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J. Biol. Chem., Vol. 276, Issue 44, 40788-40794, November 2, 2001
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From the
Received for publication, May 26, 2001, and in revised form, August 9, 2001
Single and multiple mutants of extracellular Glu
side chains of bacteriorhodopsin were analyzed by acid and calcium
titration, differential scanning calorimetry, and thermal difference
spectrophotometry. Acid titration spectra show that the second group
protonating with Asp85 is revealed in E204Q in the
absence of Cl The elucidation of three-dimensional structures of
bacteriorhodopsin (BR)1
at high resolution and the increased use of mutants have improved greatly the knowledge of the proton transport mechanism, allowing the
identification of the principal side chains undergoing
protonations/deprotonations during this process (1-4). Among key
groups, Asp85 and Asp96 are the primary proton
acceptor and proton donor of the Schiff base, respectively. An
important achievement has been the description of several water
molecules in the extracellular region, forming a hydrogen-bonded
network with key residues (2, 5, 6). This network serves most likely to
transmit protonation changes and to conduct the proton. The role of
Asp85 in the extracellular region is not limited to be the
first acceptor of the Schiff base proton. According to a coupling
model, protonation of Asp85 induces the deprotonation of
the proton release group through a relationship existing between their
pKa values (7, 8). Although this model is useful in
understanding protonation/deprotonation mutual influences between
ionizable groups, it cannot give the answer about the exact mechanism
of proton transfer steps. In fact, several important questions remain
still elusive. One of these refers to the identity of the assemblies
involved in proton release, the proton release group. Recent studies
suggest that it is a complex including at least Glu194,
Glu204, and some water molecules (9-11). It should be
noted, however, that these two Glu side chains do not behave
equivalently, as demonstrated by the inhibition of the second increase
of the Asp85 pKa during the photocycle
in the E194Q mutant but not in the E204Q mutant (12). This is a clear
indication that Glu194 forms part of X'H, the group coupled
to the Asp85 pKa.
From the time when the presence of divalent cations in the purple
membrane was described, numerous efforts have been devoted to
identify their role and location (13-26). Some authors claim a
nonspecific location of the cations in the double layer (18, 21, 22) or
in the lipid phase (26). However, the majority of works argue for the
existence of specific binding sites in the purple membrane, including a
site near Asp85 (24, 27, 28), or a more external location
(29, 30). Among the most outstanding results pointing toward specific
cation locations in the protein moiety are: (i) the evidence for
carboxyl participation in cation binding from studies using specific
reagents (13, 17); (ii) the presence of fewer cation-binding sites and
with lower affinity in the bleached and pink membranes as compared with
purple membranes (14, 16, 19), even that the electric surface potential
of bleached membrane remains unchanged (31); (iii) extended x-ray
absorption fine structure data describing a different
environment for the bound Mn2+ with respect to free
Mn2+ in water and ruling out the interaction of
Mn2+ with P or S atoms (32); (iv) Fourier transform
infrared studies showing that binding of Mn2+ to deionized
membrane induces changes in the reverse turns, located in the loops
(33); and (v) 13C NMR studies detecting changes in the
Ala196 environment upon divalent cation binding
(25).
Despite the substantial experimental background suggesting the
existence of specific cation-binding sites, none of the recent bacteriorhodopsin models reflects their presence (2, 5, 34). One
possible exception is the electron microscopy structural data of
Mitsuoka et al. (35), which detected the presence of charges
and polarized water molecules in some locations of their structure.
Recently we have described some spectral and functional properties of
the quadruple mutant E9Q/E74Q/E194Q/E204Q (4Glu), including high
hydroxylamine and Cl The construction of the mutants and their expression in
Halobacterium salinarum was done as described previously
(12, 36). The wild type and mutagenized membranes were isolated as
purple membrane sheets according to Oesterhelt and Stoeckenius (41). Deionized membranes were obtained by dialyzing the samples against a
cation exchange resin (Dowex-50W). All manipulations of deionized samples were done using well rinsed plastic materials. Concentration of
deionized samples was calculated by the method of Lowry et al. (42), modified for membrane proteins (43). Absorption spectra
were recorded on a Varian Cary3-Bio spectrophotometer in the
250-800-nm range. To avoid light-scattering artifacts, an integrating
sphere was used.
