From the Institut für Medizinische Physik und
Biophysik, Universitätsklinikum Charité, Humboldt
Universität zu Berlin, Schumannstrasse 20-21, 10098 Berlin,
Germany and ¶ Departments of Ophthalmology, Pharmacology, and
Chemistry, University of Washington, Seattle, Washington 98195
Received for publication, September 20, 2002, and in revised form, October 30, 2002
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ABSTRACT |
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Vertebrate rhodopsin consists of the
apoprotein opsin and the chromophore 11-cis-retinal
covalently linked via a protonated Schiff base. Upon photoisomerization
of the chromophore to all-trans-retinal, the retinylidene
linkage hydrolyzes, and all-trans-retinal dissociates from
opsin. The pigment is eventually restored by recombining with
enzymatically produced 11-cis-retinal.
All-trans-retinal release occurs in parallel with decay of
the active form, metarhodopsin (Meta) II, in which the original Schiff
base is intact but deprotonated. The intermediates formed during Meta
II decay include Meta III, with the original Schiff base reprotonated,
and Meta III-like pseudo-photoproducts. Using an intrinsic
fluorescence assay, Fourier transform infrared spectroscopy, and
UV-visible spectroscopy, we investigated Meta II decay in native
rod disk membranes. Up to 40% of Meta III is formed without changes in
the intrinsic Trp fluorescence and thus without
all-trans-retinal release. NADPH, a cofactor for the
reduction of all-trans-retinal to
all-trans-retinol, does not accelerate Meta II decay nor
does it change the amount of Meta III formed. However, Meta III can be
photoconverted back to the Meta II signaling state. The data are
described by two quasi-irreversible pathways, leading in parallel into
Meta III or into release of all-trans-retinal.
Therefore, Meta III could be a form of rhodopsin that is
storaged away, thus regulating photoreceptor regeneration.
Phototransduction in vertebrate rods starts with the isomerization
of the 11-cis-retinal bound to opsin and the formation of
the active photoproduct, metarhodopsin
(Meta)1 II. In Meta II, the
Schiff base linkage between the all-trans-retinal and
Lys296 is still intact but deprotonated. Catalytic
activation of the G-protein, Gt or transducin, leads to a
biochemical cascade of reactions, termed phototransduction. These
reactions culminate in the hyperpolarization of the photoreceptor cells
and ultimately in changes in the rate of neurotransmitter release at
the synaptic terminus. The signaling state of Meta II is quenched
rapidly by the action of rhodopsin kinase and arrestin. Equally
important for vision is the metabolic cycle, which enables the visual
system to take away the photolyzed chromophore,
all-trans-retinal, and replace it with
11-cis-retinal, thus regenerating the pigment. The decay of
Meta II thus provides an interlink among transduction, the quenching by
phosphorylation and capping with arrestin, and regeneration (reviewed
in Ref. 1).
During the decay of Meta II, the Schiff base linkage between the
all-trans-retinal and the opsin apoprotein
(Lys296) is hydrolyzed. A side product is the bright orange
( In addition to Meta II, three distinct signaling states are described
in the literature. First, retinal-free opsin has a measurable activity
in vitro on the order of 10 Fundamental questions about the nature of the decay process of Meta II
still remain unanswered. For example, what are the intermediates in
this process from Meta II to free opsin and
all-trans-retinal? How active are they toward G-protein?
Which products determine the sensitivity of the cell during dark
adaptation and under conditions of constant illumination? What role
does Meta III play in this process? How and when does arrestin
dissociate from the active rhodopsin?
In this study we have investigated whether a post-Meta II photoproduct
with the properties of Meta III does indeed exist. To understand how
the photolyzed chromophore is handled during Meta II decay and
regeneration, it is indispensable to know whether it remains bound to
its original binding site in a large fraction of the receptor
population. Recent accounts of the vast existing literature on the
topic come to the conclusion that during the decay of Meta II into the
470-nm absorbing species, the retinal is released from its
original binding pocket (21). Using an intrinsic fluorescence assay to
measure chromophore release, Fourier transform infrared spectroscopy to
study changes in the rhodopsin structure, and UV-visible spectroscopy
to monitor formation of spectrally distinct species, we are now able to
address the problem on a new experimental basis.
Dissection of the rather complex network of decay, regeneration, and
storage likely requires kinetic analyses of the conversions (22-24).
An appropriate biochemical system is a preparation of isolated disk
membranes, which provides the correct lipid host, an important
determinant of the formation and decay of Meta II (for review see Ref.
25). This measuring system is simplified by the lack of the metabolic
machinery of regeneration, preventing complications from these
processes on the kinetics of the decay reaction. We will present direct
evidence on the formation of a stable Meta III photoproduct and propose
a role for this form of rhodopsin in physiological conditions.
Materials--
Gt Rod Disk Membrane Preparation--
Rod outer segments were
prepared from frozen bovine retinas obtained from a local
slaughterhouse by means of a discontinuous sucrose gradient method
(27). Isolated disk membranes were prepared by repeated extraction of
rod outer segments with low ionic strength buffer (5 mM
Pipes, pH 7.0, 1 mM EDTA) as described previously (28).
