From the Centre de Biotecnologia Molecular (CEBIM), Departament d'Enginyeria Química, Universitat Politècnica de Catalunya, Colom 1, 08222 Terrassa, Catalonia, Spain
Received for publication, October 25, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Naturally occurring point mutations in the opsin
gene cause the retinal diseases retinitis pigmentosa and congenital
night blindness. Although these diseases involve similar mutations in very close locations in rhodopsin, their progression is very different, with retinitis pigmentosa being severe and causing retinal
degeneration. We report on the expression and characterization of the
recently found T94I mutation associated with congenital night
blindness, in the second transmembrane helix or rhodopsin, and
mutations at the same site. T94I mutant rhodopsin folded properly and
was able to bind 11-cis-retinal to form chromophore, but it
showed a blue-shifted visible band at 478 nm and reduced molar
extinction coefficient. Furthermore, T94I showed dramatically reduced
thermal stability, extremely long lived metarhodopsin II
intermediate, and highly increased reactivity toward hydroxylamine in
the dark, when compared with wild type rhodopsin. The results are
consistent with the location of Thr-94 in close proximity to Glu-113
counterion in the vicinity of the Schiff base linkage and suggest a
role for this residue in maintaining the correct dark inactive
conformation of the receptor. The reported results, together with
previously published data on the other two known congenital night
blindness mutants, suggest that the molecular mechanism underlying
this disease may not be structural misfolding, as proposed for
retinitis pigmentosa mutants, but abnormal functioning of the receptor
by decreased thermal stability and/or constitutive activity.
Naturally occurring mutations in rhodopsin (most of them single
amino acid replacements) are associated with retinal disease. Most of
these are the cause of retinitis pigmentosa
(RP),1 a group of inherited
retinal degenerative diseases (1-3) that leads to blindness by causing
photoreceptor cell death (4). Over 100 mutations have been found to
date in the opsin gene associated with RP, most of them being inherited
as an autosomal dominant trait (1). These are located in all the three
domains of rhodopsin, namely the intradiscal, the transmembrane, and
the cytoplasmic domains of the protein (5). Mutations in the
transmembrane and intradiscal domains of rhodopsin that cause RP have
been shown to cause misfolding of the mutant proteins (6-8). Only a
very small number of mutations have been associated with the retinal disease characterized by a congenital night blindness (CNB) phenotype. CNB appears to be a stable condition that does not seem to cause photoreceptor degeneration resulting mainly in night vision impairment. Two of these mutations were previously studied, namely G90D (9) and
A292E (10) in transmembrane helices II and VII of rhodopsin, respectively. The mechanism of action of the G90D and A292E mutations was proposed to be persistent activation of the phototransduction pathway by constitutive activity of the mutant proteins (9-11). Another possible explanation for the observed G90D mutant phenotype has
been proposed, i.e. enhanced rate of thermal isomerization due to lowered activation energy caused by the mutation (12, 13). More
recently, a third mutation associated with CNB, T94I, in the second
transmembrane helix of rhodopsin, has been described (14). The study of
mutations associated with CNB can provide important information about
the differences between RP and CNB at the molecular level. A region of
rhodopsin in the transmembrane helix II, toward the intradiscal site of
the protein, is particularly interesting to investigate these
differences. In this region, similar mutations in close proximity in
rhodopsin cause RP and CNB: G87D (RP), G89D (RP), G90D (CNB), and T94I
(CNB) (Fig. 1). Thus, although these
diseases involve similar mutations in very close locations in
rhodopsin, it is still a puzzle that their progression is very
different, with RP being responsible for a severe phenotype causing
retinal degeneration.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Secondary structure model of
rhodopsin. Two sites of RP (yellow) and CNB
(green) rhodopsin mutants in transmembrane helix II in a
secondary structure model of rhodopsin. The lengths of the
transmembrane helices (boxed) are shown as in the crystal
structure of rhodopsin (31). The helical region sequence amino acid
311-321 in the cytoplasmic region is also boxed as in the
crystal structure (31). The secondary structure shows, in addition, the
two palmitoyl groups (wiggly lines) at Cys-322 and Cys-323,
a disulfide bond between Cys-110 and Cys-187 (bolder black
circles), and the site of the PSB at Lys-296 (in blue)
and the counterion Glu-113 in helix III (in red). The
mutants related to retinal diseases RP and CNB are as follows: in
yellow circles Val-87 and Gly-89 (sites of the RP V87D and
G89D mutations), and in green squares Gly-90 and Thr-94
(sites of the CNB G90D and T94I mutants, the subject of the present
study). Additional mutations now made at the 94 site are T94D, T94S,
and T94K.