The pH of purple and deionized membranes (at a concentration of
1.5-2.0 × 10 Absorbance changes at 630 nm as a function of pH were used to monitor
the purple-to-blue transition. Experimental points were normalized with
respect to the largest value at 630 nm and fitted to the
Boltzmann equation.
Calcium binding experiments were done in darkness with deionized
samples adjusted to pH 4-4.5. Absorbance changes induced by the
addition of small quantities of calcium were monitored spectrophotometrically. BR concentration was in the 1.5-2 × 10 DSC experiments were performed using a Micro-Cal MC2 instrument. The
samples were dialyzed previously in water adjusted at pH 6.5-7, giving
a final concentration of 1.5-2 mg/ml. Experiments were done under 1.7 atm nitrogen pressure to avoid sample evaporation at high temperatures.
Scanning speed was set at 1.5 K/min. Three thermograms were registered
for each sample. The first informs about the heat released or taken by
the protein upon temperature increase. After cooling down to room
temperature, second and a third thermograms were run to check the
reversibility of the transitions. Two corrections were applied to the
first thermogram: (i) subtraction of the second thermogram, which acts
as a blank, and (ii) subtraction of the chemical base line using the
Takahashi and Sturtevant method (44). Tm was
defined as the point where the Cp value is maximal.
Thermal denaturation experiments were carried out on dark-adapted
samples in H2O and pH 7.0 by following changes in the
UV-visible absorption spectra upon temperature increase. Spectra were
taken every 5 °C in the range 250-800 nm, starting at 20 °C and
allowing 8 min for stabilization at each temperature. BR concentration was 1 × 10 Spectral Behavior of Extracellular Glu Mutants upon Acidification:
the Purple-to-Blue Transition
As has been widely described, acidification of purple membrane
samples causes the formation of the blue form, because of
Asp85 protonation (45, 46). Fig.
1 (A and B) shows
the absorbance and difference spectra of the dark-adapted E74Q mutant
in 150 mM KCl upon acidification of the medium from pH 5.7 to 2.1. As in wild type, the difference spectra of E74Q reveal the
presence of two isosbestic points at about 620 and 578 nm. The
isosbestic point at about 620 nm reflects a transition that produces a
small band broadening in the red edge of the spectrum and a small blue shift (47, 48), yielding the so-called BRacid form. The
second isosbestic point at 578 nm corresponds to the red shift of
the spectrum because of protonation of Asp85, giving the
principal transition (the purple-to-blue transition). Similar spectral
changes upon pH decrease are obtained for E74Q in H2O and
for dark-adapted E9Q and E194Q single mutants in 150 mM KCl
or in H2O (not shown).
Acidification of the dark-adapted E204Q mutant reveals more complex
spectral changes. Whereas in 150 mM KCl a similar behavior to wild type is obtained, in H2O or in 75 mM
Na2SO4 where Cl Titration of deionized wild type and deionized forms of the single
mutants E9Q, E74Q, and E194Q by increasing the pH from 4 (the initial
pH after deionization) to 8 gives only one isosbestic point at 577 nm
in the difference spectra (Fig.
2A for the spectra of
deionized E9Q). However, titration of deionized E204Q shows not only
that the red form persists but also that it is better distinguished
than in the presence of Na2SO4 (Fig.
2B). Thus, E204Q is unique among wild type and the single
Glu mutants, in that titration of the deionized sample displays changes
in the protonation state of more than one group. Moreover, the
Contribution of Extracellular Glu Residues to the Structure and
Function of Bacteriorhodopsin
PRESENCE OF SPECIFIC CATION-BINDING SITES*
§¶,¶,,
,**,,, and§§
Unitat de Biofísica, Departament de
Bioquímica i de Biologia Molecular, Facultat de Medicina,
Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del
Vallès), Barcelona 08193, Spain, the ** Departament
d'Enginyeria Química, Escola Universitària d'Enginyeria
Tècnica Industrial, Universitat Politècnica de Catalunya,
Barcelona 08036, Spain, and the Institut de
Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona,
Bellaterra (Cerdanyola del Vallès),
Barcelona 08193, Spain
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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but is not observed in the triple mutant
E9Q/E194Q/E204Q or in the quadruple mutant E9Q/E74Q/E194Q/E204Q.
