Cyclopropyl and isopropyl derivatives of 11- cis and 9- cis retinals at C-9 and C-13: Subtle steric differences with major effects on ligand efficacy in rhodopsin

Article abstract of DOI:10.1021/np100744v

Retinal is the natural ligand (chromophore) of the vertebrate rod visual pigment. It occurs in either the 11-cis (rhodopsin) or the 9-cis (isorhodopsin) configuration. In its evolution to a G protein coupled photoreceptor, rhodopsin has acquired exceptional photochemical properties. Illumination isomerizes the chromophore to the all-trans isomer, which acts as a full agonist. This process is extremely efficient, and there is abundant evidence that the C-9 and C-13 methyl groups of retinal play a pivotal role in this process. To examine the steric limits of the C-9 and C-13 methyl binding pocket of the binding site, we have prepared C-9 and C-13 cyclopropyl and isopropyl derivatives of its native ligands and of α-retinal at C-9. Most isopropyl analogues show very poor binding, except for 9-cis-13-isopropylretinal. Most cyclopropyl derivatives exhibit intermediate binding activity, except for 9-cis-13-cyclopropylretinal, which presents good binding activity. In general, the binding site shows preference for the 9-cis analogues over the 11-cis analogues. In fact, 13-isopropyl-9-cis-retinal acts as a superagonist after illumination. Another surprising finding was that 9-cyclopropylisorhodopsin is more like native rhodopsin with respect to spectral and photochemical properties, whereas 9-cyclopropylrhodopsin behaves more like native isorhodopsin in these aspects.

Full text of DOI:10.1021/np100744v

ARTICLE
pubs.acs.org/jnp
Cyclopropyl and Isopropyl Derivatives of 11-cis and 9-cis Retinals at
C-9 and C-13: Subtle Steric Differences with Major Effects on Ligand
Efficacy in Rhodopsin
,
†,‡
†
‡
‡
Willem J. deGrip,* Petra H. M. Bovee-Geurts, Yajie Wang, Michiel A. Verhoeven, and
‡
Johan Lugtenburg
†
Department of Biochemistry, UMCN 286, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical
Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
‡
Department of BioOrganic Photochemistry, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The
Netherlands
ABSTRACT: Retinal is the natural ligand (chromophore) of the verte-
brate rod visual pigment. It occurs in either the 11-cis (rhodopsin) or the
9
-cis (isorhodopsin) configuration. In its evolution to a G protein
coupled photoreceptor, rhodopsin has acquired exceptional photoche-
mical properties. Illumination isomerizes the chromophore to the all-
trans isomer, which acts as a full agonist. This process is extremely
efficient, and there is abundant evidence that the C-9 and C-13 methyl
groups of retinal play a pivotal role in this process. To examine the steric
limits of the C-9 and C-13 methyl binding pocket of the binding site, we
have prepared C-9 and C-13 cyclopropyl and isopropyl derivatives of its
native ligands and of R-retinal at C-9. Most isopropyl analogues show very poor binding, except for 9-cis-13-isopropylretinal. Most
cyclopropyl derivatives exhibit intermediate binding activity, except for 9-cis-13-cyclopropylretinal, which presents good binding
activity. In general, the binding site shows preference for the 9-cis analogues over the 11-cis analogues. In fact, 13-isopropyl-9-cis-
retinal acts as a superagonist after illumination. Another surprising finding was that 9-cyclopropylisorhodopsin is more like native
rhodopsin with respect to spectral and photochemical properties, whereas 9-cyclopropylrhodopsin behaves more like native
isorhodopsin in these aspects.
he visual pigment rhodopsin is the sensory element of the
rod photoreceptor cell, which mediates dim-light vision in
II (λmax = 380 nm), which binds and activates its cognate G
protein transducin (Gt).
11,15
T
1
-3
the vertebrate retina.
Rhodopsin is a member of the super-
The photochemical performance of the 11-cis-retinal-opsin
couple is exceptional, showing a photoisomerization quantum
yield of 0.65 ( 0.02 and a fully selective reaction pathway
(11-cis f all-trans) and generating within 200 fs a vibrationally
hot intermediate (photorhodopsin) with a highly distorted, but
already all-transoid chromophore. Within 1 ps photorhodopsin
relaxes to Batho, which still contains a highly strained all-trans
family of G protein-coupled receptors. It evolved for the recep-
tion of photons by adopting a photosensitive ligand
chromophore) that has become covalently bound to the
(
apoprotein, opsin, and provides the visual photosensitivity of
the holoprotein (λmax = 498 nm). This physiological ligand is 11-
cis-retinal (Figure 1), a derivative of vitamin A, and it is attached
in the opsin binding site to Lys-296 through formation of a
16-22
chromophore.
Isorhodopsin exhibits similar features, except
1,4-6
protonated Schiff base.
The latter is stabilized by a complex
that its photochemistry is less efficient (slower kinetics and lower
23-25
counterion positioned around a glutamate residue of opsin (Glu-
quantum yield of 0.26 ( 0.03).
