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INTRODUCTION |
Rhodopsin is the photoreceptor protein of the vertebrate retina
(1-3) belonging to the G-protein-coupled receptor
(GPCR)1 superfamily (4-6).
It is the main protein component of the rod outer segments (ROS) of the
retinal photoreceptor cells, and its easy isolation from bovine retinas
has made of this receptor a widely used model for the GPCR superfamily.
Rhodopsin is a key molecule in the biochemistry of vision and
alterations in its sequence have been associated with retinal disease.
In particular, a high number of mutations in the opsin gene have been
associated with the autosomal form of the retinal degenerative disease
retinitis pigmentosa (7, 8). A number of factors have been proposed to
be related to retinal function, and among them a possible role for
Zn2+ in the retina and its metabolism has been proposed
(9). Zn2+ is present at particularly high concentrations in
the retina (10) being a component of the disc membranes in the rod
outer segments of the photoreceptor cells (11). Zn2+ has
been histochemically localized to ROS (12) and it has been shown to
copurify with ROS proteins as well (13). Despite this presence, the
role of Zn2+ in the visual cycle and specifically in its
interaction with rhodopsin remains unclear.
Zn2+ is required for the function of numerous
proteins, serving both as a part of the active site in, for example,
metalloenzymes, and acting to stabilize protein domains, such as the
Zn2+ finger-binding motif in transcription factors (14,
15). The structure of many Zn2+-binding sites is known from
x-ray crystallography of Zn2+-binding proteins and, thus,
the geometry of the interaction between Zn2+ and different
coordinating residues is well characterized (14, 15). This, together
with the small size of the zinc (II) ion, makes artificially generated
Zn2+-binding sites a highly useful approach for probing
structure-function relationships in proteins. In GPCRs, for example,
construction of bis-His Zn2+-binding sites has led to
important information about both the organization of the transmembrane
helices and their movements during receptor activation to be obtained
(16-22). Therefore, engineered Zn2+ binding sites created
by site-directed mutagenesis is an interesting tool for probing
intramolecular interactions in these types of receptors (23).
Particularly in the case of rhodopsin, important structural information
about helical orientation, connectivities, and the conformational
change upon light activation, has been obtained from engineered
Zn2+-binding sites in this visual photoreceptor (19).
There is experimental evidence indicating the presence of naturally
occurring Zn2+-binding sites in several GPCR proteins.
These include the dopamine transporter (24), D1 dopamine
receptor (25) and all D2-like dopamine receptors:
D2L, D3, and D4 (26). Molecules
participating in the visual phototransduction process are already known
to have Zn2+-binding sites, including rhodopsin (27) and
cGMP phosphodiesterase (PDE6
) (28).
Previous reports showed direct Zn2+ binding to rhodopsin,
both in ROS membranes and in detergent-solubilized-purified protein systems, by means of 65Zn competition binding experiments,
indicating that there may be coordination sites for Zn2+ in
native rhodopsin (27). This binding occurs in the dark and it increases
upon illumination of the protein (27). Also, previous reports indicated
that Zn2+ enhanced azido-[
-32P]ATP binding
to rat ROS, suggesting a structural effect of Zn2+ on
rhodopsin (29). Four Zn2+ were included in the crystal
structure of rhodopsin at 2.8-Å resolution (30, 31). Recently this
crystal structure was refined, and seven Zn2+ were
identified, three per monomer and one which was at a boundary region of
the two monomers forming the unit cell of the crystal, as well as water
molecules that would play a functional role in rhodopsin activation
(32). Four of these ions are located in the intradiscal domain of the
receptor. Although these binding sites occur under crystallization
conditions, they also provide evidence for direct Zn2+
binding to rhodopsin.
In the present study we have undertaken the study of the
Zn2+ effect on rhodopsin structure and stability, by taking
advantage of the proposed endogenous Zn2+-binding sites in
the receptor. We find a clear specific effect of Zn2+ on
the thermal stability of rhodopsin when compared with other metal ions
tested. At the same time we can also see an effect for this metal ion
on several properties of rhodopsin like pigment regeneration and
metarhodopsin II (MetaII) stability. The results obtained indicate that
Zn2+ clearly reduces the thermal stability and regeneration
of rhodopsin in detergent solubilized form and in ROS membranes. These
results suggest that Zn2+ binding to rhodopsin results in
specific effects on the structure and stability of the receptor. This
effect alone may not be accountable for the physiological role proposed
for Zn2+ in visual function. Other effects like those
related to increased rhodopsin phosphorylation, or the proposed effect
upon other proteins of the visual phototransduction cascade, or upon
some component of the retinoid cycle, may also play a role in
vivo.