The results point to Glu9 as the second group protonating
cooperatively with Asp85. Comparison of the apparent
pKa of Asp85 protonation in water and
in the deionized forms and results of calcium titration suggest that
cation-binding sites are of low affinity in the multiple Glu mutants.
Like for deionized wild type bacteriorhodopsin, differential scanning
calorimetry reveals a lack of the pretransition in the multiple
mutants, whereas in E9Q it appears at lower temperature and with lower
cooperativity. Additionally, at neutral pH the band at 630 nm arising
from cation release upon temperature increase is absent for the
multiple mutants. Based on these results, we propose the presence of
two cation-binding sites in the extracellular region of
bacteriorhodopsin having as ligands Glu9,
Glu194, Glu204, and water molecules.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
accessibility to the Schiff base
environment and severalfold decreased effect of Ca2+ on the
regeneration of the purple color of deionized 4Glu (36). These results
suggest a more open structure for the extracellular region of the 4Glu
mutant and reduced affinity of cations for the color-controlling
binding site(s) as compared with wild type. Therefore, we proposed that
the extracellular Glu side chains could be involved in cation binding
and in the maintenance of the correct protein structure. To get further
insight on the role of extracellular Glu side chains in these issues,
as well as in proton transport, in this work we study BR mutants in
which the extracellular Glu have been substituted by Gln. Even if these changes are very conservative, Gln cannot deprotonate on one hand and
has a reduced capability of forming hydrogen bonds on the other (37).
Although some characteristics of these mutants (especially E194Q and
E204Q) have already been published (11, 26, 38-40), we complete these
studies by presenting new data in a thorough manner. Finally, we
propose a model of cation binding in the extracellular region, involved
in protein structure and function.
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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5 M) was adjusted by
microadditions of NaOH, HCl, or H2SO4
solutions. To avoid contamination, pH adjustment of deionized samples
was done using duplicates. All experiments were performed in the dark using dark-adapted samples. Because of abnormal dark adaptation kinetics of the E194Q, 3Glu, and 4Glu mutants (36), the samples were
kept in the dark for about 25 days.
5 M range.
5 M.
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (26K):
[in a new window]
Fig. 1.
Purple-to-blue transition of dark-adapted
E74Q and E204Q mutants. A, curves 1-8,
absorption spectra of the E74Q mutant from pH 5.7 to 2.1 in the
presence of 150 mM KCl. B, curves
2-8, difference absorption spectra between the sample at
pHi minus the sample at pH 5.7, where
pHi is 4.3, 3.8, 3.5, 3.0, 2.8, 2.6, and 2.1, from the
spectra in A. C, curves 1-10,
absorption spectra of the E204Q mutant from pH 7.5 to 1.7 in
H2O titrated with H2SO4.
D, curves 2-10, difference spectra between the
sample at pHi minus the sample at pH 7.5, where
pHi is 6.7, 6.1, 5.6, 4.7, 4.3, 3.9, 3.3, 2.8, and 1.7, from the spectra in C.
ions are not
present, the absorption maximum undergoes a small blue shift and band
broadening in both sides (Fig. 1, C and D). This
gives rise to two positive bands at 640 and 460 nm in the difference
spectra (Fig. 1D). Further decrease of the pH leads to the
main transition, where all species are converted to the blue form. This
produces a positive band at 628 nm and a negative band at 538 nm.
max never reaches 603 nm in any of the condition
examined (not shown).
View larger version (29K):
[in a new window]
Fig. 2.
Purple-to-blue transition of dark-adapted BR
mutants. A, curves 1-7, difference spectra
between the sample at pHi minus the sample at pH 8.8 of
deionized E9Q, where pHi is 7.6, 6.9, 6.5, 5.6, 5.3, 5.0, and 3.6. B, curves 1-9, difference spectra
between the sample at pHi minus the sample at pH 7.6 of
deionized E204Q, where pHi is 7.1, 6.6, 6.3, 5.9, 5.4, 5.2, 4.9, 4.6, and 4.0. C, curves 1-8, difference
spectra between the sample at pHi minus the sample at pH
5.9 of 3Glu mutant in 75 mM Na2SO4,
where pHi is 5.6, 5.4, 4.8, 4.4, 4.2, 3.6, 2.9, and 2.3. D, curves 1-8, difference spectra between the
sample at pHi minus the sample at pH 7.4 of E194Q/E204Q
mutant in 75 mM Na2SO4, where
pHi is 6.9, 5.2, 4.6, 4.1, 3.8, 3.5, 3.0, and 2.5.