7
-9
1
13).
Under certain pathological conditions, however, also
The crux of this top performance lies in an optimal nonbond-
ing communication between ligand and protein. This aspect has
been investigated in a large number of experimental studies,
employing modified retinals that generate (iso)rhodopsin ana-
the 9-cis configuration of retinal is observed, which generates
10
isorhodopsin (λmax = 485 nm). Both the 9-cis and the 11-cis
isomer of retinal act as potent inverse agonists of opsin, practi-
cally eliminating its basal activity, and are converted by light into
26-44
logue pigments.
These studies have vastly expanded our
2,11
insight in the binding site requirements. Relevant in the context
the all-trans configuration that acts as a full agonist.
The
photoisomerization of the ligand induces conformational
changes in the protein, which are driven by about 35 kcal of
photon energy stored in the first photoproduct, Batho.
12-14
Special Issue: Special Issue in Honor of Koji Nakanishi
These conformational steps in the protein culminate within
several milliseconds in the formation of the active state, Meta
Received: October 19, 2010
Published: February 10, 2011
Copyright r 2011 American Chemical Society and
American Society of Pharmacognosy
383
dx.doi.org/10.1021/np100744v |J. Nat. Prod. 2011, 74, 383–390

Journal of Natural Products
ARTICLE
Figure 1. Chemical structure of 11-cis- and 9-cis-retinals and R-retinals.
The structures are shown in the 12-s trans conformation, which is
dictated by the opsin binding site. The following derivatives are used in
this study: natural: R = R = methyl; 9-cyclopropyl: R = cyclopropyl,
Figure 2. Spectral properties of native and selected 11-cis- and 9-cis-
retinals in n-hexane solution (continuous curves). After illumination in
the presence of a trace of iodine the dotted curves are obtained, which
mainly represent the all-trans and 13-cis isomers.
1
2
1
R
R
2
= methyl; 9-isopropyl: R
= methyl, R
1
= isopropyl, R
= cyclopropyl; 13-isopropyl: R = methyl, R =
1 2
2
= methyl; 13-cyclopropyl:
1
2
isopropyl.
the 11-cis as well as the 9-cis configuration. The cyclopropyl and
isopropyl groups are not spherosymmetical, but the cyclopropyl
3
group is somewhat less bulky (ca. 56 versus ca. 63 Å ). We report
of this report is the outcome that the C-9 and C-13 methyl
groups are pivotal elements in this ligand-receptor communica-
tion. For instance, 9- and 13-demethylretinals still yield analogue
pigments, but with an increase in constitutive activity and/or
much reduced physiological activity.
Indeed, the crystal structure of rhodopsin as well as various
here some spectral data, the pigment formation capacity of the
various retinal derivatives, and the resulting maximal absorbance,
and where relevant some photochemical properties of the
analogue pigments and their signaling activity, measured as
capability to activate the G protein transducin.
33,34,41,45-53
biochemical and biophysical evidence agree on quite a defined
54-60
pocket for the 9-methyl substituent.
In a recent study we
’
RESULTS AND DISCUSSION
probed the spherosymmetrical boundaries of this pocket using
60
9-halogen substituents and observed that all bind readily except
Retinal Derivatives. The spectral properties of most retinal
for a sluggish reaction with the 9-iodo substituent, which, in fact, is
somewhat bulkier than the methyl group (van der Waals radii of
about 0.215 and 0.20 nm, respectively). This would agree with the
absence of any binding data for the 9-tert-butyl derivatives (van der
Waals radius of about 0.24 nm). The latter derivatives have been
prepared synthetically, and in solution they already have a distorted
derivatives used in this study were quite similar to the parent
11-cis and 9-cis compounds, shown in the top panels of Figure 2.
The native 11-cis isomer (continuous curve, top left panel) shows
a main band at 365 nm with a pronounced β-band at 250 nm,
indicative of the presence of a twisted 12-s-cis conformation
because of steric hindrance between the C-10 hydrogen and the
13-methyl group. Illumination in the presence of a trace of iodine
generates a mixture of all-trans and 13-cis isomers, which adopt
the 12-s-trans conformation, with concomitant reduction of the
β-band intensity and increase in main band intensity. The same
behavior is observed for the 11-cis derivatives, except that the
main band intensity slowly decreases and the β-band intensity
gradually increases in the order 9-cyclopropyl < 9-isopropyl < 13-
cyclopropyl < 13-isopropyl. This reflects the higher abundance of
twisted conformations and at the same time accentuates the
lower steric perturbation inflicted by the cyclopropyl substituent.
As an example, the spectrum of 11-cis-13-isopropylretinal is
presented in the lower left panel of Figure2 (continuous curve).
The 9-cis-retinals are relatively unperturbed, as demonstrated by
the absence of a strong β-band and little change in intensity of the
main band upon illumination (Figure2, top right panel), except
for the 13-isopropyl derivative, which shows a pronounced
β-band (Figure2, lower right panel). The latter probably reflects
the presence of a twisted 12-s-cis conformation due to steric
hindrance between the C-13 isopropyl group, the C-5 methyl
group, and the C-8 hydrogen.
