6-s-cis conformation and polar binding pocket of the retinal chromophore in the photoactivated state of rhodopsin

Article abstract of DOI:10.1021/ja9034768

The visual pigment rhodopsin is unique among the G protein-coupled receptors in having an 11-cis retinal chromophore covalently bound to the protein through a protonated Schiff base linkage. The chromophore locks the visual receptor in an inactive conformation through specific steric and electrostatic interactions. This efficient inverse agonist is rapidly converted to an agonist, the unprotonated Schiff base of all-trans retinal, upon light activation. Here, we use magic angle spinning NMR spectroscopy to obtain the ¹³C chemical shifts (C5-C20) of the all-trans retinylidene chromophore and the ¹⁵N chemical shift of the Schiff base nitrogen in the active metarhodopsin II intermediate. The retinal chemical shifts are sensitive to the conformation of the chromophore and its molecular interactions within the protein-binding site. Comparison of the retinal chemical shifts in metarhodopsin II with those of retinal model compounds reveals that the Schiff base environment is polar. In particular, the ¹³C15 and ¹⁵Nε chemical shifts indicate that the CdN bond is highly polarized in a manner that would facilitate Schiff base hydrolysis. We show that a strong perturbation of the retinal ¹³C12 chemical shift observed in rhodopsin is reduced in wild-type metarhodopsin II and in the E181Q mutant of rhodopsin. On the basis of the T₁ relaxation time of the retinal ¹³C18 methyl group and the conjugated retinal ¹³C5 and ¹³C8 chemical shifts, we have determined that the conformation of the retinal C6-C7 single bond connecting the β-ionone ring and the retinylidene chain is 6-s-cis in both the inactive and the active states of rhodopsin. These results are discussed within the general framework of ligand-activated G protein-coupled receptors.

Full text of DOI:10.1021/ja9034768

Published on Web 10/01/2009
6-s-cis Conformation and Polar Binding Pocket of the Retinal
Chromophore in the Photoactivated State of Rhodopsin
Shivani Ahuja,† Markus Eilers,‡ Amiram Hirshfeld,§ Elsa C. Y. Yan,|,⊥
Martine Ziliox,‡ Thomas P. Sakmar,| Mordechai Sheves,§ and Steven O. Smith*,‡
Department of Physics and Astronomy, and Department of Biochemistry and Cell Biology, Stony
Brook UniVersity, Stony Brook, New York 11794-5215, Department of Organic Chemistry, The
Weizmann Institute of Science, RehoVot 76100, Israel, and Laboratory of Molecular Biology and
Biochemistry, The Rockefeller UniVersity, 1230 York AVenue, New York, New York 10065
Received May 6, 2009; E-mail: steven.o.smith@sunysb.edu
Abstract: The visual pigment rhodopsin is unique among the G protein-coupled receptors in having an
11-cis retinal chromophore covalently bound to the protein through a protonated Schiff base linkage. The
chromophore locks the visual receptor in an inactive conformation through specific steric and electrostatic
interactions. This efficient inverse agonist is rapidly converted to an agonist, the unprotonated Schiff base
of all-trans retinal, upon light activation. Here, we use magic angle spinning NMR spectroscopy to obtain
the 13C chemical shifts (C5-C20) of the all-trans retinylidene chromophore and the 15N chemical shift of
the Schiff base nitrogen in the active metarhodopsin II intermediate. The retinal chemical shifts are sensitive
