Fluoro derivatives of retinal illuminate the decisive role of the C 12-H element in photoisomerization and rhodopsin activation

Article abstract of DOI:10.1021/ja907577p

Rhodopsin, the visual pigment of the vertebrate rod cell, is among the best investigated members of the G-protein-coupled receptor family. Within this family a unique characteristic of visual pigments is their covalently bound chromophore, 11-cis retinal,

Full text of DOI:10.1021/ja907577p

Published on Web 11/17/2009
Fluoro Derivatives of Retinal Illuminate the Decisive Role of
the C12-H Element in Photoisomerization and Rhodopsin
Activation
Petra H. M. Bovee-Geurts,† Isabelle Ferna´ndez Ferna´ndez,‡ Robert S. H. Liu,§
Richard A. Mathies,| Johan Lugtenburg,‡ and Willem J. DeGrip*,†,‡
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, Department of Chemistry,
UniVersity of Hawaii at Manao, 2545 The Mall, Honolulu, Hawaii 96822, and Department of
Chemistry, UniVersity of California, Berkeley, California 94720
Received September 7, 2009; E-mail: wdegrip@ru.nl
Abstract: Rhodopsin, the visual pigment of the vertebrate rod cell, is among the best investigated members
of the G-protein-coupled receptor family. Within this family a unique characteristic of visual pigments is
their covalently bound chromophore, 11-cis retinal, which acts as an inverse agonist. Upon illumination it
can be transformed into the all-trans isomer that acts as a full agonist. This photoisomerization process is
extremely efficient: 2 out of 3 photons are effective, full stereoselectivity is achieved, and stereoinversion
occurs within 200 fs. The mechanism behind this process is still not really understood, although the available
evidence points at the twisted C9-C13 segment of the 11-cis ligand as the quintessence. To further dissect
the role of this segment, we have generated the 10-fluoro, 12-fluoro, and 14-fluoro analogues of rhodopsin.
A fluoro substituent brings in only little more volume than hydrogen, but considerably more mass and
polarizability. The analogue pigments were compared to rhodopsin with respect to their photosensitivity
(quantum yield), light-induced structural transitions (UV-vis and FT-IR spectroscopy), and signaling activity
(G protein activation rate). We find that 14-F substitution is quite neutral, while 10-F and 12-F substitutions
exert significant but distinct effects. The 10-F pigment exhibits a quantum yield similar to that of rhodopsin
(0.65) but strongly perturbed thermodynamics of the structural transitions following photoactivation and
only 20% of the native signaling activity. The 12-F pigment exhibits a significantly decreased quantum
yield (0.47) and signaling activity (30%) but mixed effects on the structural transitions. These properties
are compared to those of the corresponding methyl derivatives. We conclude that rotation of the C12-H
bond of the rhodopsin chromophore is a major rate-limiting factor in the photoisomerization process, while
the C10-H moiety plays a dominant role in ligand relaxation and further rearrangements following
photoactivation.
formation of a protonated Schiff base1,4-6 that is stabilized by
a nearby located counterion in the form of a glutamate residue
Introduction
Rhodopsin is the visual pigment of the rod photoreceptor cells
in the vertebrate retina, which mediate dim-light vision.1-3 As
a member of the superfamily of G-protein-coupled receptors,
rhodopsin is specialized for reception of photons in such a way
that its ligand is the photosensory element (chromophore) and
has become covalently bound to the apoprotein, opsin. This
ligand is 11-cis retinal (Figure 1), a derivative of vitamin A,
which is attached in the opsin binding site to Lys-296 through
of opsin (Glu-113). The 11-cis isomer acts as a potent inverse
agonist of opsin, practically eliminating its basal activity, but
is converted by light into the all-trans configuration that acts
as a full agonist.2,7 This light-triggered isomerization of the
ligand induces conformational changes in the protein, which
are driven by about 35 kcal of photon energy stored in the
photoproduct Batho.8-10 This culminates within several mil-
liseconds via a cascade of intermediates in generation of the
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† Radboud University Nijmegen Medical Centre.
§ University of Hawaii at Manao.
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| University of California, Berkeley.
‡ Leiden Institute of Chemistry.
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A R T I C L E S
Bovee-Geurts et al.
twists during photoisomerization.17,31 Methyl substituents at these
positions moderately to severely affect the quantum yield of
the photoreaction, the thermodynamics of the subsequent thermal
reactions and the signaling activity of the photoactivated
pigment.32,33 On the other hand, the 11-19 ethano-derivative
of retinal, which severely restricts the rotational freedom of the
C10H moiety, only moderately affects the kinetics of the
photocascade.34,35 Hence, this questions whether photoactivation
triggers major structural rearrangements in the C9-C11 moiety
of the chromophore.
Figure 1. Chemical structure of 11-cis retinal (11-Z) and the fluoro
derivatives used in this study. The structures are shown in the 12-s trans
conformation, which is dictated by the opsin binding pocket.
To further elucidate the special contribution of the C10H and
C12H sites to photoactivation of rhodopsin, we introduced a
fluorine label at either position. This label is not much larger
than the hydrogen at this position but much smaller than a
methyl group (van der Waals radii of about 0.14, 0.12, and 0.20
nm, respectively36,37), while its molecular mass is even larger
than that of a methyl group (19.0 versus 15.0 Da). In addition
fluorine is a very sensitive NMR label.