Rhodopsin concentration was determined from its absorption at 500 nm
using
Membrane suspensions were sonicated briefly prior to the fluorescence
and UV-visible measurements to reduce turbidity. All measurements were
performed in isotonic buffer adjusted to the chosen pH values.
Illumination Protocol--
To follow the decay of Meta II the
reaction was started by complete bleaching of the membrane suspensions
with a 150-watt fiber optic light source filtered through a heat filter
(Schott KG2) and a 495-nm long pass filter. Samples were illuminated
(15 s) at pH 6 and 20 °C, i.e. at conditions favoring
Meta II (31). In agreement with a previous study (23) we found
formation of isorhodopsin (9-cis-retinal form of rhodopsin)
even during 15 s of illumination under conditions with significant
content of Meta I in the equilibrium (alkaline pH and/or low
temperatures) (data not shown). The given pH values of the samples were
adjusted rapidly in the dark immediately after the photoactivation.
Fluorescence Measurements--
The fluorescence assay of
illuminated rhodopsin is based on the relief from the quenching of
opsin Trp fluorescence exerted by the retinal ligand bound in its
pocket (32). The assay was applied to study the Meta II decay in
isolated disk membranes. Measurements were performed on a SPEX
Fluorolog 1680 instrument. Low light intensities were used to prevent
photolysis of rhodopsin. Slit settings for the fluorometer were 0.2 nm
for excitation (at 295 nm) and 4 nm for emission. Emission spectra were
recorded between 310 and 400 nm. For the kinetic studies, data were
recorded at 330 nm. The measurements were done in stirred membrane
suspensions (750 µl, 1 µM rhodopsin). Control
experiments were done with closed shutter during decay to exclude an
effect of the excitation light on the decay reaction (data not shown).
Reaction rates were determined by a single exponential fit of the data.
UV-visible Spectroscopy--
Absorbance spectra (see Fig. 7)
were acquired with a Cary 50 spectrophotometer (Varian). Suspensions of
isolated disk membranes (100 µl, 5 µM rhodopsin) were
used in temperature-controlled microcuvettes (1-cm path length). Opsin
membranes that closely matched the light scattering properties of
isolated disk membranes were used as a reference.
Regeneration of opsin during decay of Meta II (see Fig. 5) was followed
by recording difference spectra at pH 7.5 and 33 °C within 15 min on
a Hewlett-Packard HP 8452 diode array spectrophotometer using 1-ml
cuvettes equipped with a stirrer. 11-cis-retinal (4.3 µM) was added immediately after illumination, and pH
adjustment (see above) of the membrane suspension (4.3 µM
rhodopsin) and absorbance was set to zero. Retinylidene Schiff
base content in disk membrane samples was estimated from the formation
of the 440-nm product after acidification to pH 1.9 with 1 N HCl and solubilization of the membranes in detergent (1%
dodecyl- FTIR Spectroscopy--
FTIR samples were prepared using a
centrifugation method as described (33). In ~40 µl of washed
membranes (0.3 mM rhodopsin), the pH was adjusted by a few
microliters of diluted NaOH or HCl. Subsequently the suspension was
centrifuged for 20 min at 80,000 × g, yielding a
pellet containing 2.3 mM rhodopsin (calculated from
absorption at 500 nm). The buffer solution was removed, and the pellet
was transferred to a 30-mm diameter transmission cell with two
BaF2 windows and a 3-µm Teflon spacer.
After equilibration for 1 h at 23 °C, a set of 256 transmission
spectra was recorded. After illumination with a 150-watt fiber optic
light source filtered through a heat filter (Schott KG2) and a
495-nm-long pass filter a second set of spectra was recorded. From
these two sets of spectra the Meta II minus rhodopsin difference spectrum was generated (we use the convention that the spectra of the
conversion A Meta II Decay Monitored by Fluorescence Change--
To measure the
release of all-trans-retinal from photoactivated rhodopsin,
we have adapted the intrinsic Trp fluorescence assay (34, 35), which
was later established by Farrens and Khorana (32) as a means of
monitoring all-trans-retinal release from solubilized
rhodopsin. The data in Fig. 1 show that a
similar change in Trp fluorescence of opsin can be measured in native isolated rod disk membranes. In the lipid bilayer, within milliseconds of light activation, rhodopsin reaches an equilibrium of Meta I and
Meta II tautomers. All subsequent conversions start from the pool of
Meta intermediates, which we will term Meta I/II. The fluorescence
increased concurrently with a decrease in Schiff base concentration, as
measured at 440 nm after acid denaturation. This indicates that it is
indeed the release of the chromophore that gives rise to the
fluorescence change (32). Under these conditions the absorbance
decrease leveled out at ~25% of the starting absorbance, which is
because of the presence of other all-trans-retinal Schiff
bases formed with peripheral residues other than Lys296
(N-retinylidene opsin, NRO).