to the conformation of the chromophore and its molecular interactions within the protein-binding site.
Comparison of the retinal chemical shifts in metarhodopsin II with those of retinal model compounds reveals
that the Schiff base environment is polar. In particular, the 13C15 and 15Nε chemical shifts indicate that the
CdN bond is highly polarized in a manner that would facilitate Schiff base hydrolysis. We show that a
strong perturbation of the retinal 13C12 chemical shift observed in rhodopsin is reduced in wild-type
metarhodopsin II and in the E181Q mutant of rhodopsin. On the basis of the T1 relaxation time of the
retinal 13C18 methyl group and the conjugated retinal 13C5 and 13C8 chemical shifts, we have determined
that the conformation of the retinal C6-C7 single bond connecting the ꢀ-ionone ring and the retinylidene
chain is 6-s-cis in both the inactive and the active states of rhodopsin. These results are discussed within
the general framework of ligand-activated G protein-coupled receptors.
1. Introduction
One of the most striking features of the G protein-coupled
receptor (GPCR) superfamily is that a relatively simple archi-
tecture of seven transmembrane (TM) helices can be adapted
to specifically recognize over a thousand different signaling
ligands. The visual receptors are unique among GPCRs in that
they are activated by the photoisomerization of a covalently
bound retinal chromophore rather than by binding of a diffusible
ligand.1,2 Nevertheless, the retinal chromophores in the visual
pigments can be analyzed in much the same way as the receptor
bound ligands in the ligand-activated GPCRs. For example, in
the visual receptor rhodopsin, the 11-cis isomer of retinal
effectively functions as an inverse agonist by lowering the basal
activity of the receptor to undetectable levels3,4 (Figure 1). The
Figure 1. Molecular structures of the 11-cis retinal PSB chromophore in
rhodopsin (A) and the all-trans retinal unprotonated SB chromophore in
Meta II (B). 15N CP MAS spectra of rhodopsin (C) and Meta II (D) labeled
with 15Nε-lysine. The 15Nε-Lys296 is observed as a distinct narrow peak at
155.4 ppm in rhodopsin and shifts ∼127 ppm downfield to 282.8 ppm in
Meta II. The 15Nε resonances of the free lysines in rhodopsin are observed
as a broad peak around 8.7 ppm.
† Department of Physics and Astronomy, Stony Brook University.
‡ Department of Biochemistry and Cell Biology, Stony Brook University.
§ The Weizmann Institute of Science.
| The Rockefeller University.
⊥ Present address: Department of Chemistry, Yale University, New
Haven, CT 06520.
11-cis retinal chromophore is bound as a protonated Schiff base
(PSB) in the interior of rhodopsin and locks the receptor off
through electrostatic interactions with Glu113, a protein coun-
terion to the PSB,5,6 and through steric interactions with amino
acids in the retinal binding pocket. Photoisomerization to the
(1) Sakmar, T. P. Curr. Opin. Cell Biol. 2002, 14, 189–195.
(2) Okada, T.; Ernst, O. P.; Palczewski, K.; Hofmann, K. P. Trends
Biochem. Sci. 2001, 26, 318–324.
(3) Cohen, G. B.; Yang, T.; Robinson, P. R.; Oprian, D. D. Biochemistry
1993, 32, 6111–6115.
(4) Vogel, R.; Siebert, F. J. Biol. Chem. 2001, 276, 38487–38493.
9
15160 J. AM. CHEM. SOC. 2009, 131, 15160–15169
10.1021/ja9034768 CCC: $40.75  2009 American Chemical Society