Since methyl and fluoro derivatives at the C14 position hardly
affect the signaling properties of rhodopsin,38-40 we exploited
the 14-F analogue as a reference. The structures of native 11-
cis retinal and the three 11-cis fluororetinals used in this study
are presented in Figure 1. The 10-F, 12-F, and 14-F fluoroana-
logues of rhodopsin have been successfully prepared before,41,42
but their analysis has so far been restricted to their spectral
properties and to preliminary photochemical and solution-state
NMR studies.38,40-44
active state, Meta II, that binds and activates its cognate G
protein transducin.7,11 Next to their rate of formation, the
intermediates in this cascade (Batho T blue-shifted intermediate
(BSI) f Lumi f Meta I T Meta II) can be distinguished by
their spectral properties and can be isolated by cryotrapping.1,12-14
The photochemical performance of the 11-cis retinal-opsin
couple is quite remarkable. Photoisomerization occurs with an
unprecedented quantum yield (0.65 ( 0.02) in a truly selective
reaction pathway (11-cis f all-trans), generating within 200 fs
a vibrationally hot intermediate (photorhodopsin) with a highly
distorted but already all-transoid chromophore, which within 1
ps proceeds to Batho containing a still highly strained all-trans
chromophore.15-19
According to a variety of resonance Raman, solid-state NMR,
and computational studies, the C10H and C12H elements play special
roles in the photoisomerization of the rhodopsin chromophore.16,20-30
The crystal structure of the first photoproduct Batho also suggests
that the C10-H and C12-H bonds have proceeded through major
Our objectives are to characterize for the three fluororhodop-
sins the photoreaction (Rho f Batho) and subsequent thermal
reaction cascade generating Meta II in comparison to native
rhodopsin in order to further enlighten the contribution of the
C10H and C12H elements of the chromophore to photoisomer-
ization and activation of rhodopsin. We have determined the
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Photoisomerization and Rhodopsin Activation
A R T I C L E S
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 pigment) by incubation
for 1 h on ice. Undissolved material was removed by centrifugation
(30 min, 80,000g, 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 of inubation on ice, detergent was
extracted by addition of solid ꢀ-cyclodextrin to a slight excess over
nonylglucose, and the resulting proteoliposomes were isolated by
overnight sucrose step-gradient centrifugation at 200,000g and 4
°C as described before.53 The proteoliposome band was recovered
from the 20%/45% sucrose interface, diluted with 2 vol of doubly
distilled water, pelleted by centrifugation (60 min, 200,000g, 4 °C),
and stored in aliquots under argon 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 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.
The Meta I T Meta II equilibrium was analyzed in proteolipo-
somes 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 taken 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 photoproducts. Finally, 1 M hydroxylamine
was added to a final concentration of 50 mM to convert all
photointermediates into the all-trans retinaloxime derivative. The
relative amount of Meta I at 480 nm remaining immediately after
illumination was calculated as described before.54
FT-IR Spectroscopy. FT-IR analyses were performed on a
Bruker IFS 66/S spectrometer, equipped with a liquid nitrogen-
cooled, narrow-band HgCdTe (MCT) detector as described.32,33
In brief, sample temperature was under computer control using a
variable-temperature helium-cooled cryostat (Heliostat, APD Cryo-
genics, Inc.), covered with a set of NaCl windows in the IR light
path. Membrane films were pepared by isopotential spin drying55
2-3 nmol of pigment in proteoliposomes on AgCl windows
(Crystran Limited, U.K.). 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 fiber optics
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-1 resolution, taking about 120 s acquisition time
per block to generate a spectrum, both before and after 2 min of
illumination. 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. Habitually, difference spectra were measured
at 80, 180, 253, and 283 K, where Batho, Lumi, Meta I, and Meta
II, respectively, are sufficiently stable to allow analysis, but if
required the temperature range was expanded. Temperature stability
was (0.2 K.
quantum yield (Φ) as a measure for the efficiency and rate of
the photoisomerization reaction.16,45,46 The entire photocascade
was monitored by means of FT-IR spectroscopy at selected
temperatures, since this is the most straightforward way to
discriminate between the various intermediate states of the
cascade.47-50 The signaling activity was assayed as the G protein
activation rate per photoactivated pigment relative to that of
native rhodopsin.
Experimental Section
Materials. All chemicals were of analytical grade. Detergents
were obtained from Anatrace (Maumee, OH). Native 11-cis retinal
was provided by Dr. Rosalie Crouch (Medical University of South
Carolina, Charleston, SC) through financial support from NEI.
Synthesis of Retinals. The 10-F and 14-F derivatives of all-
trans retinal were synthetised as described before.41 The corre-
sponding 11-cis isomers were prepared by irradiation of the all-
trans isomer in acetonitrile and purified by preparative high
performance liquid chromatography.51 The 12-F derivative was
prepared in the 11-cis configuration also as described before.42 In
this case the C15 precursor was synthesized as a mixture of the
all-trans and 11-cis isomers. The 11-cis isomer was isolated by
chromatography and extended into the corresponding retinal. The
spectral and 1H NMR characteristics of the three 11-cis fluororeti-
nals fully complied with published data.38,41,42
Isolation of Bovine Opsin and Regeneration with 11-cis
Retinals. Bovine rod outer segment membranes in the opsin form
(opsin membranes) were prepared from fresh, light-adapted cattle
eyes as described.32,52 The regeneration capacity of these prepara-
tions was estimated from the A280/A500 ratio measured after
subsequent incubation with a 3-fold excess of 11-cis retinal,
whereby a ratio of 2.1 ( 0.1 was taken to represent membranes
with a maximal rhodopsin content. Rhodopsin and the fluoro-
rhodopsin analogues were prepared with opsin membranes showing
a regeneration capacity in the range 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 PIPES,
130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 0.1 mM EDTA, 1 mM
DTE, pH 6.5) with a 2- to 3-fold molar excess of the 11-cis
fluororetinal at room temperature. After 2 h a small aliquot was
assayed for the extent of regeneration by addition of 11-cis retinal
in a 1:1 molar ratio to the original opsin. With all three fluororetinals
regeneration was nearly complete; to achieve full regeneration an
additional aliquot of the fluororetinal was added, and the incubation
was continued overnight at 4 °C. Excess retinal was then converted
into the corresponding oxime by addition of a 1 M hydroxylamine
solution (pH 6.5) to a final concentration of 10 mM. After cooling
in ice and 30 min of incubation, the oxime was largely removed
by two extractions with 50 mM heptakis(2,6-di-O-methyl)-ꢀ-
cyclodextrin,32 which, however, also removes some lipids thereby
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A R T I C L E S
Bovee-Geurts et al.