The fluorescence spectra taken in the dark (rhodopsin), after
illumination for 15 s (Meta I/II), and after complete decay of
Meta I/II are shown in Fig 1B. The strong quenching of Trp fluorescence exerted by the all-trans-retinal bound in its
pocket persists with only minor change immediately after illumination. This is interesting in light of the proposed flip of the
As expected, the time course of the decay reaction depends strongly on
temperature (Fig. 2A). The
Arrhenius plot of the rate constants yields 16.0 kcal/mol as the
activation energy of the decay reaction (Fig. 2B), similar
to the 20.2 kcal/mol obtained with rhodopsin solubilized in
dodecyl- pH Dependence of Meta I/II Decay in Vitro: Formation of Meta
III--
Both the kinetics and the final level of the change of
fluorescence during Meta I/II decay depend significantly on pH (Fig. 3A). With increasing pH, the
reaction becomes slightly faster (Fig. 3D), with a half-time
of ~600 s at pH 6.0 and ~400 s above pH 8.0 (20 °C).
The maximum amplitude of the signal is reduced by a factor of 2 over
the range of pH tested (Fig. 3C). To distinguish between the
pH dependences of the actual decay reactions and of the fluorescence
monitor, the decay was analyzed in the presence of
Gt
Similar results were obtained at 33 °C (insets in Fig. 3,
A and B), except the decay is faster (half-time
of ~130 s), and the maximum fraction of Meta III formed is slightly
smaller. However, even at 33 °C, no significant loss of Meta III is
seen within 30 min, as reflected in the stable level of fluorescence.
Interestingly, the Meta II/Gt Enzymatic Reduction of All-trans-retinal Has No Effect on Meta II
Decay--
Using the fluorescence assay, we also studied the influence
of NADPH on the Meta I/II decay to opsin and Meta III. Addition of
NADPH, the cofactor of an endogenous all-trans-retinol
dehydrogenase, results in reduction of all-trans-retinal to
all-trans-retinol (see Fig. 5, B and
C). As seen in Fig. 4, neither
the time course nor the final level of the fluorescence change are
influenced by NADPH. Under the conditions of the experiments (pH 7.5, 20 °C), a significant fraction of Meta III is formed that is seen directly in the larger fluorescence increase in the presence of Gt Decay of Meta II in the Presence of 11-cis-retinal--
To probe
the accessibility of the retinal binding pocket in the decay products,
regeneration with 11-cis-retinal during Meta I/II decay was
followed by UV-visible difference spectroscopy (Fig.
5, A-C). The
records reflect an increase in absorbance around 500 nm and a parallel
decrease around 380 nm, consistent with the formation of rhodopsin at
the expense of free 11-cis-retinal. In the control membranes
(Fig. 5A), the absorption maximum of rhodopsin is apparently
blue-shifted because of the contribution of other photoproducts with
similar absorption. These include Meta III, as defined above, and
protonated Schiff bases (e.g. NRO; see "Discussion"),
which prevent a quantitative evaluation of the spectra. However,
despite the complexity of the decay reactions the spectra maintain an
isosbestic point. This implies that the species involved exhibit only
two distinct spectra. This condition is met when all conversions occur
with the same apparent rate (see "Discussion").
In Fig. 5, B and C the influence of NADPH and
Gt
Consistent with the results obtained above (see Figs. 3 and 4), more
all-trans-retinal is released and eventually reduced in the
presence of Gt Identification of Meta III by FTIR Spectroscopy--
Meta I/II
decay was also followed using FTIR difference spectroscopy. The first
record in Fig. 6, A and
B is the Meta II minus rhodopsin difference spectrum (see
"Experimental Procedures"). At pH 7.5 and 23 °C, the Meta I/Meta
II equilibrium is to 70% and at pH 5.9 and 23 °C, to more than 90%
on the Meta II side. Thus, the spectra are dominated by typical Meta II
features, including bands at 1768 cm
At both pH 7.5 (Fig. 6A) and pH 5.9 (Fig. 6B),
the decay is virtually complete after 32 min, as indicated by the
absence of the Meta II features. This difference spectrum represents a
mixture of the opsin/all-trans-retinal minus rhodopsin and
Meta III minus rhodopsin difference spectra (see "Experimental
Procedures"). There is a weakly expressed feature that arises
specifically at pH > 6.0, namely the positive band at 1348 cm
In contrast to the observation at pH 7.5, no photolysis products are
seen at pH 5.9, in conditions when virtually no Meta III is present
(Fig. 6B). This observation further supports the existence
of Meta III as a decay product. This species is the major (or even the
only) decay product that undergoes photoreversal to Meta II.
Gt
The overall time course of the decay products can be followed from the
spectral changes (Fig. 7A), yielding the same common rate
for all conversions, as was seen in the fluorescence and FTIR
experiments described above. Meta III can be identified spectrally using its photoconvertibility to Meta II (see Fig. 6A). The
experiment starts with the completely decayed sample (Fig.