Upon acid titration, the multiple extracellular Glu mutants undergo spectral changes different from those of the single mutants. The difference spectra of 3Glu or 4Glu mutants do not show the red band at about 460 nm in any of the conditions analyzed (salt, H2O, deionized; see Fig. 2C for difference spectra of 3Glu in 75 mM Na2SO4). However, acidification of the E194Q/E204Q mutant in Na2SO4 or in water gives rise to the red band (Fig. 2D), arising from a small blue shift and broadening of the absorption band, similar to the single E204Q mutant.
Determination of the Apparent Asp85 pKa
As is known, the plot of the absorbance increase at 630 nm as a function of pH yields the apparent pKa of Asp85 (45, 46). Table I shows the calculated values for pKa of Asp85 for all mutants and wild type in three different conditions: 150 mM KCl, water, and deionized membranes. Values for the Asp85 pKa similar to those found in the presence of 150 mM KCl were obtained upon titration in 75 mM Na2SO4 (not shown). In low salt concentration (150 mM KCl) all single mutants gave similar values (pKa ~ 2.7, although E194Q shows slightly lower pKa), whereas the pKa values of the multiple mutants are elevated (around one unit above wild type). In water, the absence of ions in the medium induces more negative electrical surface potential and increased proton concentration, giving rise to increased an apparent pKa value as compared with the salt-containing samples. In water, all samples give higher pKa values as compared with the same samples in salt, except the E74Q mutant (pKa about 2.8, similar to that in salt). Multiple mutants E194Q/E204Q and 4Glu have an identical and elevated pKa of 4.7, whereas 3Glu has a pKa of 5.2 (Table I).
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Analysis of deionized membrane samples permit the evaluation of the effect of mutations themselves over the Asp85 pKa, regardless of the presence or absence of endogenous cations. Additionally, comparison of the sample properties in water with those of deionized form allows estimation of the effects of the endogenous cations. As Table I shows, all deionized mutants give slightly lower pKa values as compared with deionized wild type, except for E9Q, which presents an increased pKa of 6.0. On the other hand, the absence of endogenous cations in the deionized form as compared with the sample in water cause an increase of the pKa of more than 2 pH units for wild type (13, 49) and similar values for the single mutants except for E204Q. It is noteworthy that the multiple mutants exhibit differences of less than 1 pH unit between pKa values of the deionized form and in water. This suggests that the nondeionized forms of these samples have low content of endogenous cations, thus affecting only slightly the apparent Asp85 pKa in comparison with deionized sample.
Addition of Calcium to Deionized Bacteriorhodopsin
When calcium or some other cations are added to blue deionized
samples, the purple form of bacteriorhodopsin is recovered, because of
the deprotonation of Asp85 (13, 17, 49). Fig.
3A shows typical difference
spectra obtained upon calcium addition to the wild type blue form. The presence of only one isosbestic point at 578 nm strongly indicates that
only the deprotonation of Asp85 is involved in the process,
leading to a sigmoidal curve in the plot of absorbance change as a
function of pCa (Fig. 3B).
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The pCa values for 50% of the blue-to-purple transition are shown in Table II. As can be seen, the most drastic changes in Ca2+ binding, as compared with wild type are obtained for the multiple mutants where the quadruple mutant 4Glu needs more than 10 Ca2+/BR molecule, 3Glu needs about 8 Ca2+/BR, and E194Q/E204Q needs 3.5 Ca2+/BR. Among the single mutants, only E204Q and E9Q need higher Ca2+ amounts compared with wild type.
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Thermal Denaturation Experiments
Differential Scanning Calorimetry--
It has been demonstrated
previously that BR samples with decreased cation content have lower
thermal stability (14, 50). To study the involvement of the mutated Glu
side chains on thermal stability, DSC experiments were carried out.