61-63
conformation due to severe intramolecular steric hindrance.
On the other hand, preliminary binding data have been reported for
28,64
ethyl, n-propyl, isopropyl, and even n-pentyl substituents at C-9.
This suggests that the 9-methyl binding pocket can accommodate
larger substituents, if they are not spherosymmetrical. Another
interesting aspect is that in the 9-cis-retinal configuration the
5
9,65,66
9
-methyl group does not comfortably fit into its pocket,
60
which also results in poorer binding of the 9-iodo derivative.
Remarkably, this situation is reversed in the R-retinals, where
apparently the poorer fit in the β-ionone binding pocket is partially
43,60
compensated for by the 9-cis geometry.
The 13 position has been less well investigated in this respect.
Binding data have been reported for the 13-ethyl and n-propyl
67,68
derivatives, showing decreasing affinity, respectively,
but 13-
ethylrhodopsin still having full receptor activity. The 13-n-butyl
derivative apparently is not able to generate a pigment analogue,
28
however.
In order to further clarify these issues, we have investigated the
ligation capability of C-9 and C-13 cyclopropyl and isopropyl
substituents, for both the natural retinals and the R-retinals, in
3
84
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Journal of Natural Products
ARTICLE
Table 1. Spectral and Photochemical Properties and Relative
G Protein Activation Rates of Generated Pigments
exceptional in this respect, but still significantly enhanced over the
71
free retinals (0.15-0.20). The Φ value of analogue pigments was
only determined if sufficient material was available. Conspicuous is
the marked reduction in the Φ value of the 13-cyclopropyl
derivatives and, again, the large and opposite effects of the 9-cyclo-
propyl substituent. A strong increase in Φ of isorhodopsin analogue
pigments has so far only been reported for the 7,8-dihydro
regeneration
quantum
retinal
λ
max
yield Φ
transducin
a,b,c
a,b
derivative
((10%) ((2 nm) ((0.04)
activation
1
9
1-cis unmodified
ꢀ 100
30
2
498
492
504
506
500
486
504
485
488
490
469
476
461
485
0.65
0.08
-
ꢀ 100
83 ( 7
-
42
analogue, and that value was similar to the one now observed
9
9
1
1
-cyclopropyl
for 9-cyclopropylisorhodopsin (0.39). The Φ value measured for
-isopropyl
9
-cyclopropylrhodopsin (0.08) presents a spectacular decrease
3-cyclopropyl
3-isopropyl
28
6
0.18
-
-
relative to rhodopsin itself (0.65), which is in glaring contrast to
-
the exceptional increase observed for its 9-cis counterpart.
-cis unmodified
100
36
5
0.26
0.39
-
104 ( 8
60 ( 18
-
The photoreaction of the parent and analogue pigments was
analyzed at 80 K by Fourier transform infrared (FTIR) difference
spectroscopy (Figure 3). Negative signals in these spectra
correspond to vibrational features in the pigment that change
in frequency or intensity or disappear upon formation of the all-
trans photoproduct, while the positive signals are characteristic
9
9
1
1
-cyclopropyl
-isopropyl
3-cyclopropyl
3-isopropyl
81
31
50
5
0.10
0.23
ND
-
53 ( 7
139 ( 6
ND
1
1-cis R-
R-9-cyclopropyl
-cis R-
R-9-cyclopropyl
7
2-75
for the photoproduct and can be used for identification.
In
-
the native pigments illumination at this temperature generates a
9
90
10
0.28
-
ND
1,72
stable Batho intermediate. At this stage most of the changes in
-
vibrational pattern reflect the isomerization of the chromophore
a
-
not determined because of too low production of pigment or lack of
76,77
b
c
and associated aborbance band shifts.
Most prominent are
retinal derivative. ND, not determined. The Φ value of the unmodi-
-1
23,24,69,70
those of CdC and CdN stretches (1500-1600 cm ), C-C
stretches (1100-1360 cm 7,8, 7fi9 ngerprint region), and wag
fied pigments has been determined before.
Similar behavior is observed for the R-retinals (not shown).
-1
-
1
vibrations (800-1050 cm ).
43
For spectra of R-retinals we refer to Wang et al.
Very distinct for Batho is the isolated hydrogen-out-of-plane
(HOOP) vibrational pattern between 800 and 940 cm- , which
reflects the highly strained structure of the all-trans chromophore
1
Pigment Formation and Spectral Properties. The data
obtained for the pigments discussed in this study are collected in
Table 1. We include relevant data on the R-retinals, as in that case
the opsin binding site shows clear preference for the 9-cis geometry,
18
-1
in Batho. The small tridentate feature around 1650 cm
8
0
represents small changes in protein secondary structure, in-
dicative of the very minor rearrangement of the opsin structure
occurring at this first stage of rhodopsin photoactivation.