Table 1. Spectral and Photochemical Properties and Relative G Protein Activation Rates of Rhodopsin and Its Fluoro Derivatives Discussed
in This Paper
native rhodopsin
10-F
12-F
14-F
λ
max(nm)
max (L/M·cm)
498 ( 2
40,600 ( 1,000
0.65 ( 0.02a
≡ 100
502 ( 2
40,000 ( 3,000
0.63 ( 0.12b
20 ( 10
510 ( 3
34,000 ( 4,000
0.47 ( 0.06
32 ( 5
529 ( 3
40,000 ( 3,000
0.55 ( 0.10b
g80c
ε
quantum yield
G protein activation (%)
a Native quantum yields have been reported (refs 56, 58, 59). b Quantum yields of 10-F and 14-F rhodopsin have been reported before (ref 38).
c Signaling activity of 14-substituted rhodopsins has been reported before (refs 40, 41).
Determination of Quantum Yield. The photoisomerization
quantum yield (Φ) of 12-F rhodopsin was determined relative to
rhodopsin, of which the Φ (0.65 ( 0.02) and molar absorbance
(ε498 ) 40,600 ( 500 M-1 ·cm-1) are well-established.56-59
Rhodopsin and 12-F rhodopsin were solubilized in buffer A
containing 10 mM DDM and 10 mM hydroxylamine to give an
OD·cm-1 at 497 nm of 0.150 ( 0.07. Samples were kept at 10 °C
and showed no significant decrease of A497 after 2 h in the dark.
Samples were illuminated through a 504 ( 5 nm interference filter
(Schott) such that the half time of bleaching was g30 min. Spectra
were taken at intervals of 2-10 min. The data were converted to
a straight line, the slope of which (S) is a measure of the
photosensitivity ε504 ·Φ, as described before.32,56 Using the S of
rhodopsin measured under identical conditions allows calculation
of the Φ of the analogue pigment.32
Transducin Activation. Activation of the rhodopsin-associated
G protein transducin was determined at 20 °C using a fluorescence
assay as described.60,61 A hypotonic extract of isotonically washed
bovine rod outer segments served as the source of transducin.11
Figure 2. Spectral properties of native and fluoro rhodopsins prepared as
described in the Experimental Section. Difference spectra were obtained
Samples contained 2, 5, or 10 nM pigment and about 100 nM
by subtracting the spectrum after illumination from the dark-state spectrum.
transducin in 20 mM HEPPS, 100 mM NaCl, 2 mM MgCl2, 1 mM
Illumination was performed in the presence of 10 mM hydroxylamine to
DTE, 0.01% (w/v) DDM, pH 7.4. Immediately before data
ascertain full conversion of pigment, with production of the all-trans
retinaloxime derivatives. The spectra were normalized at the maximal
acquisition a sample was illuminated for 5 min in bright white light,
and when a steady fluorescence level was reached, GTPγS was
added to a final concentration of 2.5 µM. The initial rate in the
increase in tryptophan fluorescence of the R-subunit of transducin,
induced by binding of GTPγS, was plotted against the pigment
concentration, and the slope of the resulting straight line was rated
against the slope obtained for rhodopsin in the same set of
experiments.
absorbance of the main absorbance band of the pigments near 500 nm.
absorbance (εmax) of the fluoropigments could be estimated with
about 10% accuracy. The εmax of 10-F and 14-F rhodopsin was
similar to that of the native pigment (Table 1). However, the
result for the 12-F analogue is an approximately 16% reduction
in εmax relative to rhodopsin, which in fact is in good agreement
with the 18% reduction reported before.42 A reduced oscillator
strength can be deduced from Figure 2 for the free 12-F
retinaloxime as well and hence probably is caused by a specific
interaction between the 12-F substituent and the polyene system.
Earlier we reported a 6-fold reduction in regeneration rate
for the 10-methyl pigment32 and an at least 200-fold reduction
with low incorporation level for the 12-methyl pigment.33 It is
most likely that their steric properties underlie this difference
between the fluoro and methyl pigments. In 11-cis 10-methyl
retinal it probably is due to an intrinsic ligand constraint, the
steric interaction between the C10-methyl and the C13-methyl
in the 12-s trans conformation dictated by the opsin binding
pocket. Indeed, it was shown that the torsion in the C10-C13
segment of retinal has significantly increased in 10-methyl
rhodopsin.62 In the case of 12-methyl retinal it probably reflects
steric interaction of the methyl group with the backbone CdO
oxygen of opsin residue Cys-187. According to the highest
resolution structure available (2.2 Å63), the carbonyl oxygen
C187-CdO is located at a distance of about 3.2 Å of C12
pointing toward the C12-H bond under an angle of ca. 100°,
Results and Discussion
Regeneration and Spectral Properties of the Corres-
ponding Fluoro-rhodopsins. In agreement with earlier data for
the 12-F pigment,42 fluoro substituents at position C10, C12, or
C14 only slightly reduced the rate of pigment formation, relative
to that of unsubstituted 11-cis retinal, when incubated at a 2-
to 3-fold molar excess with opsin. Exact rates were not
measured, but pigment formation was 90-95% complete after
a 2 h incubation at room temperature. When fluoropigment
formation had leveled, no additional rhodopsin was generated
upon subsequent incubation with 11-cis retinal, indicating that
maximal pigment formation was achieved. From the resulting
increase in absorbance at the peak wavelength relative to that
obtained with 11-cis retinal in parallel experiments, the molar
(56) Dartnall, H. J. A. In Photochemistry of Vision, Handb. Sens. Physiol.;
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9
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Photoisomerization and Rhodopsin Activation
A R T I C L E S
Figure 4. pH dependence of the Meta I T Meta II equilibrium in native
rhodopsin (solid line without data points) and the three fluoro analogues
(symbolized as indicated) measured at 10 °C (283 K). The Rho and 14-F
data could be fitted with a regular Henderson-Hasselbalch function. The
10-F and the 12-F data show a much steeper transition, which renders
determination of a single pKa value not reliable.