Fig. 4 shows thermograms of extracellular
mutants in H2O, obtained after correction for instrumental
and chemical base lines (see "Experimental Procedures"). A known
feature of the thermal scan of native purple membranes is the presence
of two transitions (51). The main transition at about 98 °C accounts
for the destruction of the helical interactions within the protein and
retinal release. The small and reversible pretransition at about
80 °C has been interpreted as resulting from disorganization of the
hexagonal para-crystalline arrangement (51, 52).
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As is seen in Fig. 4, the main transition of all mutants appears in the range 90-100 °C, decreasing in the order E74Q > E194Q > E9Q = E204Q > E194Q/E204Q > 4Glu. Besides that, the transitions are less cooperative except for E74Q. In comparison with the main transition, the temperature of the pretransition of the mutants is more variable. It appears at lower temperatures and with low cooperativity for E194Q, E204Q, and E9Q (at about 68 °C for this latter sample), and it is absent for all multiple mutants.
Visible Spectroscopy--
Heating the purple membrane suspension
induces first the appearance of the blue form, because of cation
release (14). This is followed by the appearance of the red form
(max at about 460 nm) and finally by the release of
retinal above 80-85 °C. Fig. 5 shows
the difference spectra obtained for E9Q and 4Glu on temperature increase, in H2O at pH 7.0. Similar to wild type, an
increase of temperature from 25 to 55 °C of E9Q causes a progressive
red shift of the absorption spectrum, which gives rise to the
appearance of the band at 630 nm in the difference spectrum. Moreover,
the thermally induced blue form is a reversible process because
lowering the temperature from 55 °C (temperature for which maximum
accumulation of blue form is observed) back to 25 °C recovers the
initial purple form. Importantly, whereas the rest of the single
mutants (E74Q, E194Q, and E204Q) also show the temperature-induced band
at 630 nm, the multiple mutants lack formation of the blue form, as
represented in Fig. 5 for 4Glu.
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DISCUSSION |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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In the first part of this work, we performed acid titration experiments to acquire new information about the influence of the extracellular Glu side chains on Asp85 protonation. Fisher and Oesterhelt (53) and Mowery et al. (47) already noted that the plot of the absorbance changes in the purple-to-blue transition of wild type BR is too steep to be considered as a transition controlled by the titration of one single group, Asp85. Later on, Váró and Lanyi (48) corroborated this observation and proposed a model postulating that more than one proton is bound cooperatively during acid titration. They identified a new species, the BRacid form, that results from BR by proton binding and appears before the blue form, BRblue. Although the additional group titrated in this process was not identified at that time, neither it is yet known whether it is worthy of mention that the authors suggested Glu9 and Glu204 residues as a possibility. However, in the majority of the following work dealing with the purple-to-blue transition, these early observations have been ignored, and only the protonation of Asp85 has been considered.
As indicated under "Results," single extracellular Glu mutants show
similar pH titrations as wild type, with the minor transition caused by
protonation of a second group remaining masked but already suggested by
the presence of two isosbestic points. The only exception to this
behavior corresponds to the E204Q mutant in the absence of
Cl ions, which shows clearly a band at 460 nm (red form).
However, in the presence of Cl ions, the red band
disappears. One reasonable explanation is that Cl binds
near Gln204, restoring the negative charge and the water
network. Therefore, unlike the rest of single mutants, E204Q senses the
presence of Cl ions in the medium and exhibits different
spectral changes upon acid titration. Although at present we are not
able to identify the second protein residue protonating cooperatively
with Asp85, comparison of titration results for single and
multiple mutants suggest that it can be one of the acidic side chains
located in the extracellular region. Moreover, the fact that the red
form is observed for E194Q/E204Q but not for 3Glu or 4Glu points to Glu9 as being responsible for the second transition.
Our data provide new evidence for cation binding to the extracellular side of the purple membrane. In low salt concentrations, the apparent pKa values of Asp85 of the single mutants are similar to that of wild type, being independent of the type of salt in the medium (sodium sulfate or chloride). However, titrations in water or in deionized membranes exhibit perturbed pKa values of Asp85 as compared with wild type. The increased pKa obtained for a particular sample in water, as compared with the same sample in salt can be explained essentially by the increased proton concentration over the negatively charged membrane surface because of the absence of counter ions. Especially in the multiple mutants, the pKa increase is considerably higher than in wild type. This effect can be attributed to a loss of endogenous cations and a subsequent increase of proton concentration on the surface. Similar pKa values found for E74Q in water and in salt can be explained by loss of a negative charge in the membrane surface upon mutation of this external residue.