We report here on the expression and characterization of the T94I CNB
mutant and other mutations at the 94 site, T94D, T94S, and T94K. Our
results are consistent with the location of Thr-94 close to the retinal
Schiff base linkage and suggest a role for this residue in keeping the
correct structure of the retinal binding pocket of rhodopsin. A very
unusual property is the extremely slow Meta II decay process of the
mutant T94I. Although no misfolding is detected for this mutant, the
high hydroxylamine reactivity indicates that its dark structure is
significantly altered. Also, the low thermal stability in the dark may
be relevant to the molecular mechanism of CNB (12, 13). The results
obtained for the Thr-94 mutants highlight the tight coupling between
the structural domains of rhodopsin in in vivo folding of
this receptor. They also provide evidence that the molecular mechanism
of CNB does not involve misfolding of the mutated proteins, as has been
proposed (or shown) for many RP mutations, but it may be related to
conformational stability changes of the mutant receptors.
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EXPERIMENTAL PROCEDURES |
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Materials-- 11-cis-Retinal was purified from illuminated all-trans-retinal using a modified version of a protocol described previously (15). Dodecyl maltoside (DM) was from Anatrace (Maumee, OH). Antirhodopsin monoclonal antibody rho-1D4 was obtained from the National Cell Culture Center (Minneapolis, MN) and was coupled to cyanogen bromide-activated Sepharose 4B (Sigma). The binding capacity of the resulting antibody Sepharose was 0.6 µg/µl of the beads.
The buffers used are as follows: buffer A, 1.8 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.2; buffer B, buffer A plus 5 mM ATP, 5 mM MgCl2, 0.1 mM phenylmethylsulfonyl fluoride, and 1% (w/v) DM; buffer C, buffer A plus 0.05% DM; buffer D, 2 mM NaH2PO4, pH 6.0, 0.05% DM; buffer E, buffer D plus 150 mM NaCl.
Cloning, Expression, and Purification of Thr-94
Mutants--
Construction of the mutant opsin genes at position 94 was
carried out by replacement of the BglII-NcoI
restriction fragment in the synthetic opsin gene in the pMT4 vector
(16). The cloning strategy involved the ligation of a large fragment
from EcoRI/NotI digestion (5128 bp), a small
fragment from EcoRI/BglII digestion (251 bp), and
a small fragment from NcoI/NotI/XmnI
digestion (752 bp) with the appropriate annealed synthetic
oligonucleotide duplexes. The synthetic oligonucleotides used were
51-mer (both strands), with the following sequence for the wild type
for the top strand 5'-gAT CTC TTC Atg gTC TTC ggT ggC TTC
ACC ACC ACC CTC TAC ACC TCT CTC-3', and 5'-CTAg gAg AgA ggT
gTA gAg ggT ggT gAA gCC ACC gAA gAC CAT gAA gA-3' for the bottom
strand. The mutants contained the following codon changes at the
threonine codon (ACC) underlined above: T94I, ACC ATC; T94D, ACC
GAT; T94S, ACC
TCT; and T94K, ACC
AAG. Plasmid DNAs were
analyzed first by restriction analysis (using NcoI enzyme)
followed by DNA sequencing by the dideoxy chain-terminated method.
The wild type and the mutant genes were expressed in transiently transfected monkey kidney cells (COS-1) as described (17). Transfected cells were harvested 50 h from the time of DNA incubation, washed twice with buffer A, and treated with 50 µM 11-cis-retinal for 12 h at 4 °C for reconstitution. The cells were then solubilized in buffer B but in the absence of 5 mM ATP and 5 mM MgCl2 (2 ml/plate) for 1 h at 4 °C. Regeneration with all-trans-retinal was carried out by addition of 50 µM purified all-trans-retinal to the COS-cell suspension for 12 h at 4 °C. Purification was carried out using rho-1D4 Sepharose in a newly designed glass microcolumn (5.6 cm × 3 mm internal diameter). The packed bed volume of the column was 400 µl of rho-1D4-Sepharose resin (binding capacity of 0.6 µg/µl). The column was first equilibrated with 10 ml of buffer A containing 1% DM, and the protein was loaded onto the column for ~2 h at a rate of 100 µl/min. The column was subsequently washed with 10 ml of buffer C, followed by 10-ml wash with buffer D at the same rate, and eluted in buffer D containing 100 µM of C'1-9 peptide, at a rate of 60 µl/min. Twenty four elution fractions, about 400 µl/each, were collected. The first 12 fractions eluted were with low salt elution (buffer D), and the next 12 fractions were with high salt elution (buffer E).