Meta II difference spectra (Figure 3), where the fingerprint
region (C-C stretch vibrations) is split up into two sets of
vibrations around 1233 and 1310 cm-1. A similar effect is
observed for the chromophore HOOP vibrations in rhodopsin
that are split in two bands at 968 and 935 cm-1. We postulate
Figure 3. FT-IR difference spectra of native (dotted lines) and 14-F
rhodopsin (continuous lines) and photoproducts generated by illumination
at the temperatures indicated. Difference spectra were constructed by
subtracting the dark-state spectrum from the spectrum obtained during 2
min after illumination. Negative bands represent vibrational bands charac-
teristic for the dark state; positive bands represent characteristic vibrations
of the photoproduct. The native Rho f Batho difference spectrum taken at
80 K exhibits the typical, isolated HOOP pattern of Batho between 800
and 950 cm-1, reflecting the highly strained structure of the all-trans
chromophore in Batho.
64-66
that the C7dC8 Au HOOP vibration remains at 968 cm-1
and that the C11dC12 A2 HOOP vibration is downshifted to 935
cm-1. The pattern of isolated HOOP vibrations in Batho between
800 and 950 cm-1 has also changed in 14-F Batho since the
C14-H HOOP is lacking, which has a frequency of about 850
cm-1 in native Batho (Figure 3, upper dotted spectrum). In
contrast, the set of small bands around 1655 cm-1, representing
the minor alterations in protein conformation accompanying the
Rho f Batho transition, shows a slightly different intensity
distribution in the 14-F pigment, but no change in frequency
distribution. Finally, it is noteworthy that the C-F stretch of
ethylenic fluoro compounds appears as a medium strong
vibration in the frequency range between 1050 and 1200 cm-1
and is also quite sensitive to H-bonding and the polarity of its
microenvironment.67-70 However, in this frequency region we
find no clear evidence for additional peaks in difference spectra
between 14-F rhodopsin and its photointermediates, indicating
that there is no obvious shift in intensity or frequency of the
C14-F vibration during the photocascade.
which allows about sufficient space for a fluoro substituent but
is clearly within van der Waals distance of a methyl group (van
der Waals radius of about 1.4 and 2.0 Å, respectively, bond
length for C-F and C-CH3 of 1.34 and 1.50 Å, respectively).
The peak absorbance in the visible region (λmax) we measure
for the fluoro-pigments (Table 1) also is consistent with earlier
data.38,41,42 All fluoro-analogues exhibit a red shift of the main
absorbance band relative to that of rhodopsin (Figure 2) that
increases in the order 10-F < 12-F < 14-F, i.e., the closer the
fluoro-substituent is located to the Schiff base, the larger the
red shift. This is most likely due to destabilization of the ground
state because of the perturbation of the charge distribution in
the polyene positron by the fluoro substituent, which will have
its largest effect at close range.
Photochemistry and Activity of 10-F Rhodopsin. In contrast
to 14-F substitution, the 10-F substituent exerts strong effects
on the signaling activity of rhodopsin (Table 1, Figures 5 and
6). The photoisomerization process is not much affected, since
the quantum yield is close to that of native rhodopsin38 and the
Photochemistry and Activity of 14-F Rhodopsin. Our FT-IR
analyses agree with earlier data in that 14-F substitution does
not significantly affect photoisomerization and signaling activity
of rhodopsin.38-40 The normal sequel of intermediates is
observed, culminating in a final intermediate that shows the
typical vibrational band pattern of Meta II in the 1550-1800
cm-1 region (Figure 3, lower 14-F spectrum). A major effect
of the 14-F substituent is the large upshift of the pKa of the
Meta I T Meta II equilibrium (Figure 4, triangles), which was
also observed for 14-F isorhodopsin.40 This can be explained
by the reduction in electronegativity of the Schiff base because
of the electron withdrawing effect of the nearby fluoro sub-
stituent.44
(64) Eyring, G.; Curry, B.; Broek, A.; Lugtenburg, J.; Mathies, R. A.
Biochemistry 1982, 21 (2), 384–393.
(65) Palings, I.; VanDenBerg, E. M. M.; Lugtenburg, J.; Mathies, R. A.
Biochemistry 1989, 28, 1498–1507.
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2787–2806.
(67) The Sadtler Handbook of Infrared Spectra; Sadtler Research Labo-
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Khim. 1969, 12, 2631–2633.
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Tarakeshwar, P.; Kim, K. S. J. Chem. Phys. 2000, 112 (4), 1844–
1858.
In addition, it is expected that a 14-F substituent will also
change the vibrational band pattern of the chromophore. This
is indeed obvious in the 14-F Rho f Batho and 14-F Rho f
(70) Kryachko, E. S.; Karpfen, A. Chem. Phys. 2006, 329 (1-3), 313–
328.
9
J. AM. CHEM. SOC. VOL. 131, NO. 49, 2009 17937

A R T I C L E S
Bovee-Geurts et al.
Figure 6. FT-IR difference spectra of native (dotted lines) and 10-F
rhodopsin (continuous lines) and photoproducts generated by illumination
at the temperatures indicated. Difference spectra were constructed by
subtracting the dark-state spectrum from the spectrum obtained during 2
min after illumination. Negative bands represent vibrational bands charac-
teristic for the dark state; positive bands represent characteristic vibrations
of the photoproduct. The native spectra were taken at pH 5.5 to accentuate
the differences between Meta I and Meta II. The 10-F spectra were taken
at pH 6.5. The Meta I to Meta II difference spectra are stable over at least
12 min and hence represent the equilibrium state.
Figure 5. FT-IR difference spectra of native (dotted lines) and 10-F
rhodopsin (continuous lines) and photoproducts generated by illumination
at the temperatures indicated. Difference spectra were constructed by
subtracting the dark-state spectrum from the spectrum obtained during 2
min after illumination. Negative bands represent vibrational bands charac-
teristic for the dark state; positive bands represent characteristic vibrations
of the photoproduct. The native Rho f Batho difference spectra taken at
80 K shows the typical isolated HOOP pattern of Batho between 800 and
950 cm-1, reflecting the highly strained structure of the all-trans chro-
mophore in Batho. This pattern represents the HOOPs from C11-H (921
cm-1), C10-H (874 cm-1), C12-H (854 cm-1), C14-H (848 cm-1), and
HC7dC8H (838 cm-1).