Comparison of the pKa of the purple-to-blue transition in water with that of the deionized sample gives an indication of the effects of bound cations on this transition and thus reveal their affinities, analogous to the comparison of pKa values between salt and water. Reasonably, large differences in pKa between a sample in water and in the deionized state reflect mainly strong affinity of cations, whereas small differences reflect low affinity of cations. Therefore, the difference of about 2.3 pH units found for wild type indicates the presence of cations bound with high affinity. This difference is strongly reduced to less than 1 pH unit for all multiple mutants, indicating clearly that cations have a low affinity in these samples (Table I). On the other hand, Ca2+ binding experiments to deionized samples give further support to the suggested role of extracellular Glu side chains in cation binding. As described under "Results," the single mutant E9Q and the multiple mutants require higher cation concentrations for reaching the 50% of the blue-to-purple transition as compared with wild type. Thus, both acid titration and Ca2+ binding experiments point to Glu9, Glu194, and Glu204 as being involved in cation binding.
Thermal denaturation experiments can provide further information about
the role of extracellular side chains in cation binding. Therefore, we
analyzed thermal behavior of extracellular mutants by DSC experiments
and UV-visible spectrophotometry. In some mutants, the DSC main
transition appears at slightly lower temperature as compared with wild
type, indicating somewhat decreased stability of the secondary and/or
tertiary structures. However, the most interesting effect was obtained
for the pretransition, resulting from disorganization of the hexagonal
para-crystalline arrangement (51, 52). As presented under
"Results," there is a concurrence between the lack of the
pretransition and the absence of cations. Particularly, the deionized
wild type membrane lacks the pretransition but, upon cation addition of
at least 2 Mn2+/BR, it is partly recovered (50). On the
other hand, the recently reported decrease in the content of the
BR-specific 
II helical structure upon temperature
increase (54) is likely to be due also to cation release. First, there
is a coincidence of the temperature of II decrease with
the temperature of the pretransition (54); second, removal of cations
by deionization causes a decrease in the II content as
has been reported previously by Duñach et al. (33).
Therefore, much experimental evidence indicates that the DSC
pretransition is caused by cation release as temperature increases,
which in turn induces the cooperative disorganization of the
para-crystalline arrangement.
Appearance of the pretransition at lower temperatures and with lower cooperativity for the single mutants E194Q, E204Q, and especially E9Q point out that these mutations induce a loosening of the hexagonal disposition of trimers. Furthermore, the mutants E194Q/E204Q, 3Glu, and 4Glu do not show the pretransition, indicating either that the hexagonal arrangement is strongly perturbed in these mutants or that the disorganization occurs continuously as temperature increases, thus lacking any cooperativity and becoming unobservable by DSC. Most likely, these effects do not come directly from the loss of the Glu negative charges themselves but from the loss of cation-binding sites. There is a complete correlation between DSC and calcium titration data; as more calcium is needed to recover the purple form in the different mutants, the DSC pretransition is less evident and appears at lower temperatures.
Importantly, for the multiple mutants, UV-visible spectrophotometry establishes the absence of the blue form ensuing from cation release upon temperature increase. These results, which are a sign of the absence of cations in these multiple mutants, correlate closely with the observed lack of pretransition.
In agreement with the location of cations in the extracellular region, previous DSC results established the absence of the pretransition when the extracellular BC loop is cleaved but its presence when the cytoplasmatic loop EF is cleaved (55). On the other hand, normal pretransition is obtained for the cytoplasmatic mutants D36N/D38N and D102N/D102N,2 indicating that neutralization of Asp side chains in the cytoplasmatic side do not affect the pretransition, thus leaving intact the para-crystalline arrangement.