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EXPERIMENTAL PROCEDURES |
Materials--
Bovine retinas were from J. A. Lawson Co.
(Lincoln, NE). n-Dodecyl
-D-maltoside (DM)
detergent was from Anatrace (Maumee, Ohio, USA).
11-cis-Retinal was a gift from Prof. P. P. Philippov (Moscow State University).
Water used for buffer preparation was Milli-Q (Millipore) quality and
was received with 10-18 m
. Traces of zinc were detected using a
polarographic instrument (Metrohm Ltd., Herisau, Switzerland). The zinc
concentration was determined to be <5 × 10
2
µM by the inverse voltammetric (anodic stripping) method,
using the hanging mercury drop electrode.
ROS membranes were prepared under dim red light from frozen bovine
retinas using a sucrose gradient method. The membranes were suspended
in isolation buffer (buffer A): 70 mM potassium phosphate,
1 mM MgCl2, and 0.1 mM EDTA (pH
6.9) and pelleted by centrifugation. The membrane pellets were
resuspended in 5 mM Tris-HCl (pH 7.5) and 0.5 mM MgCl2 (hypotonic buffer). Two alternating washes with these buffers were carried out to decrease the amount of
any further contaminating proteins. Finally, ROS membranes were split
in several aliquots and stored in the dark at
20 °C. Typically, 1 mg of ROS membranes was solubilized in 2 ml of buffer A containing 1%
DM. Solubilization was done at 4 °C in the dark for 1 h (33).
The solution was then centrifuged for 30 min in a T865 Sorvall rotor at
30000 rpm, and the supernatant was used for the assays as solubilized
rhodopsin (Rho). Rho was usually stored in the dark at 4 °C or kept
at
20 °C for long term storage. Rho concentration was determined
by using a molar extinction coefficient value
500 of
40,600 M
1 cm
1, and a molecular
weight of 40,000 Da. The preparations showed UV/vis absorbance ratios
(A280/A500) in the
1.8-2.0 range.
Sample Preparation--
Zinc binding to Rho was carried out in
the dark. Rho was diluted with 20 mM HEPES, containing 145 mM NaCl, 2 mM MgCl2, 0.5 mM CaCl2, and 0.012% DM in the
spectrophotometric cuvette to a final DM concentration of 0.02% DM.
These preparations were monitored by UV/vis spectroscopy. A stock
solution of 1 M ZnCl2 was prepared in the same
dilution buffer. Small aliquots of the stock zinc solution were added
to obtain the final desired zinc concentration in the sample. The
sample was then incubated for 1 h at 15 °C or 20 °C in the
dark, and it was monitored by recording UV/vis spectra at 5-min
intervals over cycle mode to ensure that there was no spectral change
with time. The procedure for the control experiments was the same, but
zinc addition was omitted. Sample preparation for ROS rhodopsin in
membranes (unsolubilized samples) was carried out essentially as
described for the detergent-solubilized samples. Other metal ions
(Ca2+, Co2+, Cd2+, and
Cu2+) were assayed as chloride salts to rule out unspecific
charge effects. In these cases, sample preparation was carried out in the same way as with zinc. Choline was used as a control model compound
to completely rule out the possible unspecific electrostatic nature of
the observed effects.
UV/Vis Spectra of Rhodopsin--
All measurements
were made on a Cary 1E spectrophotometer (Varian, Australia), equipped
with water-jacketed cuvette holders connected to a circulating water
bath. Temperature was controlled by a Peltier accessory connected to
the spectrophotometer. All spectra were recorded, in the 250-650-nm
range, with a bandwidth of 2 mm, a response time of 0.1 s, and
scan speed of 250 nm/min. The protein sample binding reaction was
carried out in the spectrophotometric cuvette during 1 h at
20 °C and was monitored by UV/vis spectroscopy as described above.
These reaction samples were used for photobleaching, thermal stability,
and regeneration experiments. The experiments were performed in
triplicate. Fourth derivative spectra were obtained, from five
absorption spectra coadded to improve the signal-to-noise ratio, by
using the Savitzsky-Golay algorithm and 200 points with the Grams/32
software (Galactic Industries).
