by the results of the 13C NMR study.18 Apart from the state of the Asp-85 side chain, it is noted that the bands at 1742 and 1735 cm-' of D85N have comparable intensities in Figure 4, whereas the corresponding bands of bR in Figure 2 are unequal in intensity. Since each of these two bands arises from one COOH group, the D85N case is more understandable than the bR case. The origin of the unbalanced intensities for bR needs further study in the future, which may lead to a better understanding of the microenvironment of the Asp-115 side chain. Mention should also be made of the weakness of the 1754-cm-' band, in comparison with 1741cm-' band, in the spectrum of deionized bR (Figure 2e), which also presents a problem for further studies. It is important to note that the appearance of the 1754-cm-' band in Figure 2 and the disappearance of the 1405-cm-' band in Figure 3 are observed when the Ca2+/bR*ratio changes from 2:1 to 1:1. In both of these two samples, Ca2+ is bound to the first affinity site.l0 On going from the 2:l Ca2+/bR* ratio to the 1:l Ca2+/bR* ratio, one Ca2+ is released from the second high affinity site in most of the protein molecules. Accordingly, the binding of Ca2+ to the first high affinity site does not affect the state of the Asp-85 side chain. The protonation of the Asp85 side chain is concomitant with the loss of Ca2+ from the second high affinity site. This does not directly mean that Ca2+ is bound to the Asp-85 COO- group. However, complexation of the Asp-85 COO- group with Ca2+ in metal-ion-bound bR is likely in view of the fact that its 1405-cm-' band is definitely higher in position than the bands arising from the side-chain COO- group of aspartate and aspartylphenylalaninemethyl ester (vide ante). 2. Role of the Asp-85 Side Chain in Color Regulation and the L to M Transformation. As described in the Introduction, the binding of Ca2+ to the second high affinity site causes the color transition from blue to purple. The present infrared results establish that this blue to purple transition is f i d y 'correlated with the deprotonation of the Asp-85 COOH group. Regeneration of deionized bR with metal cations release several protons from a molecule of bR.6 On this basis, it has been considered6 that several acidic side chains are protonated in deionized bR. It has also been s u g g e ~ t e dthat ~ ~ .the ~ ~Asp85 side chain is deprotonated and has a negative charge in metalion-bound bR. The COO- group of this residue is located at a distance of about 3 A from the Schiff-base nitrogen atom' and is one of the candidates for the counterion of the protonated Schiff base. The retinal visible absorption of D85N, which has no negative charge in the position of the Asp-85 side chain, is similar to that of deionized bR. Hence, it has been prop ~ s e d ~ , that ' ~ , 'the ~ Asp-85 side chain is important for the color regulation of bR, and it is protonated in deionized bR. This proposal now has additional experimental evidence as described above. Genvert et aLZ9employed infrared difference spectra to study the blue to purple transition initiated by exposing deionized bR to NH3. In their study, a broad band at 1723 cm-' was observed in the deionized bR side, and two bands at 1571 and 1396 cm-'
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7780 J. Phys. Chem., Vol. 99, No. 19, 1995
in the NH3-regenerated bR side. The 1723-cm-' band was assigned to the CEO stretch of the COOH group, and the 1571and 1396-cm-' bands were assigned to the COO- antisymmetric and symmetric stretches, respectively. According to Gerwert et al., these bands arise from the water-exposed acidic side chains (for example, those located in the C-terminal region), and no acidic side chains in the interior of the protein change their states during the blue to purple transition. Their observation does not agree with ours. The discrepancy between the two infrared studies may be due to the difference in the hydration states between their and our samples. Their sample was once dried and rehydrated with water vapor,2gwhereas our sample contained about 90% (w/w) bulk water. In the L to M transformation of native bR, the Schiff base becomes deprotonated and the Asp-85 side chain becomes protonated.28 It has been considered3that the Schiff-base proton is transferred to the Asp-85 side chain. As mentioned in the Introduction, deionized bR does not undergo the L to M transformation. On the other hand, the 1800-1700-cm-' region of the L minus BR infrared difference spectrum of deionized bR is identical with that of native bR.30 This means that the state of the Asp-85 side chain in BR is kept unchanged in L of both metal-ion-bound and deionized bR; in other words, the Asp-85 side chain is in the COO- form in both BR and L of metal-ion-bound bR, and it assumes the COOH form in both BR and L of deionized bR. Therefore, it is impossible for the Schiff-base proton in deionized bR to be transferred to the Asp85 side chain which is protonated. This seems to give a good explanation for why the L to M transformation is blocked in deionized bR. The peak position of the C=O stretch of the Asp-85 COOH group in BR of deionized bR (1754 cm-') is almost identical with that of N (1756 cm-') and 8 cm-' lower than that of M (1762 cm-') of metal-ion-bound bR.28 This may indicate that the environment around Asp-85 in deionized bR is close to that in N rather than M of metal-ion-bound bR. The upshift of the Asp-115 band upon deionization is analogous to the shift of the corresponding band on going from BR to N of metal-ionbound bR.28 At present, the correlation between the peak position of the C-0 stretch of the Asp COOH group and the microenvironment around this group is not clear. Further studies are required for understanding implications of the band shifts. 3. Secondary Structure Change upon Deionization. The deconvoluted spectra of metal-ion-bound and deionized bR in the amide-I region (1700-1600 cm-l) are shown in Figure 6. The same kind of spectra for D85N are shown in Figure 7. The deconvoluted spectrum of native bR (Figure 6a) reproduces the result reported by Cladera et al.,38 except for the relative intensities of the individual bands. Upon deionization, the relative intensities of the bands at 1665 and 1658 cm-' are reversed, and the band at 1657 cm-' losses its intensity. This indicates that the secondary structure of bR changes upon deionization. Interestingly, D85N shows far smaller spectral changes upon deionization (Figure 7). The spectra of both metal-ion-bound and deionized D85N resemble that of deionized bR (Figure 6), except that, in the former, the 1657-cm-' band is missing and the intensities of the 1643- and 1650-cm-' bands are decreased. The position of the 1665-cm-' band of bR, as well as that of the 1663-1662-cm-' band of D85N, is slightly higher than the region expected for the amide-I band of a typical a-helix (1660-1650 cm-1).31-33 The origin of this highwavenumber amide-I band has been the subject of many s t ~ d i e s . ~The ~ ~ present ~ ~ ~results ~ ~ -add ~ ~ some new data for future studies on the assignment of this band.
Lu 0
z
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WAVENUMBER I cm-' Figure 6. Deconvoluted infrared spectra in the amide-I region of (a) metal-ion-bound bR and (b) deionized bR.
17b0
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WAVENUMBER / cm-' Figure 7. Deconvoluted infrared spectra in the amide-I region of (a) metal-ion-bound D85N and (b) deionized D85N.
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