UV-visible Absorption Spectroscopy--
Absorption spectra were
recorded with a PerkinElmer Life Sciences 6 spectrophotometer
equipped with water-jacketed cuvette holders connected to a circulating
water bath. All spectra were recorded with a bandwidth of 2 nm,
response time of 1 s, and scan speed of 480 nm/min at 20 °C.
For photobleaching experiments, the samples were illuminated with a
150-watt fiber optic light equipped with a 495-nm long pass filter for
10 s, and spectra were immediately recorded. In some cases the
bleaching was carried out for further periods of time up to 2 min.
Hydroxylamine treatment was performed by adding an aliquot of a 1 M NH2OH, pH 7.0, stock solution to the sample,
to a final concentration of 30 mM. Thermal bleaching
experiments involved measuring the decrease of absorption at
max in the visible with time at 37 °C.
Rate of Metarhodopsin II (MetaII) Decay as Measured by Retinal
Release--
The rate of MetaII decay was measured, after illumination
of the samples for 30 s, by means of fluorescence spectroscopy
(18). Briefly, 2 µg of protein in 200 µl of buffer D were allowed
to equilibrate at 20 °C for 30 min, then bleached for 30 s, and
the fluorescence increase measured. The excitation and emission
wavelengths were 295 (slit = 0.25 nm) and 330 nm (slit = 12 nm), respectively. Spectra were normalized and fitted to single
exponential functions using SigmaPlot to derive the
t1/2 values.
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RESULTS |
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Purification and Spectral Properties of Thr-94 Rhodopsin Mutants in
the Dark--
The expressed opsin proteins from T94I, T94D, T94S, and
T94K were purified (Fig. 2) using
separation on a rho-1D4 Sepharose column as under "Experimental
Procedures." A selective elution chromatographic method (19) was used
to separate the folded (retinal-binding species; eluted at pH 6, no
salt) and misfolded fractions (non-retinal binding species; eluted at
pH 6, 150 mM NaCl) of the different recombinant rhodopsins
studied. This method was previously used in the study of mutants
associated with RP (6-8). Similar elution profiles were obtained for
the wild type and the T94I and T94D mutants (Fig. 2, A-C)
with most of the protein eluting with pH 6 buffer, with no salt, in the
third fraction, and only a very small amount of non-retinal binding
fraction eluting with added salt. In the case of the T94S mutant the
elution behavior was similar to that for the wild type, but, in
addition, a significant amount of protein eluted with 150 mM salt (Fig. 2D). In contrast, the protein from
T94K mutant eluted predominantly at high salt conditions with very
little protein being eluted at low salt condition indicating that most
of the protein is misfolded.
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UV-visible absorption spectra in the dark for the wild type and for the
low salt eluting fractions from the Thr-94 mutants are shown in Fig.
3. Thus, all the rhodopsin mutants
(A-D), except for T94K (E), showed
rhodopsin-like chromophores with
A280/A500 ratios in the
1.7-1.9 range (WT and T94D, 1.7; T94I, 1.9; and T94S, 1.8). T94K
(E) formed very little A500 absorbing
chromophore and showed some absorption at about 380 nm. In the case of
the mutant T94S there was some amount of misfolded protein eluting at
high salt (trace II, D). The amount of misfolded opsin
eluted at high salt conditions was very high for the mutant T94K
indicating that most of this mutant protein was misfolded (trace II,
E). T94I formed chromophore like the wild type protein, the
slightly higher A280/A500
ratio being presumably due to a change in the of the maximum of the
visible absorption band for the mutant protein. All the mutants studied
showed blue-shifted visible absorption maxima, especially the T94I
mutant with an absorption maximum in the visible region at 478 nm
(Table I). The shift for the other
mutants is less pronounced, with
max of 494 nm for T94S and 496 nm for T94D, respectively.