Similar observations were made for the Meta I to Meta II
transition. Normally, Meta I decays into Meta II at temperatures
over 255 K, under formation of a Meta I T Meta II equilibrium
with a temperature- and pH-dependent equilibrium constant.7,73,74
However, 10-F Meta I is still stable at 10 °C and a pH of 6.5
(283 K; Figure 6) as evident from the typical Meta I vibrations
at 1665, 1538, and 943 cm-1. Formation of 10-F Meta II is
only clearly observed at temperatures g20 °C (293 K; Figure
6). Probably, the activation energy for the Meta I to Meta II
transition has increased significantly, reducing the proportion
of Meta II in the relevant temperature range relative to that in
the native system. This aberrant behavior is further reflected in
the pKa of the Meta I T Meta II equilibrium, which is
significantly downshifted relative to native rhodopsin (Figure
4, filled circles). The decrease in pKa, observed for the 10-F
analogue, in combination with the stronger temperature depen-
dence of the Meta I T Meta II equilibrium will be responsible
for the strong reduction in signaling activity per photolyzed
rhodopsin, to about 20% of that of native rhodopsin (Table 1).
The signaling activity is assayed at 20 °C and pH 7.4, and under
these conditions the 10-F analogue will strongly prefer the Meta
I state.
FT-IR difference spectrum of the 10-F Rho f Batho transition
(Figure 5, middle spectrum) is very similar to that of native
rhodopsin, except for downshifts of HOOP vibrations and lack
of the C10-H HOOP, which is represented by the 874 cm-1
vibration in the native spectrum (Figure 5, top spectrum). Again,
we do not find evidence for additional peaks that may arise in
a change of the C10-F vibration between 10-F rhodopsin and
photoproducts.
The effects of the 10-F substituent are most conspicuous in
the temperature dependence of the thermal transitions (Batho
f Lumi f Meta I T Meta II) and in the pKa of the Meta I T
Meta II equilibrium (Figures 4, 5, and 6). An upshift in all
transition temperatures is observed. Typical Batho signals,
normally decaying into Lumi at temperatures over 135 K, are
still completely intact at 180 K in the case of the 10-F analogue
(Figure 5). This increase in stability of the 10-F Batho
intermediate corroborates earlier observations by electronic
cryospectroscopy,43 except that in that case 10-F Batho already
started to decay near 150 K. Probably, this discrepancy
originates in the different conditions applied. While our
vibrational data were obtained for native membrane preparations,
the visible spectroscopy was done on digitonin extracts in the
presence of 66% glycerol, and a detergent environment usually
reduces the activation barrier for conformational changes in
membrane proteins.71,72
The change in properties induced by 10-F substitution
(thermal upshift of reaction cascade transitions and delayed
formation of Meta II, leading to a significant lower G protein
activation rate) is remarkably similar to those reported for 10-
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Photoisomerization and Rhodopsin Activation
A R T I C L E S
methyl rhodopsin.32 Here as well, Batho is stable up to about
200 K, at pH 6.5 Meta II formation is only observed at
temperatures g20 °C, and the G protein activation rate at 20
°C is strongly reduced, again to about 20% of native rhodopsin.
Since a fluoro substituent is not much larger than hydrogen,
but much smaller than a methyl group, it seems unlikely that
steric effects are an important factor in the mechanism underly-
ing the higher thermal stability of both the 10-F and 10-methyl
photoproducts. Examination of the available Batho structure
(PDB 2G8717) also does not reveal obvious candidates among
the protein residues that would obstruct further relaxation of
the twisted chromophore due to steric crowding with the
additional methyl group at C10. The latter may have to cope
with quite a weak interaction with the ring of Try-265, but that
certainly would not be an issue for a fluoro substituent at C10.
On the other hand, the fluoro and methyl substituent have in
common a much higher molecular mass relative to hydrogen,
and a mass effect therefore could be a common denominator in
their effect on the photocascade. That may imply that relaxation
of the twisted all-trans chromophore in the Batho to BSI
transition would involve further localized rotation of the C10-H
element. It has been argued that the additional mass of the
methyl group in 12-methyl rhodopsin would slow down the
rotational movement of the C12-H element and thereby the rate
of photoisomerization, which can explain the decrease in
quantum yield.33 In a similar way, the additional mass in 10-F
and 10-methyl Batho may increase the activation barrier for
further relaxation of the chromophore. This would corroborate
time-resolved resonance Raman observations, indicating that
decay of Batho involves further relaxation of the C10-C12 unit
of the chromophore.16,75 It would be interesting to see whether
computational and molecular dynamics studies will be able to
shed more light on this issue.
Figure 7. FT-IR difference spectra of native (dotted lines) and 12-F
rhodopsin (continuous lines) and photoproducts generated by illumination
at the temperatures indicated. Difference spectra were constructed by
subtracting the dark-state spectrum from the spectrum obtained during 2
min after illumination. Negative bands represent vibrational bands charac-
teristic for the dark state; positive bands represent characteristic vibrations
of the photoproduct. Native Rho f Batho difference spectra taken at 20 or
80 K are identical and exhibit the typical, isolated HOOP pattern of Batho
between 800 and 950 cm-1, reflecting the highly strained structure of the
all-trans chromophore in Batho. The 12-F spectra present unique, strong
vibrations at 1395 and 1098 cm-1, which derive from the 12-F rhodopsin
state, and are further discussed in the text.
Remains the observation that the 10-F and 10-methyl substituents
also have profound effects on the temperature dependence of the
Meta I T Meta II equilibrium. This transition involves a variety
of protein conformational changes,2,10,48,49,74,76-85 and it is hard
to imagine that the additional mass of a fluoro or methyl
substituent in the ligand could play a major role here, unless
additional rotational rearrangement in the ligand is involved,
for which there is some evidence.86 Alternatively, these sub-
stituents could be at a critical position in the ligand to impair
by steric interaction (methyl substituent) or by dipolar interaction
(fluoro substituent) the H-bonding network rearrangements
accompanying Meta II formation.