The isorhodopsin to Batho transition (labeled 9-Z-Rho)
presents the same distinct HOOP pattern for Batho, but the
other changes show a different pattern, reflecting the fact that
here it concerns a 9-cis to all-trans isomerization. For instance, the
43
as well. However, we observed that the 9-cyclopropyl and
9-isopropyl derivatives of R-retinal are very poor ligands. In fact,
the 9-isopropyl-R-retinals did not generate detectable quantities of
analogue pigment. We have therefore restricted the analysis of R-
pigments and did not test R-retinal modified at C-13.
The general finding is that, as expected, the less bulky
cyclopropyl derivatives present a larger rate of pigment forma-
tion and a higher incorporation level. It should be noted that in
the case of 11-cis-retinal the 9- and 13-cyclopropyl derivatives are
almost equally effective, and the 9- and 13-isopropyl derivatives
almost equally ineffective. In the case of 9-cis-retinal, the C-13
derivatives are much more effective than the C-9 derivatives and
also much more effective than the C-13 derivatives of 11-cis-
retinal. The 9-cis-13-cyclopropylretinal derivative stands out in
this respect, being capable of regenerating about 80% of the
available aporeceptor into photopigment, while the other cyclo-
propylretinal derivatives do not reach further than about 30%.
In addition, we observed that most derivatives effectuate no or
a small red-shift in the main absorbance band of the analogue
pigments relative to that of the parent pigment, as is evident from
their λmax. Exceptions are the 9-cis-9-cyclopropyl derivatives of
retinal and R-retinal, which induce red-shifts of about 20 nm, and
the 11-cis-9-cyclopropylretinal, which induces a small blue-shift
-1
combination C-H wag of the polyene chain absorbs at 959 cm in
-
1
isorhodopsin and at 967 cm in rhodopsin. In fact, the protein
-
1
pattern around 650 cm is still small but also clearly different
from the rhodopsin to Batho transition.
The difference spectra of the analogue pigments globally show
the same pattern. The 9-cyclopropylrhodopsin analogue pro-
duces a very typical Batho spectrum. Small changes like the
upshift and split of the polyene C-H wag to 977 and 988 cm-
reflect an effect of the cyclopropyl group. Interestingly, the
structural changes in the protein seem to be somewhat enlarged.
The isorhodopsin analogues show a typical 9-cis to all-trans
difference pattern in the fingerprint region and protein changes
quite similar to the parent pigment. The polyene C-H wag is
1
-
1
again shifted, from 959 to 974 cm , and the CdC stretch is split.
In the isorhodopsin analogues modified at C-13 the HOOP
pattern of the photoproduct is less intense and shifted, probably
due to the large C-13 substituent, but we are confident to identify
it as the corresponding Batho intermediate. The 9-cyclopropyl
analogue of isorhodopsin produced very noisy spectra, which
may be due to large spectral overlap between the pigment and the
photoproduct. Most features look similar to the C-13 analogues,
except for a seemingly different pattern in the HOOP region,
which however suffers from a very poor signal-to-noise ratio.
Signaling Activity. In the majority of analogue pigments the
transducin activation rate (the signaling activity toward the
(
Table 1).
Primary Photochemistry. The primary photoreaction of
selected analogue pigments was documented by their photoi-
somerization quantum yield (Table 1) and the photoproduct
generated at 80 K, where native pigments produce a stable Batho
1
intermediate (Figure 3).
Rhodopsin is distinguished by an unusually high photoisome-
23,24,69,70
rization quantum yield (Φ),
while isorhodopsin is less
3
85
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Journal of Natural Products
ARTICLE
Figure 4. pH dependence of the Meta I T Meta II equilibrium in native
rhodopsin (solid line without data points) and selected derivatives
(
9-cyclopropylrhodopsin, 13-cyclopropylrhodopsin, and R-rhodopsin)
measured at 10 °C (283 K). The rhodopsin and 9-cyclopropylrhodopsin
data could be fitted with a regular Henderson-Hasselbalch function,
a
yielding a pK of 7.3 and 7.4, respectively. R-Rhodopsin behaves
irregularly, while the 13-cyclopropylrhodopsin data suggest a significant
upshift of the pK , but this could not be experimentally verified because
a
of the low stability of Meta I at higher pH.
as a “superagonist”. Owing to limited quantities we have not
been able to determine the pK of the Meta I T Meta II equili-
a
brium, but this analogue pigment certainly deserves further
investigation.
Figure 3. FT-IR difference spectra of selected 11-cis and 9-cis pigments
and photoproducts generated by illumination at 80 K. Difference spectra
were constructed by subtracting the dark-state spectrum from the
spectrum obtained during two minutes after illumination. Negative
bands represent vibrational bands characteristic for the pigment; positive
bands represent characteristic vibrations of the photoproduct. The
native rhodopsin f Batho and isorhodopsin f Batho difference spectra
present the typical, isolated HOOP pattern of Batho between 800 and
While the signaling activity of the active analogue pigments is
not much reduced relative to rhodopsin, Φ is markedly lower,
varying from 0.08 to 0.39, i.e., 13% to 60% of that of rhodopsin.