7A, trace f). Addition of
Gt The visual process in the vertebrate retina depends not only on
the signal transduction machinery in rods and cones and on neuronal
processing but also on an extended network of metabolic reactions. They
produce the necessary energy, intermediates, and 11-cis-retinal to regenerate bleached visual pigments to
their light-sensitive ground state. This regeneration process was
underscored recently, when it was shown that a direct, light-induced
reversal from all-trans- to 11-cis-retinal
(so-called photoregeneration) does not occur to any measurable degree
(37). Light absorption of the active Meta II state of the rhodopsin in
the rods does not, as in cyclic proteins such as sensory rhodopsin in
bacteria, lead back to photoregenerated rhodopsin but to new forms with properties different from native rhodopsin (37). Replacement of
all-trans-retinal with fresh 11-cis-retinal
requires that the retinylidene Schiff base be hydrolyzed and the
all-trans-retinal be released from the opsin chromophore
pocket. All-trans-retinal release occurs by decay of the
active, G-protein binding Meta II intermediate, in which the Schiff
base is intact but deprotonated.
The conversions of Meta II have been the subject of intense
investigations, mainly in intact retina and in rod outer segment preparations. An exceedingly complex picture emerged from these studies, introducing elements of retinal metabolism (e.g.
all-trans-retinol dehydrogenase, the retina-specific ABC
transporter) and regulation (including Gt, rhodopsin
kinase, and arrestin). Even in membrane preparations, which contain
only the pigment and no additional protein, several products of thermal
decay appear in variable amounts. These include non-covalent
opsin/all-trans-retinal complexes (16, 20) and Meta III-like
pseudo-photoproducts (N-retinylidene-opsin, i.e.
opsin/all-trans-retinal complexes with Schiff bases; see Refs. 5 and 11). All these forms have the potential to serve as a
"storage" for the photolyzed chromophore.
The goal of this study was to identify some of the intermediates that
arise during Meta II decay. Using a preparation of isolated photoreceptor disk membranes and biophysical techniques, we present conclusive evidence for the parallel decay of the active Meta II into
two species: (i) Meta III, an inactive storage form with all-trans-retinal bound in or near the original binding
site, and (ii) an all-trans-retinal opsin complex, in which
the all-trans-retinal has been translocated to second
binding site(s). Both products are formed in parallel through
essentially irreversible pathways, i.e. they remain present
at constant concentration when the decay reaction has gone to
completion. We will first discuss the species involved and then the way
they are concatenated.
Metarhodopsin III--
Definitions of Meta III based on visible
absorbance around 460-470 nm are ambiguous because of the various
products that form on a quite different chemical basis but with similar
UV-visible properties. The UV-visible spectrophotometric results are
consistent with the presence of a protonated Schiff base. This, by
definition, is the classical Meta III product (see Refs. 21 and 25).
However, previous work has suggested the existence of protonated Schiff bases that are formed with Lys residues other than the active Lys296, and the photolyzed chromophore is also known to
form adducts with phosphatidyl-ethanolamine (38). Our results, based on
the combined application of fluorescence and FTIR assays, allow us to
identify the decay products and to separate products that are isochromic in the UV-visible range. Therefore, we now define Meta III
as the fraction of decay products that (i) does not go through the
fluorescence change, (ii) is resistant to NADPH-dependent reduction, and (iii) is photoconverted to a Meta II-like intermediate (37). Thus, the most obvious assumption is that Meta III contains the
all-trans-retinal in the original site (37).
All-trans-retinal/Opsin Complexes--
According to the
classical scheme, the main path of Meta II decay leads via Schiff base
hydrolysis to opsin and the photoisomerized chromophore,
all-trans-retinal. There is general agreement that this
conversion is irreversible (39, 40). In apparent contradiction to this
notion, recombination of purified opsin with exogenous all-trans-retinal leads to substantial activity toward the
G-protein (16-20), rhodopsin kinase (11, 41), and arrestin (11).
However, the activity toward Gt was also seen with a
permethylated active site (16), and regeneration with
11-cis-retinal was not inhibited in the presence of
all-trans-retinal added in excess (20). This has led to the
conclusion that a non-covalent complex is formed in which the
all-trans-retinal has virtually no access to the original
binding site occupied in Meta II (16, 20). Whether the residual
activity, which is measured after completion of Meta II decay (data not
shown) (42), is because of the formation of similar non-covalent
complexes remains to be elucidated.
Reaction Scheme--
To derive a reaction model for Meta II decay,
we start from two experimental findings: (i) Schiff base hydrolysis
and/or all-trans-retinal release are irreversible, and (ii)
Meta III is not affected by NADPH-dependent reduction of
all-trans-retinal to all-trans-retinol (Fig. 4).
We therefore conclude that the decay of Meta I/II comprises two
parallel conversions that are irreversible within the time window (1 h)
of the experiments (shown in Reaction 1).