Photochemistry and Activity of 12-F Rhodopsin. The most
diverse intriguing effects are observed upon 12-F substitution
(Figures 7 and 8) and involve perturbation of photoisomerization
as well as of signaling activity of rhodopsin. The quantum yield
shows the largest decrease among the three fluororhodopsins
studied here (Table 1). This is a strong indication that the rate
of the photoisomerization process is significantly reduced only
in the 12-F analogue. In addition, the G protein activation rate
is strongly reduced as well, to about 32% of that of rhodopsin
(Table 1).
The quantum yield we measure for the 12-F pigment (0.47)
is substantially lower than that of the 12-methyl pigment
(0.55).33 An important factor is probably the larger mass of the
fluoro substituent in comparison to a methyl group (19 and 15
Da, respectively), which according to the same argument
mentioned above and detailed before33 would further slow down
the out-of-plane rotational movement of this C12 bond, essential
for driving the photoisomerization process, and hence would
further reduce the quantum yield. It is questionable however,
whether this could lead to the substantial decrease in quantum
(75) Pan, D. H.; Ganim, Z.; Kim, J. E.; Verhoeven, M. A.; Lugtenburg,
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A R T I C L E S
Bovee-Geurts et al.
knowledge. We reasoned that it might represent a transition state
between Batho and the next intermediate. Therefore we
performed similar analyses for native and 12-F rhodopsin at
130 and 135 K, a temperature traject where Batho was reported
to directly decay into Lumi.12,13 The BSI intermediate has been
observed by cryotrapping only in rhodopsin analogues so far,
since in rhodopsin the Batho to BSI transition is entropy-driven
and at low temperatures the equilibrium strongly prefers the
Batho state.87-91 Unexpectedly, we encountered a condition
where illumination of native rhodopsin (2 min, OG530 cutoff
filter, 130 K) produced a photoproduct with a, compared to
Batho, slightly changed protein pattern around 1650 cm-1, a
quite different CdC stretch pattern around 1550 cm-1 and a
shifted and strongly reduced HOOP pattern between 800 and
950 cm-1 (Figure 7, bottom spectrum). This typical pattern had
fully developed at about 4 min after illumination and remained
stable for at least 6 min, and is quite reminiscent of that observed
for the BSI intermediate in analogues of rhodopsin by FT-IR
cryospectroscopy and in native rhodopsin by time-resolved
resonance Raman spectroscopy at 7 °C.75,88,92 Hence, we assume
this is the first demonstration of native BSI by cryotrapping.
Interestingly, applying the same conditions to the 12-F pigment
resulted in a photoproduct with very similar characteristics to
native BSI, taking the strong, 12-F inflicted downshift of the
HOOP vibrations into account. Therefore, we assign this to the
12-F BSI intermediate, implying that illumination of 12-F
rhodopsin generates a 12-F Batho analogue that is stable at 20
K but not at 80 K and at 130 K rapidly decays into the 12-F
BSI analogue. When comparing the 12-F difference spectra at
20, 80, and 130 K (Figure 7), only minor changes in protein
pattern around 1650 cm-1 and in fingerprint pattern around 1200
cm-1 are obvious, while there is a clear frequency shift in the
CdC pattern near 1540 cm-1 and a frequency shift and intensity
change in the HOOP pattern between 800 and 870 cm-1. The
overall pattern at 80 K seems to be a mixture of that at 20 and
at 130 K, as is evident in particular in the CdC and fingerprint
patterns. Our interpretation is that 12-F Batho is in equilibrium
with 12-F BSI with a temperature-dependent equilibrium
constant, which is still very small at 20 K and has become very
large at 130 K. Similar to the native system the transition from
12-F Batho to 12-F BSI mainly involves vibrational alterations
in the chromophore, reflecting further relaxation of the highly
strained conformation.14,75 The temperature dependence of this
equilibrium is strongly affected by the 12-F substitution,
however, which suggests that the enthalpic component of this
equilibrium has increased its weight. It points at a higher thermal
stability of 12-F BSI relative to that of 12-F Batho as compared
to the native situation, which may also bear on the higher
stability of the 12-F Lumi intermediate relative to native Lumi,
described below.
Figure 8. FT-IR difference spectra of native (dotted lines) and 12-F
rhodopsin (continuous lines) and photoproducts generated by illumination
at the temperatures indicated. Difference spectra were constructed by
subtracting the dark-state spectrum from the spectrum obtained during 2
min after illumination. Negative bands represent vibrational bands charac-
teristic for the dark state; positive bands represent characteristic vibrations
of the photoproduct. The 12-F spectra present unique, strong vibrations at
1395 and 1098 cm-1, which derive from the 12-F rhodopsin state, and are
further discussed in the text.
yield we have measured (0.55 f 0.47). An additional factor
may be dipolar and/or H-bonding interaction of the fluoro
substituent with its microenvironment, for which we have
indirect evidence (see below), that might dampen or counteract
this out-of-plane rotation and thereby also impair the rate of
isomerization.
We observe mixed effects of 12-F substitution on the
photocascade. At 80 K no typical Batho pattern was observed,
in as far as the characteristic and stable HOOP vibrations in
the 800-930 cm-1 region are absent (Figure 7, middle 12-F
spectrum). Instead, this region was quite noisy and not very
reproducible between experiments. However, we did observe
stable HOOP vibrations when we cooled down to 20 K before
illumination. In fact, the 20 K photoproduct of 12-F rhodopsin
has all the characteristics of Batho. Typical for 12-F Batho are
the downshifted HOOP vibrations and the absence of the 12-
HOOP, in native Batho present as an 850 cm-1 vibration (Figure
5, top spectrum). For instance, the strong Batho 11-HOOP has
shifted from 921 to 830 cm-1 (Figure 7, top 12-F spectrum).