Photobleaching and Acidification of Rho--
Samples were
bleached with a 150 watt power source equipped with an optic fiber
guide and using a 495 nm cut-off filter. Dark-adapted Rho samples with
50 and 100 µM zinc or without zinc were illuminated for
10 s to ensure complete photoconversion to 380-nm absorbing species. Acidification was carried out, immediately after
photobleaching, by addition of 2 N
H2SO4 to a final pH of 1.9 (1% of the sample volume), and after 2 min to allow for stabilization an absorption spectrum was recorded.
Photobleaching Efficiency and Metarhodopsin III (MetaIII)
Formation--
In the case of the photobleaching efficiency
experiments, dark-adapted Rho samples with 100 µM zinc or
without zinc were illuminated with subsaturating light (with a 25 watt
light source and a distance between the sample and the light source of
5 cm) at 5-s intervals. Immediately at the end of each bleaching
interval, changes in the absorption spectra were recorded until
complete photobleaching was achieved (complete disappearance of the
500-nm chromophoric band).
The formation of MetaIII was measured, after Rho illumination for
60 s (saturating illumination), in the presence of different zinc
concentrations or without zinc (control) at 15 °C. Complete absorption spectra in the range studied were recorded starting immediately after illumination and at 2.5-min intervals thereafter. MetaIII formation was monitored at A480. All
absorbance values were normalized to the same initial
A500 nm before photoconversion.
Thermal Stability Assay--
Rho thermal stability in the dark
was followed by monitoring the rate of A500
loss, and the appearance of A380 as a function of time, at constant temperature. Rho, in the presence of 5, 15, 30, 50, 100, and 200 µM zinc, or in the absence of zinc
(control), was thermally bleached in the dark at 55 °C. The process
was carried out in a similar way as previously described (34) over
3 h. Complete absorption spectra were recorded at 5 min intervals. Other experiments, using the same conditions but with 50 µM calcium and other metal ions, were performed to rule
out a possible unspecific charge effect. Thermodynamic parameters for
the dark thermal bleaching process were determined as a function of
temperature. This method regards thermal bleaching as an irreversible
process (34). The thermodynamic parameters Ea,
G
,
H
, and
S
were calculated from the decay rate
constants versus 1/T (Arrhenius plot) using the values for
the slopes of these plots, which were derived from least-squares
analysis. For this purpose, Rho samples in the dark and in the presence
of 50 µM zinc (or without zinc in the control sample)
were thermally bleached in the dark at different temperatures in the
range of 53 to 58 °C.
Regeneration of Rhodopsin--
The regeneration of Rho was
carried at 15 °C in the dark. 11-cis-Retinal in an
ethanolic stock solution was added to a 2.5-fold molar excess over dark
Rho in the presence of 50, 100, and 200 µM zinc or
without zinc (control). Then, the sample was illuminated for 60 s
(light-saturated), and absorption spectra were immediately recorded at
2.5-min cycles over 4 h. Rho regeneration rate was monitored at
500 nm. Samples were eventually acidified to confirm the presence of
Schiff base-linked species. Regeneration experiments were also carried
out with ROS rhodopsin samples essentially in the same way as for the
detergent-solubilized samples.
 |
RESULTS |
UV Spectroscopic Characterization of the Zn2+-Rhodopsin
Complex--
The presence of Zn2+, at a concentration of
200 µM, did not affect the spectral properties of
rhodopsin in the dark or in the light (Fig.
1). The characteristic 500-nm visible
chromophoric band remained unchanged in the Zn2+ containing
sample (compare Fig. 1, A and B). The presence of Zn2+ did not interfere with the ability of rhodopsin to
form the 380-nm band, corresponding to the MetaII intermediate, upon
illumination under light-saturating conditions (Fig. 1B)
indicating the same photobleaching behavior than the non-zinc
containing sample (Fig. 1A). In both cases the presence of a
protonated Schiff base linkage was detected by acidification of the
illuminated samples, with the corresponding shift of the band to 440 nm, confirming that the 380-nm band can be attributed to the MetaII
species, and indicating that the protonation status of the Schiff base
linkage was not affected by Zn2+ (Fig. 1B).