Kinetic theory says that parallel reactions proceed with one common
observed reaction rate. In the simplest case (shown in Reaction
2), the observed reaction rate
(kobs) is the sum of the individual reaction rates (kobs = k1 + k2) (43).
Thus Meta III formation and all-trans-retinal release must
necessarily occur with the same observed reaction rate. The scheme also
implies that the relative amounts at which the two species are formed
depend on kinetic competition, i.e. the intrinsically faster
pathway produces the higher yield. This is, under our conditions, always the formation of all-trans-retinal/opsin. The higher
the pH, the more the decay runs into Meta III formation, with a maximum of ~35% at pH > 8.0 (inset of Fig. 3). The reaction
scheme (Reaction 1) does not exclude more complicated schemes that
arose from detailed analyses of UV-visible difference spectra (24). In
particular, we cannot exclude the existence of isochromic species
formed during the decay of Meta II. However, the results of this study
expand existing approaches by showing that a substantial amount of the species formed is Meta III, in which the chromophore is still bound to
its original binding pocket. Meta III is a stable species, formed in a
quasi-irreversible, although pH-dependent, manner. Even at
33 °C, the stable level of fluorescence (Fig. 3) indicates that the
Meta III, as defined above, is stable within the physiologically relevant time range of 30 min.
The stability of Meta III is in apparent conflict with the partial loss
within minutes of Meta III-like absorbance in membrane preparations
(23, 24). We can now suggest that these unstable species were not Meta
III but rather NRO-like species, as discussed above. Under cellular
conditions, Meta III eventually decays to opsin and
all-trans-retinal (2-5). Here, other factors
(e.g. Gt) come into play that may prevent (see
this work and Ref. 10) or even reverse (4, 42) Meta III formation. In
addition, direct hydrolysis of the Schiff base in Meta III itself was
suggested (3, 5, 44) to explain the loss of 470-nm absorbance.
Mechanism of Schiff Base Hydrolysis--
The decay reaction
proceeds from a pool of Meta I/II. Because these species rapidly
interconvert on a millisecond time scale, our kinetic data do not allow
us to decide whether the decay reactions start from Meta I or Meta II.
It is known that hydrolysis of free Schiff bases requires their
protonation. If this is true for the all-trans-retinal
Schiff base in rhodopsin, one would argue that hydrolysis cannot start
from Meta II. On the other hand Gt Structural Basis of the Fluorescence Change--
Rhodopsin
contains five Trp residues in positions 35, 126, 161, 175, and 265 (Fig. 8A). These residues are
located in different parts of the molecule and play different roles.
Trp126 is sequestered within a hydrophilic part of the
transmembrane bundle of helices (Fig. 8, A and
B), in close vicinity of Glu122. The
substitution of this Glu residue resulted in changes in both the decay
rate of Meta II and the rate of regeneration (48, 49). In addition, it
was postulated that changes in helix III are associated with formation
of Meta II (reviewed in Ref. 50). Trp126 is too distant
from the chromophore to be impacted when chromophore is released.
Trp265, located on helix VI, which is critical for the
activation process (50), is closest to the Physiological Implications--
Our finding that
NADPH-dependent removal of all-trans-retinal
does not accelerate the decay of Meta II extends previous results that
the reduction of all-trans-retinal limits the rate of
regeneration in mice (8). The present study now provides the evidence
that the actual rate-limiting step for the reduction, and thus the pacemaker for the overall regeneration process in the vertebrate retina, is an intrinsic transformation in the active photopigment itself. It is the step that makes all-trans-retinal
available for all-trans-retinal reduction and/or transport
(reviewed in Ref. 1). Parallel to this reaction, the Meta III form
arises to an amount dictated by kinetic competition. Meta III has the properties to be a storage form of rhodopsin that has a photoisomerized chromophore but is inactive for Gt stimulation. Factors
that can regulate the level of Meta III formation include
Gt and green light. Storing aside rhodopsin that is in Meta
III could lower the probability of quantum catch of the rod cell, if
the quantum efficiency of Meta III is reduced compared with rhodopsin.
Reducing further excitation of already light-saturated photoreceptor
cells would contribute significantly to light adaptation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
max ~ 470 nm) Meta III, which slowly replaces
the pale yellow color of the Meta II product (
max = 380 nm). Although it is not clear whether Meta III represents one
homogeneous species, one may define it as the late product in which the
chromophore is still bound to its original binding site. In the
isolated retina and in intact rod outer segment preparations, Meta III
eventually decays into all-trans-retinal and opsin (2-5).
Compared with Meta II, opsin and Meta III adopt a more rhodopsin-like
conformation (6, 7) and are either inactive or marginally active toward
the G-protein. In the native system of mice, there is evidence that
all-trans-retinal can accumulate (8), although the nature of
the product between the photoisomerized chromophore and opsin has not
been elucidated. In vivo, the decay of the Meta species is
the first step in the retinoid cycle, and all-trans-retinal
is released from opsin directly to either the cytoplasmic surface or to
the intradiskal side, from where it is flipped to the cytoplasm
(9). Next, all-trans-retinal is reduced to
all-trans-retinol by a dehydrogenase (reviewed in Ref. 1).