On the basis of our findings, we propose a model of the extracellular
region containing two cation-binding sites: one linked to
Glu194/Glu204 and the other linked to
Glu9 (Fig. 6). In this model,
Glu194 is postulated to control the Asp85
pKa in the resting state as in the photocycle,
participating in the diffuse water moiety that shares the proton
that is released to the bulk (represented in the model by a protonated
water molecule). Mainly from DSC and calcium titration results, it can
also be suggested that Glu204 is interacting more strongly
with the cation and is less linked with the water network. Thus, it is
not coupled with Asp85 (or only weakly coupled). The
different role envisaged for Glu194 and Glu204
is in keeping with our recent data demonstrating that, in the presence
of Cl ions, the Asp85 pKa
during the photocycle depends on Glu194 but not on
Glu204 (12).
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The two cations placed in the extracellular side may have not only structural significance by helping to maintain the correct structure in the extracellular region (36) and the para-crystalline arrangement, but most probably they are also involved in the functioning of the protein. Especially, the cation linked to Glu194/Glu204 may be implicated in the maintenance of the optimal spatial relationship between these two residues and the water molecules that form the hydrogen-bonded network. This cation may constitute an essential element of the proton release machinery, by providing the electrical potential necessary to release the proton. In other words, the cation may help to regulate the pKa of the proton release group for it to be decreased sufficiently in the proton release step. What is more, the cation may have a gate-like function, allowing the proton release group to be reprotonated from the interior of the protein in the last steps of the photocycle but keeping it isolated from the exterior. In this context, it is known that the blue form does not have a normal photocycle, because of the presence of the already protonated Asp85 (13, 47). On the other hand, the purple-deionized form has a normal-like photocycle (56) with a yield of M intermediate around 50% (57), but its proton transport capacity has not been measured yet. The possibility of a normal photocycle lacking proton transport has been raised before based on electro-optical studies of purple deionized BR (58).
An intriguing question is why the endogenous cations are not identified in the high resolution crystallographic structures published so far. One reason may be that the methods applied for crystal preparation cause loss of bound cations, which are then substituted by monovalent cations. This is likely to occur, because purple membrane is first delipidated by detergent solubilization and finally placed in a highly concentrated Na/K-Pi (59). It is well established that to recover purple membrane from blue, about 50 times more monovalent than divalent cations are needed (17). Moreover, it is known that monovalently regenerated BR turns blue by dilution (49). These facts indicate that monovalent cations have low affinity for BR and thus have high mobility around the binding sites, making them unlikely to be observed by diffraction techniques.
Finally, it should be indicated that other workers have also proposed
the occurrence of cation-binding sites in the extracellular region. A
site near Glu194 was proposed based on 13C NMR
studies (25). Cation-binding site(s) near the membrane surface was
proposed by Fu et al. (29), and a site involving Glu204 was anticipated because of theoretical
considerations (30). While this article was in the revision process, a
new work appeared claiming for a cation site located at less than 9.8 Å from Glu74 (60). Thus, although no cations have been
seen in the crystallographic structures, accumulated evidence suggests
the presence of cation-binding sites in BR. Our results place two of
them in the extracellular region, linked to Glu9,
Glu194, Glu204, and several water molecules.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Joaquim Villaverde for advice on DSC experiments and for many helpful discussions, to Dr. Richard Needleman for the generous gift of the plasmid pXLNovR and pSI-bop, containing the bop gene, and to Elodia Serrano and Yolanda Moreno for skillful technical assistance.
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FOOTNOTES |
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* This work was supported by Dirección General de Investigación Grant BMC2000-0121, Secretaría de Estado de Educación y Universidades Grant SAB1999-0102, and Direcció General de Recerca Grant 1999SGR-102 and fellowships (to S. E. and to M. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Biochemistry, University of Cambridge, 80 Tennis Court Rd., Cambridge CB2 1GA, UK.
¶ These authors contributed equally to this work.
Present address: Dept. de Chimie, Université de
Montréal, C.P. 6128, Montréal, PQ H3C 3J7, Canada.
§§ To whom correspondence should be addressed. E-mail: esteve.padros@uab.es.
Published, JBC Papers in Press, August 27, 2001, DOI 10.1074/jbc.M104836200
2 C. Sanz and E. Padrós, unpublished experiments.
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ABBREVIATIONS |
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The abbreviations used are: BR, bacteriorhodopsin; 3Glu, the bacteriorhodopsin mutant E9Q/E194Q/E204Q; 4Glu, the bacteriorhodopsin mutant E9Q/E74Q/E194Q/E204Q; DSC, differential scanning calorimetry.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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