From the
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 H
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
The binding of the chromophore to this
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
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.
The most general case
of a system of n components A
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
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
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.
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.
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 (
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 H
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
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.
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 (
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
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 H
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
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
One interesting region that shows significant
sensitivity to D
The FTIR spectra
indicate a major change in the negative peak at 1653
cm
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.
Dedicated to Prof.
Koji Nakamishi on his 70th birthday.
We thank Profs. R. Needleman and J. Lanyi for their
generous gift of bR mutants.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
O and D
O, 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.
),(
)
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) .
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.
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).
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.
,
A
, . . . A
in which
first-order reactions of the following type may occur between any two
components is shown in Equation 1.
=
-k
except when i =
j, which gives Equation 3.
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.
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 D
O, 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
D
O (
) 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 D
O, 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, D
O;
C, 14,15-
C retinal in
H
O.
Figure 10:
Difference FTIR spectra of 390 to 570 nm
transition of two mutants D96N and D115N.
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%.
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.
O there was an
increase in the time of formation of 470 in D
O 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.
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.
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.
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.
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.
O
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
.
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.
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
D
O. 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 D
O 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.
O 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 D
O.
, 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 D
O, 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 D
O. An additional interesting observation that
can be deduced from this sensitivity of the amide II to D
O
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.
Table:
Calculated rate constants for formation of the
470 nm species
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.