Moreover, all proteins that interact with photoactivated rhodopsin
(e.g. Gt, rhodopsin kinase, and arrestin) are
expected to inhibit the decay reaction (10-13).
6 that of Meta II
(14); at very low pH, opsin changes its conformation toward an active
state (15). Second, a signaling state is achieved by formation of a
reversible Schiff base between all-trans-retinal and
peripheral lysine group(s) of opsin, leading (preferentially at pH > 8.0) to reversible pseudo-photoproducts (11, 16). These
products interact with arrestin and rhodopsin kinase, but an
interaction of these products with Gt was not measured.
Finally, a non-covalent opsin/all-trans-retinal complex can
form between opsin and all-trans-retinal (16-20). It has a
substantial activity toward the G-protein, three to four orders of
magnitude higher than the empty apoprotein. In this complex, the
binding site for the all-trans-retinal appears to be
different from that of Meta II (16, 20).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(340
350)-high
affinity analog-peptide (Gt
-HAA), VLEDLKSCGLF (26), was
synthesized by Dr. Peter Henklein (Humboldt University).
11-cis-retinal was a generous gift from Rosalie Crouch and
NEI, National Institutes of Health; the concentration was determined at
380 nm using
= 24.400 M
1cm
1. Isotonic buffer contains
130 mM NaCl, 1 mM MgCl2, and 20 mM bis-tris-propane adjusted to pH 7.5, if not otherwise
noted. Phosphate buffer contains 50 mM potassium phosphate,
80 mM NaCl, and 1 mM MgCl2, pH
6.5.
= 40.600 M
1cm
1
(29). Opsin membranes were prepared by retinal extraction with bovine
serum albumin (fatty acid-free) and hydroxylamine using illuminated
disk membranes as described previously (20, 30). Opsin concentration
was determined from its absorption at 280 nm using
= 81.200 M
1 cm
1 (19). All membrane
suspensions were stored at
80 °C in isotonic buffer containing 0.3 M sucrose.
-maltoside).
B are calculated as B
A and termed B
A
difference spectrum). The sample was then allowed to decay for 2 h
at pH 5.9 and 7.5. The decay was followed by recording sets of spectra
every 2 min after the first illumination. After 2 h, final spectra
of the decay product were taken, and the sample was illuminated with
green light (495 nm) for 30 s. Subtraction of the decay product
spectra ("after" minus "before" illumination) yielded the Meta
II minus Meta III FTIR difference spectrum.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Change of intrinsic fluorescence monitoring
the decay of photoactivated rhodopsin (R*) in bovine rod disk
membranes. A, kinetics of fluorescence increase
(a) after saturating photoactivation of rhodopsin (1 µM) in disk membranes ( ex = 295 nm,
em = 330 nm, pH 6.5, 20 °C). The amount of protonated
Schiff base left at the indicated times (b) was estimated
from the absorption at 440 nm after acid denaturation (pH 1.9) and
solubilization of the samples. B, fluorescence emission
spectra (
ex = 295 nm) of rhodopsin membranes (pH 6.5, 20 °C) in the dark (a), immediately after photoactivation
(b) and after 60 min (c).
-ionone ring out of its location in the ground state (see "Discussion"). After complete decay of Meta I/II, the fluorescence of the resulting opsin is twice as large, as compared with the initial Meta II (the
dominant species at pH 6.5).
-maltoside (32). The results validate the rhodopsin
fluorescence assay of all-trans-retinal release in
membrane-bound rhodopsin (Fig. 1A).
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Fig. 2.
Temperature dependence of fluorescence
change. A, fluorescence increase after photoactivation
of rhodopsin (1 µM) at different temperatures (pH 6.5).
B, Arrhenius plot of the rates of fluorescence increase.
Solid line is a linear least squares fit to the data.
-HAA. This peptide binds to the Meta II conformation
(26, 36). The Gt
-HAA strongly reduces the pH dependence
of the fluorescence change (Fig. 3, B and C).
Because of the fact that Meta II decays quantitatively to opsin in the
presence of the peptide (see below), the residual pH dependence is
likely reflecting the pH effect on Trp fluorescence. The difference
between the fluorescence changes with and without Gt
-HAA
yields the relative fraction of a photoproduct in which the Trp
fluorescence is quenched persistently. From the resulting curve, shown
in the inset of Fig. 3C, it can be estimated that
this photoproduct is formed with an apparent pKa
of 7.0. Under the conditions of the experiments, its maximum fraction is 33% of the initially bleached rhodopsin. Anticipating the results described below, this photoproduct contains
all-trans-retinal bound via a protonated Schiff base to
Lys296 and is here defined as Meta III.
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Fig. 3.
pH dependence of fluorescence change.
Families of fluorescence traces measured at 20 °C and the
indicated pH in the absence (A) and in the presence
(B) of 100 µM Gt -HAA. Samples
(1 µM rhodopsin in disk membranes) were illuminated at pH
6.0 for 15 s and immediately adjusted to the respective pHs.