Thus, when these analogues would need to function in a native
setting, i.e., the rod photoreceptor cell, their physiological activity
would exhibit a shift in their stimulus/response curve relative to
rhodopsin. Their maximum response would probably not be
much lower than in the case of native rhodopsin, but they would
show a lower photosensitivity; that is, they would need brighter
light to elicit the same response.
-
1
930 cm , reflecting the highly strained structure of the all-trans
chromophore in Batho.
cognate G protein transducin) is reduced; that is, the ligand
behaves as a partial agonist at most. Usually this at least partially
entails a marked downshift of the pH dependence of the Meta I
T Meta II equilibrium, which leads to a much lower production
of the active state (Meta II) at physiological pH and hence a
Interpretation. The C-9 and C-13 methyl groups of 11-cis-
retinal play a pivotal role in the exquisite interaction between
ligand and receptor responsible for the exceptional photochemi-
33,34,41,45-53
cal performance of this photoreceptor couple.
This
suggests the presence of well-defined binding pockets for these
structural elements as part of the global ligand binding site. There
are several lines of evidence supporting such a pocket for the
11,75
lower signaling activity.
We determined the pH dependence
of this transition for some analogues (Figure 4). Both the 11-cis
and 9-cis R-pigments exhibit a significant downshift, with quite a
steep pH dependence, reminiscent of the profile reported for the
54-57,59,60
9-methyl group,
requirements has been described using 9-halogen substituents.
and a first delineation of its steric
60
44
1
0-F and 12-F analogues. The 9- and 13-cyclopropylrhodop-
From the latter study one can estimate an upper limit of ca. 2.2
nm for the radius of a spherical ligand. Nevertheless, preliminary
data on pigment formation indicate that this pocket also accom-
sins behave quite differently. The profile of the 9-cyclopropyl
analogue is very similar to the parent compound with pK ’s of 7.4
a
2
8
and 7.3, respectively. The 13-cyclopropyl analogue actually
produces very little Meta I even at higher pH, and because of
modates isopropyl and n-pentyl substituents at C-9. Hence,
this pocket may not be spherosymmetrical but more elongated in
shape. Evidence for a defined pocket for the 13-methyl group is
the low stability of Meta I at higher pH, a pK could not be
a
determined. This is reminiscent of the behavior of 14-F
pigments.
less substantial. The 13-ethyl and 13-n-propyl groups can be
4
0,44
67,68
accommodated, albeit with much lower binding kinetics,
but
28
Considering that the pK of the Meta I T Meta II equilibrium
the 13-n-butyl group does not seem to give a stable pigment.
a
is independent of whether originating in rhodopsin or in
isorhodopsin, we would expect that, except for the R-analogues,
the signaling activity of the (iso)rhodopsin analogues would
approach that of the parent pigment. The transducin activation
rate of the cyclopropyl analogues indeed is quite high for an
analogue pigment, ranging from 50% to 80% of that of the native
pigments (Table 1). Surprisingly, the activity of the 13-isopropyl-
isorhodopsin analogue pigment is significantly higher than the
parent compound (ca. 140%), classifying its all-trans chromophore
To further clarify these issues, we have prepared the potential
ligands with cyclopropyl and isopropyl substituents at C-9 or
C-13, presented in Figure 1. The cyclopropyl and isopropyl
groups both are not spherosymmetical, but the cyclopropyl
group is somewhat less bulky (ca. 56 versus ca. 63 Å ) and hence
slightly more asymmetric.
The spectral properties of the prepared retinals are outlined in
Figure 2 and are in line with the properties of the substituent. A
tert-butyl substituent at C-9 or C-13 generates substantial
3
3
86
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Journal of Natural Products
ARTICLE
intramolecular steric hindrance, thereby stabilizing a highly
induces a small blue-shift, but with the 9-cis geometry it gives rise
to large red-shifts of 18 and 24 nm. We offer the following
explanation: In the 9-cis geometry the polyene chain is straight
from C-9 on. An electron-dense substituent like the cyclopropyl
group then is in an optimal position to conjugate with the
61,63
twisted conformation.
This effect will increase in the succes-
sion cyclopropyl < isopropyl < tert-butyl and explains the gradual
intensity changes in the β-band and main band of the 11-cis-
retinal analogues. The 9-cis configuration is less perturbed by
these modifications, but does show a pronounced β-band in the
case of the 13-isopropyl substituent, probably reflecting torsion
around the 12-s bond to minimize steric hindrance between the
C-13 isopropyl, the C-5 methyl, and the C-8 hydrogen.
85
polyene system, which has positron properties, and to induce
a significant bathochromic shift. Such a strong effect cannot
occur with the 11-cis geometry. The small blue-shift, observed
instead for the 11-cis-retinal derivative, must arise in a specific
interaction with the protein, since the same derivative in 11-cis-R-
retinal again shows a small red-shift like most other analogue
pigments in Table 1.