Likewise, the C7dC8 and C11 HOOPs in 12-F rhodopsin have
shifted to 884 and 836 cm-1. This is most obvious in the 12-F
difference spectra at 80 and 283 K (Figure 7, top 12-F and
Figure 8, bottom 12-F spectrum, respectively), since in the other
spectra the 836 cm-1 peak is less evident because of partial
overlap with positive peaks in the photoproduct.
Our data can explain results from an early study on the
photocascade of 12-F rhodopsin, employing UV-vis cryospec-
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The unstable pattern between 800 and 900 cm-1 in the 12-F
photoproduct at 80 K has not been described before, to our
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Photoisomerization and Rhodopsin Activation
A R T I C L E S
troscopy, which concluded that progress and temperature
dependence of the photointermediates were very similar to that
of rhodopsin, converse to our results.43 In this case this
discrepancy is probably not mainly due to the less natural
experimental conditions employed in that study (digitonin
solubilized pigment, cryoprotected with 66% glycerol). In fact,
close inspection of the published data reveals that at 82 K, the
lowest temperature investigated, the λmax of the photosteady-
state mixture is red-shifted by only 3 nm relative to that of 12-F
rhodopsin, much less than in the case of native rhodopsin (ca.
14 nm), and the extent of 12-F Batho formation seems to be
much smaller than with native rhodopsin. In line with our data,
we can now interpret this in the sense that at 82 K a mixture of
12-F rhodopsin, 12-F Batho, and 12-F BSI is present, which
reduces the red shift of the λmax of the photomixture as well as
the proportion of Batho, relative to the situation for native
rhodopsin where BSI is not generated. At temperatures over
130 K, 12-F BSI will then decay into 12-F Lumi. In this way,
the results of both studies can be reconciled.
At 180 K the 12-F photoproduct exhibits a typical Lumi
pattern, similar to native (Figure 8, two top spectra). This is
for instance apparent in the change in CdC and fingerprint
patterns and in the simple HOOP pattern, as compared to Batho,
reflecting a more relaxed all-trans chromophore, and in the
strong 1655 cm-1 signal reflecting changes in the interaction
between the protonated Schiff base and the counterion
(Glu-113).93-95 Remarkably, in the 12-F system this pattern
persists at 253 K, where normally Meta I has been formed
(Figure 8, two middle spectra). This indicates that 12-F Lumi
is thermally more stable than native Lumi. Since a similar
situation seems to pertain to 12-F BSI, one explanation could
be that the 12-F substituent in the more relaxed all-trans
chromophore is interacting weakly with protein elements lining
the binding site. This will be discussed in more detail below.
At temperatures over 273 K a typical pH-dependent Meta I T
Meta II equilibrium is observed for the 12-F system by UV-vis
spectroscopy (Figure 4), and at 283 K (10 °C) and pH 5.5 the
typical Meta II pattern is clearly present in FT-IR difference
spectra (Figure 8, two bottom spectra). We conclude that the
temperature dependence of the Meta I T Meta II equilibrium
is not markedly perturbed by 12-F substitution.
temperature dependence of the Meta I T Meta II equilibrium
in the 10-F pigment that will only slowly increase the proportion
of Meta II at increasing temperature.
Among the three fluoroderivatives studied in this paper, the
12-F pigment shows two unique features in the FT-IR difference
spectra with its photoproducts. Those are the strong rhodopsin
peaks at 1395 and 1098 cm-1 (Figures 7 and 8). We have not
been able to assign the 1395 cm-1 vibration yet and cannot
exclude that it reflects an increase in intensity of the small peak
at about the same frequency already present in the native FT-
IR difference spectra, which probably represents the 13-methyl
symmetric deformation.66 The strong vibration at 1098 cm-1 is
a novel feature, however, and most likely represents the C-F
stretch vibration in the rhodopsin state. In free 12-F 11-cis retinal
we have observed an among retinals unique medium strong
vibration at 1084 cm-1 (not shown) and tentatively assign this
to the C12-F stretch. The shift in frequency and strong intensity
in rhodopsin can be due to several factors. For instance, in
rhodopsin the chromophore is twisted around the C11dC12 and
the s-trans C12-C13 bonds. Further, we modeled a fluoro-atom
at position C12 in the rhodopsin structure (1U1963) and this
seems to experience quite a polar environment with a distance
of 2.5-3 Å to a nearby water molecule (wat2014) and about
2.5 Å to the carbonyl oxygen of Cys-187. Dipolar and/or H-bond
interaction with these constituents in the protein structure may
also explain the relative strong downshift reported for the 12-F
resonance in early F-NMR studies (chemical shift of -92 ppm
for 12-F rhodopsin versus -107 ppm for free 11-cis 12-F
retinal42,99). During the photoisomerization process the C-F
bond rotates and the fluoro substituent moves away from this
polar environment. Taking the published Batho structure
(2G8717) we estimate that the F atom has moved away over at
least 1 Å from the water molecule and the C187 CdO and has
moved toward the Cꢀ of Ala-117. Rearrangements in at least
three internal water molecules during the rhodopsin to Batho
transition have been well documented.100 A corresponding
change in dipolar interactions may contribute to a frequency
shift and/or change in intensity of the C-F vibration.
In addition, close juxtaposition of the 12-F atom to the Cꢀ of
Ala-117 may destabilize the 12-F Batho state, facilitating the
transition to 12-F BSI. However, in the absence of sufficiently
high-resolution structures of Batho and later intermediates, the
molecular mechanism underlying the various effects of 12-F
substitution on the photocascade remains speculative.