These results are in agreement with studies using wild-type recombinant
rhodopsin in which even in the presence of 5 mM
Zn2+ similar results were obtained (19), suggesting that
the rhodopsin-Zn2+ complex is functional, although some
differences in the transducin activation ability were detected in the
presence of zinc (19). The photobleaching efficiency is not altered by
zinc when rhodopsin is illuminated with subsaturating light intensities
(Fig. 2A). However, MetaIII
formation is decreased in the presence of zinc when compared with the
non-zinc-containing sample (Fig. 2B). Furthermore, when Rho
samples are acidified at different times after photobleaching a 2-fold
decrease under the studied conditions in the MetaII stability can be
detected (the determined t1/2 values are 30 and 15 min in the absence and in the presence of Zn2+,
respectively).

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Fig. 1.
UV/vis spectroscopic characterization of the
Zn2+-rhodopsin complex. A, rhodopsin in the
absence of Zn2+; B, rhodopsin in the presence of
200 µM Zn2+. UV/vis absorption spectra of
rhodopsin were measured in buffer A with 0.02% DM at 20 °C.
Absorption spectra were measured before (dark spectra) and
immediately after illumination for 10 s with a fiber-optic light
equipped with a >495-nm long-pass filter (light spectra).
Acid spectra were recorded after the addition of 2 N
H2SO4 (to a final pH 1.9) to the illuminated
samples. Comparison of the spectral properties of Zn2+- and
non-Zn2+-containing samples indicates that conversion of
rhodopsin to MetaII and the acidification behavior (protonation of the
Schiff base) were not affected by the formation of the
Zn2+-rhodopsin complex.
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Fig. 2.
Time-course of rhodopsin photobleaching and
MetaIII photoproduct formation. A, relative
photobleaching of rhodopsin in the absence ( ) and in the presence of
100 µM Zn2+ ( ). Samples were illuminated
with subsaturating light at 5-s intervals, and the decay of the band at
500 nm was then recorded as a function of time. B, MetaIII
formation was measured at 480 nm in the absence ( ) and in the
presence of 200 µM Zn2+ ( ). Rhodopsin
samples were illuminated for 10 s using >495-nm light and 150 watts. UV/vis scans were recorded at 15 °C in the dark each 2.5 min,
and the absorbance at 480 nm was normalized to the initial
A500 nm before illumination. Values are
mean ± S.E. of experiments performed in triplicate.
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Fourth Derivative of the UV Absorption Spectra--
Fourth
derivative spectroscopy allows separation of the different electronic
components corresponding to Phe, Tyr, and Trp residues in proteins.
This allows a detailed analysis of the structural environment of these
aromatic amino acids to be performed (35). The fourth derivative
represents the best compromise between the best resolution and the best
signal-to-noise ratio (35). This technique has been successfully
applied to the study of conformational changes in native and
recombinant proteins (36). No significant differences were detected in
the fourth derivative UV spectra of rhodopsin in the dark upon
Zn2+ addition to the sample
(Fig. 3, A and B
and Table I). The wavelengths of the
fourth derivative peaks at 285.6 and 292.2 nm and troughs at
289.0 and 295.5 nm remained unchanged upon zinc addition to the sample
(compare a traces in Fig. 3, A and B;
see Table I). However, some differences were detected after
illumination of the control rhodopsin sample in the absence of zinc
(compare a and c traces in Fig. 3A;
see Table I). These differences were increased when Zn2+
was present in the sample (compare Fig. 3, A and
B; see Table I) with larger blue-shifts in the 275-300-nm
region and increased intensity changes in some of the bands. In
contrast, peaks in the 240-275 nm underwent only minor changes when
compared with the spectra of the non-zinc-containing samples.

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Fig. 3.
Fourth derivative of the UV absorption
spectra of rhodopsin. A, rhodopsin in the absence of
Zn2+. B, rhodopsin in the presence of 200 µM Zn2+. The first spectrum corresponds to
dark rhodopsin (dark, a). Spectra recorded at different
times after illumination are shown (traces b and
c). Spectrum labeled as light (trace
c) was obtained 9 h after illumination. Fourth derivative
spectra were obtained from five coadded absorption spectra by using the
Savitzky-Golay algorithm as described under "Experimental
Procedures."