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Photobleaching and MetaII Decay Rates--
The photobleaching of
the T94I and T94S mutants showed wild type-like behavior on
illumination (Fig. 4). Thus, after
10 s of illumination with light >495 nm formed the characteristic
380 nm-absorbing species with concomitant disappearance of the visible band (Fig. 4, A-C). In contrast, T94D showed a very
abnormal behavior (Fig. 4D). After 10 s of
illumination, the product showed max at 480 nm,
presumably a MetaI-like product that changed little upon further
illumination for up to 2 min and then being kept in the dark for 1 h. Altered photobleaching has also been reported for the G90D mutant
(9), but in this case the Meta I-like species formed upon illumination
decayed in the dark in a 1-h period even at the lower temperature of
15 °C (9). Altered photobleaching has been also reported for other
mutants involving the introduction of Asp, like the RP G89D mutant (8).
In this case, the species formed upon illumination decayed in the dark
to free opsin plus all-trans-retinal which is different from
the behavior observed for the T94D mutant we describe here which does
not decay in the dark in the same period of time.
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An aspect relevant to the functionality of the mutant proteins is the stability of their photoactive conformations, i.e. their MetaII species. The decay of the photointermediate MetaII formed upon rhodopsin illumination was examined for the wild type and the Thr-94 mutants by fluorescence spectroscopy. The fluorescence curves were fitted to a single exponential function, and the t1/2 for the processes was derived (Table I). The t1/2 values for the MetaII decay for the wild type rhodopsin is typically about 15 min under the conditions of the assay. All the mutants studied showed very abnormal values compared with wild type. T94D and T94I showed very slow decay processes, being about 2- and 6.5-fold slower than the wild type, respectively. In the case of the T94I this is the slowest MetaII decay process reported for a mutant so far. T94S showed a 2-fold faster MetaII decay than wild type rhodopsin.
Thermal Stability and Reactivity to Hydroxylamine in the Dark-- Although the purified mutants showed good A280/A500 ratio indicating the presence of correctly folded proteins, it was important to investigate the stability of these proteins in the dark state and their Schiff base accessibility. To this goal, assays were carried out to measure the chromophore stability in the dark (thermal bleaching) and the accessibility of the protonated Schiff base (PSB) in the dark (hydroxylamine reactivity).
Thermal bleaching was followed by monitoring the decay of the visible
absorption band with time at 37 °C in the dark (Fig. 5A). The results obtained show
that wild type rhodopsin and the mutant T94S had the same behavior in
their stability in the period studied. The mutant T94D was less stable
(20% decrease in 5 h), and T94I, in particular, showed reduced
thermal stability (loss of 70% absorption in 5 h, Fig.
5A). The accessibility of the PSB toward hydroxylamine was
also measured in the dark (Fig. 5B). Again, wild type
rhodopsin and the mutant T94S were completely stable to
NH2OH reactivity in the dark for 2 h, and the
susceptibility of the mutants T94D and T94I to hydroxylamine again
followed the order they showed for thermal instability. After 2 h
about 45% of the mutant T94D had survived, although the mutant T94I
had reacted with hydroxylamine more than 80%.
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All-trans-retinal Binding by Mutant T94I--
As seen in Fig.
6, the T94I mutant also bound
all-trans-retinal forming a chromophore with its visible
absorption band located at 466 nm, close to the 478 nm band observed
for the same mutant regenerated with 11-cis-retinal (Fig.
6). Because the max for the PSB under denaturing
conditions is 440 nm, the chromophore for
all-trans-retinal-constituted pigment reflects most likely the presence of a PSB linkage. The presence of the PSB was confirmed by
acidification of the sample that resulted in the formation of the
440-nm characteristic absorption (data not shown).
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DISCUSSION |
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RP mutations in rhodopsin have been shown to affect correct folding of the receptor resulting in impaired 11-cis-retinal binding (6-8). This misfolding would be caused by structural uncoupling between the packing of the helices and the formation of the tertiary structure in the intradiscal domain of the protein (8). Rhodopsin mutations associated with CNB have been proposed to act by a constitutive activity mechanism involving disruption of the electrostatic salt bridge between Glu-113 and the PSB (9, 11). In contrast, it has been shown that constitutive activity is not the cause of photoreceptor degeneration in transgenic mice carrying RP mutations (20). An alternative mechanism, involving thermal instability of the mutant rhodopsins, has also been proposed for CNB mutations, like G90D (12, 13). Thus, different molecular mechanisms underlying RP and CNB have been outlined previously (34). We wanted to provide new insights into the differences between the molecular mechanisms of RP and CNB by studying the phenotypic features of the recently found T94I mutant associated with CNB, and comparing them with those observed for other CNB and RP mutants reported previously (6-13, 36).