Another major difference with the native system lies in the
large shift in the pH dependence of the 12-F Meta I T Meta II
equilibrium (Figure 4, open circles). Such shifts are observed
for many retinal derivatives,32,33,40,50,92,95-98 and it explains the
lower signaling activity of 12-F rhodopsin relative to native
rhodopsin, since this activity is assayed at 20 °C and pH. 7.4,
conditions where the relative proportion of 12-F Meta II is much
lower than that of native Meta II. At 10 °C the pH dependence
curve of the 12-F Meta I T Meta II equilibrium is even further
shifted away from native than that of 10-F. Nevertheless, the
signaling activity of 12-F rhodopsin surpasses that of 10-F
rhodopsin (Table 1). The reason probably lies in the unfavorable
In contrast to substitution at C10, quite different patterns are
observed for methyl and fluoro substitution at C12. As discussed
above, we attribute the difference in the photoisomerization
process of the 12-methyl and 12-F pigments to the distinction
in mass and polarity of the two substituents. The remarkable
deviation in the subsequent reaction cascade of the 12-methyl
pigment,33 we rather attribute to steric effects. A methyl group
at C12 will encounter severe steric interaction in the Batho state
with Cꢀ of Ala-117 and also with the backbone CdO of the
same residue. This will probably force the photoactivated
binding pocket to first relay photon energy into a different strain
distribution in the chromophore and subsequently dissipate it
through an altered conformational pathway in the protein moiety.
The markedly distinct FT-IR difference spectra observed through
the entire 12-methyl photocascade33 would concur with such a
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J. AM. CHEM. SOC. VOL. 131, NO. 49, 2009 17941

A R T I C L E S
Bovee-Geurts et al.
situation. For instance, at 80 K the vibrational patterns of both
protein and chromophore are markedly altered as compared to
those of the native as well as the 12-F rhodopsin, and there is
no evidence for intensified HOOP vibrations in the photoprod-
uct. Both the protein and chromophore patterns remain very
atypical at later stages in the 12-methyl photocascade, a normal
Meta II intermediate is not observed and signaling activity is
practically absent.33 A novel feature, a medium strong positive
peak at 1003 cm-1, was observed at early stages in the 12-
methyl cascade and was tentatively assigned to a C12-methyl
wag.33 This would agree with our present perspective that steric
interaction of the C12-methyl group with protein residues
dominates the fate of the photocascade of 12-methyl rhodopsin.
structure, which may further slow down the out-of-plane rotation
and reduce the quantum yield.
The reaction cascade following photoisomerization into Batho
is affected both by 10-F and by 12-F substitution. The effect of
12-F substitution is relatively minor, however. It is probably
connected to specific interactions of the 12-F moiety with its
environment that affect the thermal stability of Batho and Lumi,
but eventually a Meta II state is generated in the normal
temperature range. On the contrary, 10-F substitution has
pronounced effects in the sense that the temperature range of
all transitions is shifted upward substantially. Considering the
small additional size of a fluoro substituent over hydrogen it is
unlikely that steric crowding is a major issue here. Further, the
same phenomenon is also observed with the 10-methyl pig-
ment,32 and the most obvious property these substituents have
in common, is their much larger mass than hydrogen. This would
suggest that specific conformational relaxation and rearrange-
ment steps continue in the chromophore up to formation of Meta
II, and that a local increase of mass may put an additional energy
requirement in the early transitions of the reaction cascade.
Summary
Inspired by the strongly perturbing action of 10-methyl and
12-methyl substituents on photocascade and signaling activity
of rhodopsin, we here report on the corresponding fluoro
derivatives that also add significant mass to the chromophore,
but are much smaller in size. The 14-F derivative did not
significantly impair rhodopsin properties, in agreement with
earlier studies,38,40,41 indicating that fluoro substitution by itself
need not have a perturbing action. However, both 10-F and 12-F
substitution have complex effects on several rhodopsin properties.
A common effect is that they both significantly reduce the
signaling activity to 20 and 32% of native rhodopsin, respec-
tively. As such, the 10-F and 12-F all-trans chromophore both
classify as partial agonists. A major cause of this lower activity
is a significant downshift of the pH-sensitivity curve of the Meta
I T Meta II equilibrium (Figure 4), resulting in a substantial
decrease in the proportion of the active Meta II state generated
at physiological pH. Such a downshift is a common feature in
modified chromophores, but the underlying molecular mecha-
nism is still unclear and more detailed structural information
on all photointermediates is needed.
Our data again illustrate the very subtle interplay between a
ligand and its cognate G protein coupled receptor and extend
the examples that small modifications in the ligand can modify
this interplay and thereby redirect the conformational space and
at the same time the activity profile of the receptor. In addition
our results allow the conclusion that the C12-H element of the
rhodopsin chromophore is a major rate-limiting factor in the
photoisomerization process leading to a strained 11-trans
chromophore in Batho, in support of current theories on the
molecular mechanism of this process.15,16,25,26,28,29,33,46 On the
other hand, the C10-H element seems to play a dominant role in
the relaxation and further rearrangement of the strained chro-
mophore during the thermal reactions following decay of Batho.
Quite different are the effects of 10-F and 12-F substitution
on the photochemical properties of rhodopsin. Since speed and
efficiency of the photoisomerization process are closely
correlated,16,45,46 the quantum yield is a good measure for the
overall performance of the process. In view of this it appears
that photoisomerization is only substantially perturbed by 12-F
substitution. We propose that this is largely due to a mass effect,
requiring more energy to perform the out-of-plane rotational
movement of the C12-F bond that initiates a coherent path over
the excited state energy surface toward a perturbed 11-transoid
configuration. This is similar to our explanation for the decrease
in quantum yield of the 12-methyl pigment.33 An additional
factor in the 12-F pigment, we propose, is dipolar interaction
between the fluoro substituent and nearby elements in the protein
Acknowledgment. We thank Dr. Rosalie Crouch (Medical
University of South Carolina, Charleston) for a generous gift of
11-cis retinal. Dr. Gert Vriend (Centre for Molecular BioInformatics
CMBI, Radboud University Nijmegen Medical Centre, The Neth-
erlands) is acknowledged for expert assistance with interpretation
of the rhodopsin and bathorhodopsin structures using What-If-
YASARA (www.yasara.org). We thank Dr. Arthur Pistorius (Rad-
boud University Nijmegen Medical Centre) for generating rhodopsin
models containing the fluoro substituents. This research was
supported by The Netherlands Foundation for Scientific Research
through its Chemical Council (NWO-CW) and by the EC (E-MeP,
contract LSHG-CT-2004-504601).
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17942 J. AM. CHEM. SOC. VOL. 131, NO. 49, 2009