Pigment formation with this panel of retinal analogues yielded
several unexpected results. None of the analogues were able to
bind the full complement of aporeceptor present, even when
added in 10-20-fold molar excess (Table 1). The data in this
table are based on the assumption that the molar absorbance of
32,39,42,44,68,86
Analogue pigments usually show a decrease in Φ,
probably reflecting suboptimal cooperation with the protein in
the photochemical isomerization process. Hence, it is not
surprising that most analogue pigments we report here comply
with this general behavior. In calculating the Φ value we again
assume the same molar absorbance for all pigments in Table 1,
but also in this case an increase of 40% for the analogue data
would not change the general trend. The most striking observa-
tion is again the quite opposite effect of the 9-cyclopropyl group
in the 9-cis configuration (large increase relative to the native
pigment) versus the 11-cis configuration (dramatic decrease).
The large decrease for the 11-cis derivative is difficult to explain.
Since the FTIR analysis indicates that upon Batho formation in
this analogue pigment, somewhat larger conformational changes
occur in the protein elements lining the binding site (Figure 3,
second spectrum from the top), an additional energy barrier
might kick in and lead to the observed effect. The large increase in
the case of the 9-cis derivative is even more difficult to explain.
The high Φ value in native rhodopsin is facilitated by the fixed
torsion in the C-10-C-13 segment of the polyene chain of its
-
1
the analogue pigments is similar to that of rhodopsin (40 600 M
-
1
cm ). However, even if this value would be 40% lower (24 000
-
1
-1
M
cm
is the lowest value reported for an analogue
3
2
pigment ), all retinal analogues in Table 1 would still yield less
than 50% regeneration, except for the 9-cis-9-cyclopropyl deri-
vative. The phenomenon of only partial pigment formation is
28,46,81
quite common in the retinal analogue field.
This is not just
a matter of affinity or equilibrium distribution, since it does not
depend on the molar excess of analogue, and sometimes the
remaining opsin can still bind 11-cis-retinal to generate native
rhodopsin. Lower pigment formation usually is accompanied by
a substantially lower binding rate, and that is also what we
observe for these retinal analogues. We have extensively dis-
60
cussed this aspect before and have suggested that the binding
process is more complicated and may involve at least a two-step
process involving secondary binding sites, for which there is
8
2-84
some evidence.
Nevertheless, a lesser fit of the ligand in the
binding site will manifest itself as a lower rate of pigment
formation and, hence, a lower extent of pigment formation.
Since we observe strongly reduced rates of pigment formation for
all analogues, we assume that the data given for the percentage of
regeneration are a good approximation.
2
2,54,85,87
chromophore.
This torsion is largely absent in the C-8-
C-11 segment of the chromophore of native isorhodopsin.
Possibly, the bulky 9-cyclopropyl substituent is tightly fixed in
9
its binding pocket and can induce a twist in the Δ double bond
As such, it can be concluded that the combination of an
R-ionone ring with a cyclopropyl or isopropyl moiety at C-9 is
poorly accommodated by the binding site. In the case of the
normal retinals the cyclopropyl group is much better accepted for
binding than the isopropyl group. This again demonstrates that a
small difference in steric properties can make a large difference in
ligand effectivity and further supports the concept that the
and/or facilitate isomerization electronically. Femtosecond
vibrational and electronic spectroscopy and molecular dynamics
will hopefully shed more light on these fascinating effects.
The present evidence indicates that the retinal analogues in
this study that generate fair levels of analogue pigment do not
downshift the pK of the Meta I T Meta II equilibrium
a
(Figure 4) and have relatively high partial agonist activity. A
remarkable exception is the 13-isopropylisorhodopsin analogue,
the only isopropyl derivative that can be reasonably well accom-
modated in the binding site. This analogue induces a transducin
activation rate that is about 140% of that of the parent com-
pound, and hence its all-trans photoproduct can be classified as a
superagonist. This analogue pigment certainly deserves more
detailed investigation, as well.
9
-methyl binding pocket is not spherosymmetrical. It is therefore
not surprising that the 9-n-propyl derivative can generate a
6
4
28
pigment analogue. Binding of the 9-n-pentyl analogue re-
mains surprising, however, and deserves a more detailed study.
With respect to the 13-substituent it is obvious that the binding
site can only with some difficulty accommodate an isopropyl
group, and it is not surprising that an n-butyl group is not
accepted. The most salient is our observation that the 13-
cyclopropyl group is much better accommodated in the opsin
binding site in combination with the 9-cis configuration than in
combination with the 11-cis configuration. Possibly, the slight
structural adaptation the binding site has to undergo to accom-
2
8
’
CONCLUSION
To examine the steric limits of the 9-methyl and 13-methyl
binding pocket of opsin, we have prepared cyclopropyl and
isopropyl derivatives of its native ligands, 11-cis- and 9-cis-retinal,
at C-9 and C-13, and of R-retinal at C-9. Most isopropyl
analogues show very poor binding, except for 9-cis-13-isopro-
pylretinal. Most cyclopropyl derivatives exhibit intermediate
binding activity, except for 9-cis-13-cyclopropylretinal, which
presents good binding activity. In general, the opsin binding site
has preference for the 9-cis over the 11-cis analogues. One of
54,59
modate 9-cis-retinal
may lead to small rearrangements in the
protein surface near C-13. Hence, this would suggest a defined
binding site for the 13-methyl group, as well.