The T94I mutant protein could be eluted at low salt conditions
indicating that the protein is folded and forms the retinal binding
pocket. However, in the case of T94S, a small amount of misfolded
protein could be eluted at high salt conditions indicating the presence
of some misfolded opsin. T94K was mostly misfolded because most of the
protein was eluted at high salt conditions, and only a very small
amount could be eluted at low salt conditions. The mutants that formed
chromophore showed blue-shifted max in the visible
region. The observed blue shifts in the
max in the visible absorption bands, particularly for T94I, involving a change from a polar to a hydrophobic side chain, indicate that Thr-94 in
rhodopsin is located in proximity to the retinal binding pocket. The
results obtained indicate that the dark state conformation of the T94I
mutant must be significantly different from that of wild type
rhodopsin. This conclusion can be reached from the following results.
(i) This purified mutant protein showed reduced thermal stability in
the dark, and even at the moderate temperature of 37 °C the decay of
the chromophoric band was greatly accelerated compared with that of the
wild type rhodopsin (mutant T94I samples stored at 4 °C also decayed
in several weeks). In contrast, T94S mutant shows the same stability as
wild type rhodopsin. (ii) T94I mutant protein shows reactivity toward
hydroxylamine in the dark. This may be interpreted as an increase in
the accessibility of the PSB by the reagent, reflecting structural
alterations in the PSB environment and in the intradiscal domain, in
the dark state. (iii) T94I mutant is able to regenerate chromophore
with all-trans-retinal to give a PSB.
A different behavior can be observed for the mutants upon illumination. T94I and T94S showed the same photobleaching behavior than wild type, whereas T94D showed a very abnormal photobleaching pattern. Both T94I and T94S showed the formation of the 380-nm Meta II conformation. T94D showed the formation of a MetaI-like photoproduct at 480 nm that is stable at 20 °C for at least 1 h. This result is in contrast with those found for other mutants, like the RP G89D mutant (8) and the CNB G90D mutant, which showed also an altered photobleaching (with formation of the of the MetaI-like species), but the resulting species did decay in the dark in 1 h in these latter cases (even at 15 °C in the case of G90D (9)). Thus, mutations involving the introduction of an Asp residue in the transmembrane domain show an altered photobleaching pattern, but the T94D mutation here described is different in the very slow decay of the species formed after bleaching.
The T94I mutant can regenerate chromophore with all-trans-retinal to form a PSB linkage in the dark. Several mutant rhodopsins have been described previously that can bind all-trans-retinal to form stable complexes. Among them, E113Q, involving mutation at the site of the counterion to the PSB which alters the pKa of the PSB, was found to be able to bind all-trans-retinal to form a 380 nm absorbing species (21). More recently, mutations at position 257, M257A for example, have been shown to bind all-trans-retinal to form a covalent complex that could be purified in detergent solution (22). The ability to bind all-trans-retinal has been correlated to the constitutive activity of the Met-257 mutants in the opsin state (23). However, in all these previously reported cases, all-trans-retinal-bound forms of the mutants had absorbance maxima at 380 nm reflecting the presence of an unprotonated Schiff base linkage.
In our case, the all-trans-retinal-bound mutant conformation resulting from photobleaching of the T94I (originally regenerated with 11-cis-retinal) mutant must be different from the all-trans-retinal form obtained from regenerating the mutant T94I opsin with all-trans-retinal, because both conformations show very different spectra, with the former showing an absorption band at 380 nm and the latter at 466 nm. It is interesting to note that a different binding site for all-trans and 11-cis conformations of retinal in rhodopsin has been proposed (24). Thus, Thr-94 could be related to the all-trans-retinal-binding site proposed.
Although T94S and T94I showed the same photobleaching behavior, the stability of the MetaII conformation formed was dramatically different. Whereas T94S showed a 2-fold faster decay than wild type rhodopsin (Table I), T94I showed a dramatically slower MetaII decay, this being 6.5-fold slower than that of the wild type. T94D also showed a slower decay than wild type rhodopsin.