Insets, fluorescence traces measured at 33 °C and pH 6.4 (red traces) and pH 8.1 (black traces).
C, pH dependence of the maximum amplitude of the
fluorescence increase (20 °C) with and without Gt
-HAA
(open and closed circles, respectively).
Solid lines are fits to the titration data using linear
hyperbolic functions. The difference between the fitted curves yield
the pH dependence of Meta III formation (inset).
D, pH dependence of the reaction rates of fluorescence
increase (20 °C) with and without Gt
-HAA
(open and closed circles, respectively).
-HAA complex decays above
pH 6.0 in a pH-independent manner, with a half-time of 620 ± 17 (S.E.) s (20 °C). A similar rate is found for the Meta I/II decay
without the peptide between pH 6.0 and 6.5. Although the peptide mimics the effect of Gt on Meta II stabilization, it does not
significantly influence the fluorescence change and, hence, hydrolysis
of the Schiff base and loss of the chromophore from the binding pocket (see "Discussion").
-HAA-peptide (see Figs. 3 and 4). The lack of any
additional opsin formation in the presence of NADPH indicates a
(quasi-)irreversible formation of Meta III.
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Fig. 4.
Effect of enzymatic reduction of
all-trans-retinal on the changes in fluorescence.
Fluorescence increase of illuminated disk membranes (pH 7.5, 20 °C)
in the absence of NADPH (black), in the presence of 70 µM NADPH (red), and in the presence of both 70 µM NADPH and 100 µM Gt -HAA
(blue), respectively.
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Fig. 5.
UV-visible difference spectra recorded during
decay of Meta II with concurrent regeneration of opsin to rhodopsin
with 11-cis-retinal. Time course of spectral
changes during rhodopsin decay and regeneration at pH 7.5 and 33 °C
without NADPH (A), with 70 µM NADPH
(B), and with both Gt -HAA-peptide (100 µM) and NADPH (70 µM) (C). Disk
membranes (4.3 µM rhodopsin) were fully bleached at pH
6.0 and adjusted immediately to pH 7.5. After addition of equimolar
11-cis-retinal, the absorption was set to zero. The
difference spectra were taken at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 10, 12, 14, and 15 min, respectively.
-HAA-peptide on Meta I/II decay and opsin
regeneration, respectively, is shown. Reduction of
all-trans-retinal by endogenous all-trans-retinol dehydrogenase is seen directly as an increase in absorbance at 320 nm
(all-trans-retinol formation). The loss of an isosbestic point in these spectra indicates that the additional reaction introduced, namely the reduction of all-trans-retinal after
its release from photoactivated rhodopsin, becomes kinetically visible. This is readily explained by a transient accumulation of free all-trans-retinal and/or its complexes with opsin
(all-trans-retinal/opsin and NRO). Eventually, the pool of
released all-trans-retinal is reduced, which is seen as a
further decrease in absorbance at 380 nm
(all-trans-retinal/opsin). In addition, the absorption shifts toward 500 nm in the late spectra, indicating complete removal
of transiently formed NRO.
-HAA-peptide, which makes opsin available that would have otherwise escaped into the Meta III storage (further decrease in 380 nm absorption). Any additional absorption change in the
470-500-nm range is masked by a simultaneous loss of Meta III, but a
final shift toward 500 nm is evident (Fig. 5C).
1 and 1745 cm
1 (indicating a change in hydrogen bonding of
Asp83 and Glu122) and 1713 cm
1
(indicating the protonation of the Schiff base counterion,
Glu113).
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Fig. 6.
Characterization of Meta III by FTIR
spectroscopy. FTIR difference spectra were recorded immediately
after Meta II formation (Meta II minus rhodopsin) and after the times
indicated (decay products minus rhodopsin) at 23 °C, pH 7.5 (A) and pH 5.9 (B). A second illumination with
green light after 120 min yields the difference spectra between a form
of Meta II (see text and Ref. 37) and Meta III (Meta II-like minus Meta
III).
1, which was recently assigned to Meta III formation
(37). When the sample is illuminated after 2 h of decay, a Meta II
minus Meta III difference spectrum can be recorded. This spectrum shows the Meta III band at 1348 cm
1 with negative polarity and
the features around 1700 to 1750 cm
1 with positive
polarity, indicating the formation of Meta II (or Meta II-like product
in terms of protein conformation) (Fig. 6A), as described
previously (37). This identifies Meta III as the only decay product
that can be converted by light to the active species Meta II.
-HAA-Peptide Prevents Meta III
Formation--
UV-visible spectroscopic data on the formation and
decay of Meta I/II are shown in Fig. 7.