Most analogues have no effect on the main absorbance band of
the resulting photopigment or generate a small red-shift
(Table 1). In this context, a cyclopropyl group at C-9 presents
a striking exception. In combination with the 11-cis geometry it
3
87
dx.doi.org/10.1021/np100744v |J. Nat. Prod. 2011, 74, 383–390

Journal of Natural Products
ARTICLE
several surprising findings was that 9-cyclopropylisorhodopsin is
more like native rhodopsin with respect to spectral and photo-
chemical properties, whereas 9-cyclopropylrhodopsin behaves
more like native isorhodopsin in these aspects.
pigment) by incubation for 1 h on ice. Undissolved material was
removed by centrifugation (30 min, 80000g, 4 °C), and the supernatant
was mixed with a solution of asolectin (100 mg/mL in 50 mM
nonylglucoside in buffer A) to achieve a 50-fold molar excess of asolectin
with respect to pigment. After 15 min incubation on ice detergent was
extracted by addition of solid β-cyclodextrin to a slight excess over
nonylglucose, and the resulting proteoliposomes were isolated by over-
night sucrose step-gradient centrifugation at 200000g and 4 °C as
described before. The proteoliposome band was recovered from the
20%/45% sucrose interface, diluted with 2 volumes of doubly distilled
2
H O, pelleted by centrifugation (60 min, 200000g, 4 °C), and stored in
aliquots under Ar in a light-tight container at -80 °C.
UV/Vis Spectroscopy. The spectral properties of the pigments
were determined in micellar solution, by solubilization to about 2.5 μM
in 20 mM dodecylmaltoside (DDM) in buffer A containing 10 mM
hydroxylamine. The wavelength of maximal absorbance in the visible
region (λmax) was determined as the peak position in the difference
spectrum obtained after subtraction of the spectrum after illumination
(300 s; 150 W halogen light through Schott OG530 cutoff filter and fiber
optics) from the dark-state spectrum.
Our data are in line with the growing body of evidence that the
interplay between a receptor and its ligand is very finely tuned.
Small modification of a ligand can already alter this interplay and
thereby redirect the conformational space of a receptor, leading
to a different activity profile. Thus, full agonist activity can readily
be modified into partial agonist or even neutral antagonist
9
4
3
9,88,89
activity.
This emphasizes the need of very accurate map-
ping of the in situ ligand structure and nonbonding ligand-
protein communication to allow proper structure-based drug
design.
’
EXPERIMENTAL SECTION
Materials. All chemicals were of analytical grade. Detergents were
obtained from Anatrace (Maumee, OH, USA). Native 11-cis-retinal was
provided by Dr. Rosalie Crouch (Medical University of South Carolina,
Charleston, USA) through financial support from the National Eye
Institute (NEI).
The Meta I T Meta II equilibrium was analyzed in proteoliposomes
using the end-on photomultiplier setup of a Perkin-Elmer Lambda 18
spectrophotometer. Pigments were suspended to about 1 μM in buffer
of various pH as indicated in the text using MES, MOPS, or bis-tris
propane as buffering compound. Samples were maintained at 10 °C by
means of a circulating water bath. Spectra were recorded before and after
illumination (10 s; conditions as above) and then every 5 min up to 30
min after illumination to check for the stability of the Meta photo-
products. Finally, 1 M hydroxylamine was added to a final concentration
of 50 mM to convert all photointermediates into the all-trans-retinalox-
ime derivative. The relative amount of Meta I at 480 nm remaining
Synthesis of Retinals. The 9-cyclopropyl and 9-isopropyl deri-
vatives of retinal and R-retinal were prepared in their isomerically pure
90
9
-cis or 11-cis configuration as described before. The 13-cyclopropyl
and 13-isopropyl derivatives were obtained as a mixture of isomers,
employing a C15 þ “C ” scheme with isopropyl- or cyclopropyl-
5
5
functionalized trifluoroethyl C -phosphonates to increase the yield of
91
the 11-cis isomers. The corresponding 11-cis and 9-cis isomers were
92
purified by preparative HPLC. The purity of the compounds was
1
13
95
verified using H and C NMR spectroscopy and was always better than
8%. UV-vis absorbance spectra were recorded in n-hexane solution on
immediately after illumination was calculated as described before.
9
FT-IR Spectroscopy. FT-IR analyses were performed on a Bruker
a Perkin-Elmer lambda 18 double-beam spectrophotometer at a con-
centration between 2 and 4 μM.
IFS 66/S spectrometer, equipped with a liquid-nitrogen-cooled, narrow-
3
2,39
band HgCdTe (MCT) detector as described.