An electrostatic switch at Glu-113, in addition to the steric switch
provided by photoisomerization, has been proposed as a critical
determinant of rhodopsin activation (25). Our results suggest that
Thr-94 may be part of this electrostatic switch between the active and
inactive conformations of rhodopsin. Molecular models place Thr-94
close to the Glu-113 counterion (26) and to the water molecule proposed
to be in the vicinity of the PSB (27-30). More importantly, the OH
group of Thr-94 has been located within 3.4 Å of one of the oxygen
atoms of the Glu-113 counterion in the recently published crystal
structure of rhodopsin (31, 32). A water molecule is also proposed to
be in the vicinity of Thr-94 in the crystal structure (33). Our results
would be compatible with the structural effect of Thr-94 being exerted by forming a hydrogen bond with this water molecule or by direct interaction with Glu-113 (34). A structural model (based on recent
crystal structure data) of the different amino acid substitutions studied is depicted in Fig. 7, showing
the location of Thr-94 and providing the structural basis for the
results obtained. The structural model for wild type rhodopsin shows
close proximity of Thr-94 to the PSB at 296 and to
11-cis-retinal (Fig. 7A). In the case of the
mutant T94I, the hydrophobic side chain of Ile at position 94 is seen
close to Glu-113 (Fig. 7B). Substitution of the polar Thr by
the hydrophobic Ile would plausibly explain the marked blue shift in
the max of T94I relative to wild type rhodopsin, as well
as the stability and conformational specific properties of this mutant
when compared with wild type rhodopsin. The T94K mutant shows the
-amino group of Lys-94 extending to Lys-296 (site of the PSB) with
steric and electrostatic effects (Fig. 7C) that would
explain the extensive misfolding observed for the mutant protein. The
model for the mutant T94D shows close proximity of a carboxyl oxygen of
Asp with the oxygen of the Glu-113 counterion (Fig. 7D).
This proximity would cause a change in the electrostatic interactions
network around the PSB and would account for the observed effects on
the spectral and bleaching properties of this mutant rhodopsin. It is
also interesting to note that a similar mutation to the T94I studied,
T90A, has been recently shown to affect the properties of
bacteriorhodopsin by affecting the Schiff base environment of this
bacterial rhodopsin (35).
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From the reported results, taken together with those reported
previously for G90D (9) and A292E (10), it was foreseen that T94I
mutant could be also constitutively active. In fact constitutive
activity of the T94I mutant has been recently
observed.2 These results
indicate that mutations at the same site can cause either constitutive
activity (T94I) or misfolding (T94K). These point mutations in the
transmembrane domain can affect the coupling between the packing of the
transmembrane domain and the formation of either the structure in the
cytoplasmic domain or that in the intradiscal domain. RP mutations like
V87D and G89D (6-8), in the second helix of rhodopsin, cause
misfolding and result in the formation of an abnormal disulfide bond
between Cys-185 and Cys-187 at the intradiscal side of the protein (8,
36). The RP mutations V87D and G89D are located in helical-helical
interacting regions. In contrast, T94I and other CNB mutants like G90D
are located in the vicinity of the retinal Schiff base and cause
structural instability of the dark state of the mutant proteins. This
decrease in stability may be relevant to a proposed molecular mechanism for CNB (13).
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ACKNOWLEDGEMENTS |
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We thank Jong Kim and John Hwa for helpful discussions.
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FOOTNOTES |
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* This work was supported by Grant PM98-0134 from the Dirección General de Enseñanza Superior e Investigación Científica and in part by grants from FUNDALUCE and Dirección General de la ONCE.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: CEBIM, Dept.
d'Enginyeria Química, Universitat Politècnica de
Catalunya, Colom 1, 08222 Terrassa, Catalonia, Spain. Tel.:
34-93-7398044; Fax: 34-93-7398225; E-mail: pere.garriga@upc.es.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M210929200
2 Gross, A., Rao, V., and Oprian, D. (2003) Biochemistry, in press.
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ABBREVIATIONS |
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The abbreviations used are:
RP, retinitis
pigmentosa;
CNB, congenital night blindness;
DM, n-docecyl
-D-maltoside;
MetaI, metarhodopsin I;
MetaII, metarhodopsin II;
PSB, protonated Schiff base;
WT, wild type.
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