Illumination of rhodopsin at pH 6.0 and 20 °C results in an almost
quantitative formation of Meta II (Fig. 7A, traces
a and b). After adjusting the same sample to pH 8.0 (to
avoid photoconversion of Meta I to isorhodopsin; see "Experimental
Procedures"), Meta I is formed rapidly at the expense of Meta II
(Fig. 7A, trace c). The subsequent decay of Meta
I/II results in only minor spectral changes (Fig. 7A,
traces c-f) because of the spectral overlap
between reactants and products. Species in the 360-380-nm range
include Meta II, free all-trans-retinal, and
opsin/all-trans-retinal complex with unprotonated Schiff
base. In the 460-480-nm range are Meta I, Meta III, and all other
protonated Schiff bases (reviewed in Ref. 25).
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Fig. 7.
Characterization of Meta II decay products by
UV-visible spectroscopy. The measurements were carried out as
described under "Experimental Procedures." A,
spectrophotometric characterization of the formation and decay of Meta
I/II. Absorption spectrum of a disk membrane suspension in the dark
(a) and after 15 s of illumination at pH 6.0 (b). After adjusting the sample to pH 8.0 by addition of
NaOH, further spectra were recorded 1.5 (c), 5 (d), 15 (e), and 35 (f) min after
illumination of the suspension. Note the shift of the baseline upon
addition of NaOH. B, after complete decay of Meta II (40 min) 100 µM Gt -HAA was added to the sample
shown in A, and spectra were taken before (a) and
after (b) a second illumination of the suspension with green
light. A second sample was allowed to decay at pH 8.0 in the presence
of 100 µM Gt
-HAA-peptide, and a spectrum
was taken after 45 min (c).
-HAA-peptide yields trace a in Fig.
7B; it coincides with trace f in Fig.
7A apart from a small dilution effect. Subsequent illumination results in a spectrum (Fig. 7B, trace
b) that is very similar to the spectrum obtained from an aliquot
that had decayed in the presence of Gt
-HAA (Fig.
7B, trace c). The experiment confirms that the
peptide blocks formation of Meta III.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Reaction 1.
View larger version (5K):
[in a new window]
Reaction 2.
-HAA-peptide stabilizes Meta II but has only a minor effect on
all-trans-retinal release and thus Schiff base hydrolysis
(see Figs. 3, 4, and 7). A solution for the problem would be an
additional active, peptide-binding intermediate with reprotonated
Schiff base (45-47) as the actual precursor of hydrolysis. In the
scheme (Eq. 1), we consider all these species in a pool of
conformations that form and equilibrate within milliseconds after light
activation. They all have in common the photoisomerized chromophore,
all-trans-retinal, that is still in its original binding
site, with the Schiff base bond to Lys296 intact.
-ionone of the
chromophore (51) (Fig. 8, A and C). By virtue of
this location and isomerization, Trp265 should undergo the
most profound changes during the activation process. It was noted that
isomerization of the chromophore may cause changes in the location of
the
-ionone during formation of Meta II (52, 53). Based on molecular
dynamics, Röhrig et al. (54) proposed that such
changes occur even earlier, just nanoseconds after photon absorption.
In the new orientation, Trp265 comes closer to
Phe261, and such interaction would have a similar quenching
effect on the Trp emission as one provided by
-ionone in rhodopsin.
Therefore, changes in fluorescence during formation of Meta II are
small (only ~10% difference between Meta II and rhodopsin) and could be attributed to Trp265 and possibly Trp126, in
agreement with Trp absorbance studies (55). Upon chromophore release,
large fluorescence changes could result from relaxation of
Trp265, now in the empty binding pocket of rhodopsin. Meta
III would have preserved quenching properties either by the chromophore or by Phe261. These assertions require further experimental
proof.
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Fig. 8.
Model of rhodopsin. The model is based
on a high resolution crystal structure (1HZX) (56). a,
rhodopsin with indicated positions of Trp residues. b,
close-up of the vicinity of Trp126. c, close-up
of the vicinity of Trp265.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Peter Henklein for providing the
peptide. We also thank Jana Engelmann and Ingrid Semjonow for excellent
technical assistance. We thank S
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 366) and from the Fonds der Chemischen Industrie (to K. P. H.), National Institutes of Health Grant EY09339, a grant from Research to Prevent Blindness, Inc. (RPB) (to the Dept. of Ophthalmology at the University of Washington), and grants from Foundation Fighting Blindness, Inc. and the E. K. Bishop Foundation (to K. P.).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.
§ To whom correspondence should be addressed. Tel.: 49-30-450-524111; Fax: 49-30-450-524952; E-mail: martin.heck@charite.de.
RPB Senior Investigator and recipient of the Humboldt Research
Award for Senior U. S. Scientists.
Published, JBC Papers in Press, November 9, 2002, DOI 10.1074/jbc.M209675200
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ABBREVIATIONS |
---|
The abbreviations used are:
Meta, metarhodopsin;
FTIR, Fourier transform infrared;
Gt, G-protein of the rod, transducin;
Gt-HAA, Gt
(340
350)-high affinity analog;
NRO, N-retinylidene opsin;
bis-tris-propane, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol;
Pipes, 1,4-piperazinediethanesulfonic acid.
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