In brief, sample
Isolation of Bovine Opsin and Generation of Analogue
Pigments. Bovine rod outer segment membranes in the opsin form
temperature was under computer control using a variable-temperature
helium-cooled cryostat (Heliostat, APD Cryogenics Inc.), covered with
a set of NaCl windows in the IR light path. Membrane films were
(
opsin membranes) were prepared from fresh, light-adapted cattle eyes
32,93
96
as described.
The regeneration capacity of these preparations was
prepared by isopotential spin drying 2-3 nmol of pigment in
estimated from the A280/A500 ratio measured after subsequent incuba-
tion with a 2-3-fold excess of 11-cis-retinal, whereby a ratio of 2.1 ( 0.1
was taken to represent membranes with maximal rhodopsin content.
Rhodopsin, isorhodopsin, and the analogue pigments were generated
with opsin membranes, showing a regeneration capacity in the range
proteoliposomes on AgCl windows (Crystran Limited, UK). The
membrane film was rehydrated, sealed using a rubber O-ring spacer
and a second AgCl window, and screwed tight in the sample holder of
the cryostat. Samples were illuminated in the spectrometer using a
modified fiberoptics ring illuminator (Schott) fed by a 150 W halogen
light filtered through a 488 ( 10 nm interference filter and a long-pass
filter (Schott). Routinely, six consecutive blocks of 1280 scans each were
recorded at 4 cm resolution, taking about 120 s acquisition time per
block to generate a spectrum, both before and after 2 min of illumina-
tion. No differences in pattern for the subsequent spectra were detected,
and routinely the last dark-state spectrum was subtracted from the first
spectrum of the photoproduct to generate a difference spectrum.
Difference spectra were measured at 80 K, where Batho is sufficiently
stable to allow analysis. Temperature stability was (0.2 K.
90-100%. Regeneration and further manipulations with the pigments
were done under dim red light (>620 nm, Schott RG620 cutoff filter).
Analogue pigments were generated by incubating a suspension of
opsin membranes (50-100 μM opsin in buffer A: 20 mM piperazine-N,
-
1
0
N -bis(2-ethanesulfonic acid) (PIPES), 130 mM NaCl, 5 mM KCl,
2
2
mM CaCl , 0.1 mM EDTA, 1 mM dithioerythritol, pH 6.5) with a
5-10-fold molar excess of the retinal derivative at room temperature.
After two hours a small aliquot was assayed for the extent of regeneration
by addition of 11-cis-retinal in a 2:1 molar ratio to the original opsin.
With most retinal analogues regeneration had not yet reached its
maximal level. Thus, an additional aliquot of the retinal derivative was
added, and the incubation continued for at least overnight at 4 °C until
pigment formation had leveled. Excess retinal was then converted into
the corresponding oxime by addition of a 1 M hydroxylamine solution
Determination of Quantum Yield. The photoisomerization
quantum yield (Φ) of selected analogue pigments was determined
relative to rhodopsin, of which the Φ (0.65 ( 0.02) and molar
-
1
-1
absorbance (ε
hed.
4
98
= 40 600 ( 500 M cm ) are well establis-
2
3,24,69,70
Rhodopsin, isorhodopsin, and analogue pigments were
(
pH 6.5) to a final concentration of 10 mM. After cooling on ice and 30
solubilized in buffer A containing 10 mM DDM and 10 mM hydro-
xylamine to give an OD/cm at 500 nm of 0.100 ( 0.07. Samples were
kept at 10 °C and showed no significant decrease of A500 after 2 h in the
dark. Samples were illuminated through a 497 ( 5 nm interference filter
(Schott) such that the half-time of bleaching was g30 min. Spectra were
recorded at intervals of 2-10 min. The data were converted to a straight
min incubation, the oxime was largely removed by two extractions with
5
also removes some lipids, thereby perturbing the Meta I to Meta II
transition. To restore a native lipid/protein ratio, the membrane pellet
was dissolved in 20 mM nonylglucoside in buffer A (to ca. 50 μM of
32
0 mM heptakis(2,6-di-O-methyl)-β-cyclodextrin, which, however,
3
88
dx.doi.org/10.1021/np100744v |J. Nat. Prod. 2011, 74, 383–390

Journal of Natural Products
ARTICLE
line, the slope of which (S) is a measure of the photosensitivity ε497Φ, as
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32,69
described before.
Using the S of rhodopsin measured under identical
conditions allows the calculation of the Φ value of the analogue
pigment.
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AUTHOR INFORMATION
Corresponding Author
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’
ACKNOWLEDGMENT
We thank Dr. R. Crouch (Medical University of South
(
Carolina, Charleston, USA) for a generous gift of 11-cis-retinal.
Dr. G. Vriend (Centre for Molecular BioInformatics CMBI,
Radboud University Nijmegen Medical Centre, The Nether-
lands) is acknowledged for expert assistance with interpretation
of the rhodopsin and bathorhodopsin structures using What-If-
YASARA (www.yasara.org). This research was supported by a
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DEDICATION
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Dedicated to Dr. Koji Nakanishi of Columbia University for his
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