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Table I
Changes in Tyr and Trp environment
The wavelength for the maximum and minimum peak values ( max
and min) were obtained from UV fourth derivative spectra
(see Fig. 3). Tyr and Trp residues of rhodopsin (control, in the
absence of Zn2+) in the dark and after bleaching have different
wavelength location reflecting a blue-shift ( ). In the presence
of Zn2+ (200 µM Zn2+), these peaks become
further blue-shifted reflecting changes in Tyr and Trp residues of
rhodopsin to a more polar environment.
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Zn2+-induced Decrease in the Thermal Stability of
Rhodopsin--
The thermal stability of Rho in the presence of
different concentrations of Zn2+ was determined at 55 °C
in the dark. The kinetics of the thermal bleaching process of rhodopsin
in the dark was spectroscopically monitored by measuring the decay of
the 500-nm chromophoric band at constant temperature (55 °C) as a
function of time (Fig. 4A). At
this temperature, a progressive faster decay of this band was observed
as a function of Zn2+ concentration with regard to the
control sample without Zn2+. This reflects a decrease in
the thermal stability of rhodopsin in its ground state (dark state)
with increasing Zn2+ concentrations. Thus, the thermal
process is accelerated in the presence of Zn2+ with
k rate constants of 0.028 ± 0.002 min
1
(control, 0 µM Zn2+) and 0.056 ± 0.003 min
1 at 50 µM Zn2+ (Fig.
4A, inset). It corresponds to thermal bleaching
processes with t1/2 values of 24.4 ± 1.6 min and 11.8 ± 0.1 min, respectively. Thermodynamic parameters
derived from Arrhenius plots obtained from experiments performed at
different temperatures (Fig. 4B) are shown in Table
II. Activation energy (Ea), and the
H
component were significantly increased (by about 58%) for the thermal
bleaching process of rhodopsin in the presence of Zn2+. A
G
decrease of 17% was also observed in
the presence of Zn2+. In the case of the entropic
component, this showed an increase of about 63% in the zinc-containing
sample.

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Fig. 4.
Thermal bleaching of rhodopsin in DM
solution. A, kinetics of the thermal decay processes of
rhodopsin in the presence of different concentrations of
Zn2+ ( , 0 µM (control); , 15 µM; , 50 µM Zn2+) at
55 °C in 0.02% DM detergent in the dark. The time course of the
decay of the 500-nm absorption band with time was followed as a measure
of the thermal stability of rhodopsin. Inset, rate constant
(obtained from the decay curves) versus [Zn2+]
is shown for the Zn2+ concentration-dependent
decay of ground-state rhodopsin. Rate constants were determined from
the corresponding experimental data fitted to a single exponential
function. Values are mean ± S.E. (n = 3).
B, Arrhenius plots of the rate constants (k)
versus 1/T. k was determined from thermal decay
curves in the dark at different temperatures. Rhodopsin in the absence
( ) or in the presence of 50 µM Zn2+ ( ).
Values are mean ± S.E. (n = 3).
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Table II
Thermodynamic parameters for the thermal bleaching of rhodopsin
These parameters were determined from experiments in the absence and in
the presence of 50 µM Zn2+. The
Ea parameter was determined using values from the
slopes of the Arrhenius plots (Fig. 4B), which were derived
from least-squares analysis. H = Ea RT; In k =  G /
RT; and G /RT = H /RT + S
/R. All values were derived for T = 55 °C: R = 8.3 J·mol 1·K 1. These parameters
were significantly different for the Zn2+-rhodopsin complex (*,
p < 0.05).
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The previous experiments were done with Rho samples. We also performed
similar experiments with rhodopsin in its native membrane environment
(ROS membranes). Although rhodopsin in membranes is more stable than
Rho (compare Fig. 5 and Fig.
4A), a decrease in stability is also observed upon addition
of 100 µM zinc when compared with the sample with no zinc
added (the t1/2 derived for the process is 121.6 min
in the absence of zinc and 53.0 min in the presence of 100 µM zinc) (Fig. 5).

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Fig. 5.
Thermal bleaching of rhodopsin in ROS
membranes. Thermal bleaching of rhodopsin in native ROS membranes
in 20 mM HEPES (pH 7.4) containing 145 mM NaCl,
2 mM MgCl2, at 55 °C in the dark. Rhodopsin
in the absence of zinc ( ), and in the presence of 100 µM Zn2+ ( ). Values are mean ± S.E.
(n = 3).
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Specific Effect of Zn2+ on the Thermal Stability of
Rhodopsin--
In order to assess the presumed specificity of zinc on
the thermal stability of rhodopsin several other metal divalent cations were tested. To this aim the decrease of the visible band at 500 nm
with time was recorded as a measure of the thermal stability of the
protein as described under "Experimental Procedures." The results
indicate that in the presence of Cd2+, Co2+,
and Ca2+ the thermal stability of rhodopsin was similar to
that of rhodopsin alone (Fig. 6). Among
the different metal ions tested, only Cu2+ showed a
significant effect with a similar decay to that observed in the case of
Zn2+. This result is in agreement with the reported ability
of Cu2+ to compete with Zn2+ for rhodopsin
binding, both in membranes and in purified form in detergent (27). We
also tested the model compound choline to rule out a mere possible
electrostatic effect. Choline increased the thermal stability of
rhodopsin and this effect could be related to interactions of the
molecule with rhodopsin micelles.

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Fig. 6.
Thermal bleaching of rhodopsin in the
presence of different metal ions. Rhodopsin was incubated in 20 mM HEPES (pH 7.4) containing 145 mM NaCl, 2 mM MgCl2, and 0.02% DM. The metal ion was
added (as the corresponding chloride salt) to a final concentration of
50 µM. Choline was assayed at a concentration of 100 µM. The stock solution volume of metal ion added did not
exceed 1-2% of the rhodopsin sample volume. Thermal bleaching of
rhodopsin, in the dark-state at 55 °C, was followed by monitoring
the time course decay of the 500-nm absorption band. Values are
mean ± S.E. (n = 3).
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Regeneration of Rhodopsin in the Presence of
Zn2+--
The effect of zinc on pigment regeneration with
11-cis-retinal after rhodopsin photobleaching is shown in
Fig. 7A. A clear decrease is
observed for maximal rhodopsin regeneration with increasing zinc
concentration. This decrease is about 30% at 200 µM
added zinc. Higher zinc concentrations tested indicated further
reduction of the regeneration percentage (to very low regeneration
level), but the corresponding values could not be reliably obtained due to a high aggregation propensity of the samples with time. Regeneration experiments carried out with rhodopsin in ROS membranes
(non-solubilized samples, Fig. 7B) indicated a similar
behavior to that found for Rho samples (compare Fig. 7, A
and B). In the case of ROS membranes the maximal
regeneration observed was slightly lower than that observed for the Rho
samples, but the effect of zinc addition was comparable.

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Fig. 7.
Regeneration of rhodopsin in the presence of
Zn2+. A, rhodopsin in 20 mM HEPES
(pH 7.4) and 0.02% DM was preincubated with different concentrations
of Zn2+, then illuminated for 10 s, and
11-cis-retinal was immediately added. The plot shows the
time course of rhodopsin regeneration by monitoring absorption increase
at 500 nm. Values are mean ± S.E. (n = 3).
B, same as in A but with rhodopsin in ROS
membranes in 20 mM HEPES (pH 7.4) containing 145 mM NaCl, 2 mM MgCl2. Rhodopsin in
the absence of zinc ( ) and in the presence of 200 µM
Zn2+ ( ). Values are means of two different experiments.
Temperature is 20 °C.
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 |
DISCUSSION |
Previous studies have reported specific Zn2+ binding
to purified rhodopsin and disc membranes in both dark and light
conditions (27, 29). Furthermore, Zn2+ was shown to enhance
rhodopsin phosphorylation (29). Structurally bound zinc has been also
found in the recent crystal structure of rhodopsin (30-32). Despite
these reports, a detailed study of the effect of Zn2+ on
rhodopsin properties has not been previously reported. We find a clear
effect of zinc on several rhodopsin properties. Our spectroscopic
results indicate that zinc does not alter the retinal binding pocket of
rhodopsin and the formation of the MetaII intermediate. Also, the
status of the protonated Schiff base is not affected by the presence of
zinc ions.
Under physiological conditions, inactive dark state rhodopsin changes
almost entirely to the active MetaII conformation upon photon
absorption. In vitro there is an equilibrium between the MetaI and MetaII conformations that is modulated by several
experimental conditions (37-40). It has been recently shown that this
equilibrium is also influenced by salts, which shift the equilibrium to
the MetaII side (41-43). In our case and under our experimental
conditions, zinc does not seem to alter the MetaI-MetaII equilibrium.
We find that the MetaII intermediate of the Zn2+-rhodopsin
complex formed upon illumination is destabilized, and MetaIII formation is reduced. These two facts could be reconciled if we assume as a
likely interpretation that the faster MetaII decay is also accompanied by direct formation of opsin and free all-trans-retinal
(without going through a MetaIII intermediate).
Detailed analysis of the fourth derivative results indicates small but
significant differences as a consequence of illuminating the rhodopsin
sample. Changes in the amplitude of the fourth derivative peaks
together with blue-shifts of their maxima can be interpreted as
reflecting exposure of Trp and Tyr residues to a more hydrophilic microenvironment upon the rhodopsin-MetaII conformational transition. This is consistent with previous studies on UV photobleaching difference absorption spectra of ROS rhodopsin (44) and rhodopsin tryptophan mutants (45). The spectroscopic differences observed upon
rhodopsin photobleaching are increased, mainly in the Tyr-Trp region,
when Zn2+ is present in the illuminated sample. The
observed effect suggests differential Zn2+ binding to dark
and illuminated rhodopsin. Zinc ions found in the crystal structure of
rhodopsin are mainly located at the intradiscal side of the protein
(32). In particular, one of these zinc ions is located close to Tyr-96
in chain B of the receptor (32).
Thermodynamic parameters measure macroscopic changes representing the
sum of all reaction events of the Zn2+-rhodopsin complex
formation. The thermal bleaching process is accompanied by a decrease
in
G
and an increase in
H
and
S
for
the Zn2+-containing samples when compared with the values
in the absence of Zn2+. Changes in enthalpy can be
interpreted as reflecting conformational changes, and entropy effects
can be interpreted as a reordering of water molecules as proposed
previously (23, 24). Similar interpretation has been provided for the
binding of zinc to the D2-like dopamine receptor. In this latter case,
the fact that the reaction was entropy driven suggested that zinc was
essentially chelated by dopamine receptors, possibly resulting in
the reordering of hydrogen bonds (23). Thermodynamic changes could
reflect a structural rearrangement of native rhodopsin, during the
formation of the Zn2+-rhodopsin complex, without affecting
the retinal binding pocket and the protonated Schiff base linkage in
the dark state of rhodopsin. The observed effect, reflecting reordering
of water molecules, may be significant in the case of rhodopsin where a
functional role for water molecules has been recently proposed (32). In this latter study more than ten water molecules are found structurally associated with each one of the two monomers, and two of these water
molecules are in the intradiscal domain of the receptor, where four of
the seven zinc atoms are located (32).
The preceding section of the discussion has dealt with results obtained
with Rho (rhodopsin solubilized in DM detergent). However, it is known
that rhodopsin is more stable in native ROS membranes than in
detergent-solubilized form. Thus, it could be possible that the effect
observed with Rho would be different when rhodopsin was assayed in disc
membranes. Our results show that, although the rhodopsin samples are
more stable in membranes, the differential effect found in the case of
the zinc-containing sample is maintained, i.e. rhodopsin in
membranes with zinc has lower thermal stability than control samples
without zinc. Furthermore, the same behavior is detected in the case of
the pigment regeneration experiments. In that case the reduction in
rhodopsin regeneration induced by zinc is also observed when rhodopsin
is found in its native membrane. These findings argue for the
physiological relevance of the results obtained.
A structural effect of Zn2+ on rhodopsin was also detected
in a previous study, using engineered Zn2+-binding sites.
In this case Zn2+ binding to wild-type recombinant
rhodopsin resulted in a somewhat reduced transducin activation (19). We
find that zinc specifically alters several rhodopsin properties: a
definite reduction in thermal stability of ground state dark rhodopsin,
reduced stability of the active MetaII conformation, altered pathway of
photointermediates after MetaII formation, and reduced pigment
regeneration with 11-cis-retinal. These effects are
accompanied by conformational changes of the protein that are shown to
be specific of zinc and not of other divalent metal ions studied. The
effect of zinc on retinal function seems to strongly depend on the
local concentration and the ratio of free to bound cation (10). The
proposed effect of Zn2+ on visual function may be also
related to effects upon other proteins of the visual phototransduction
cascade (28) or upon some component of the retinoid cycle. Furthermore,
the proposed increase in rhodopsin phosphoylation caused by
Zn2+ (29) could be also related to other processes, like
receptor internalization, necessary for its recycling, thus changing
the normal turnover of the receptor in the membrane.