Solid-State 13C NMR of Retinal in Metarhodopsin II
A R T I C L E S
all-trans configuration and deprotonation of the PSB rapidly
converts the chromophore from an inverse agonist to a full
agonist. Using solid-state 13C and 15N NMR, we report on the
structure and environment of the all-trans retinal unprotonated
Schiff base (SB) chromophore in metarhodopsin II (Meta II),
the photoactivated state of the rhodopsin, and describe how the
all-trans retinal molecule functions as the agonist for rhodopsin
activation.
The C6-C7 bond divides the plane containing the ꢀ-ionone
ring from the two planes containing the retinylidene chain. In
bacteriorhodopsin, Harbison et al.17 used three independent
NMR measurements (the 13C5 chemical shift, the 13C8 chemical
shift, and the 13C18 T1 relaxation time) to establish a 6-s-trans
conformation of the ꢀ-ionone ring about the C6-C7 single bond.
On the basis of a number of retinal derivatives,17 the T1
relaxation times were observed to be on the order of seconds
for C18 methyl groups in a planar 6-s-trans conformation due
primarily to the steric interaction between the C18 methyl group
and the proton on C7. On the other hand, the T1 relaxation time
for the C18 methyl group in a skewed 6-s-cis conformation was
found to be much shorter, on the order of milliseconds.
On the basis of 13C chemical shift measurements, we
originally assigned the retinal C6-C7 conformation in rhodopsin
as 6-s-cis.18 However, the assignment was subsequently chal-
lenged by experimental19 and computational20 studies. More
recently, several studies have revisited the C6-C7 single bond
conformation.21-24 However, to date, the distinctive relaxation
measurements described for bacteriorhodopsin have not been
repeated on rhodopsin or its photoreaction intermediates.
Much less is known about the conformation of the retinal in
the active Meta II intermediate than in the inactive, dark state
of rhodopsin. The crystal structure of a photoactivated inter-
mediate of rhodopsin having an unprotonated Schiff base has
been reported.25 However, the diffraction data are of low
resolution for both the dark state and the photointermediate,
making it difficult to establish how the chromophore or the
receptor changes structure upon illumination. In contrast, solid-
state NMR spectroscopy is well suited for structural studies that
target specific regions of membrane proteins in native membrane
environments. High-resolution magic angle spinning (MAS)
NMR methods have been used to investigate ligand conforma-
tion in ligand-activated GPCRs, such as the histamine26 and
the neurotensin receptors,27 as well as the structure of the retinal-
containing membrane proteins.28,29
The retinal chromophore in the dark state of rhodopsin can
be conceptually divided into three distinct planes broken by the
C6-C7 and the C11dC12-C13 bonds. These planes are twisted
relative to one another to fit the retinal PSB chromophore into
a tight receptor binding site.7 Specific packing interactions
between the retinal and protein have two consequences. First,
binding of the 11-cis retinal PSB is responsible for lowering
the basal activity of the apoprotein opsin. For example, the
bound 11-cis retinal restricts the motion of Trp265, a highly
conserved aromatic amino acid on TM helix H6 that rotates
toward the extracellular surface upon receptor activation.8,9
Second, NMR,7,10 crystallographic,11 and computational12 stud-
ies argue that the protein binding site imparts a pretwist about
the C11dC12 bond. The ground-state twist about the C11dC12
bond is thought to be required for the extremely fast and
selective photoreaction to the all-trans conformation of the
chromophore.13
Protein-retinal interactions are also responsible for the high
quantum yield of the 11-cis to all-trans photoreaction. The
quantum yield is controlled in part by Glu113 on TM helix H3,
the counterion for the retinal PSB. Interestingly, Glu113 has
also recently been shown to be responsible for the high quantum
yield in UV-absorbing pigments where the retinal SB is
unprotonated,14 suggesting that Schiff base protonation and
associated π-electron delocalization are not necessary for
maintaining a high quantum yield. A second glutamic acid
residue, Glu181 on the second extracellular loop (EL2), is in
close proximity to the retinal and may also contribute to the
rapid and selective photoisomerization of the C11dC12 double
bond. The side chain of Glu181 is oriented into the retinal
binding site with the glutamate carboxyl group near C12 of the
retinal. The occurrence of a negative charge directed at C12
was first proposed by absorption measurements using dihydro
derivatives of retinal15 and later confirmed by measurement of
the retinal 13C NMR chemical shifts.16
In this Article, we characterize the retinal 13C chemical shifts
in the active Meta II intermediate to address how the all-trans
retinal SB functions as a full agonist for receptor activation.
Comparison of the Meta II chemical shifts with those of the
(17) Harbison, G. S.; Smith, S. O.; Pardoen, J. A.; Courtin, J. M.;
Lugtenburg, J.; Herzfeld, J.; Mathies, R. A.; Griffin, R. G. Biochemistry
1985, 24, 6955–6962.
(18) Smith, S. O.; Palings, I.; Copie, V.; Raleigh, D. P.; Courtin, J.; Pardoen,
J. A.; Lugtenburg, J.; Mathies, R. A.; Griffin, R. G. Biochemistry 1987,
26, 1606–1611.
(5) Zhukovsky, E. A.; Oprian, D. D. Science 1989, 246, 928–930.
(6) Sakmar, T. P.; Franke, R. R.; Khorana, H. G. Proc. Natl. Acad. Sci.
U.S.A. 1989, 86, 8309–8313.
(19) Gro¨bner, G.; Burnett, I. J.; Glaubitz, C.; Choi, G.; Mason, A. J.; Watts,
A. Nature 2000, 405, 810–813.
(7) Struts, A. V.; Salgad, G. F. J.; Tanaka, K.; Krane, S.; Nakanishi, K.;
Brown, M. F. J. Mol. Biol. 2007, 372, 50–66.
(20) Singh, D.; Hudson, B. S.; Middleton, C.; Birge, R. R. Biochemistry
2001, 40, 4201–4204.
(8) Crocker, E.; Eilers, M.; Ahuja, S.; Hornak, V.; Hirshfeld, A.; Sheves,
M.; Smith, S. O. J. Mol. Biol. 2006, 357, 163–172.
(21) Fujimoto, Y.; Ishihara, J.; Maki, S.; Fujioka, N.; Wang, T.; Furuta,
T.; Fishkin, N.; Borhan, B.; Berova, N.; Nakanishi, K. Chem.-Eur. J.
2001, 7, 4198–4204.
(9) Patel, A. B.; Crocker, E.; Reeves, P. J.; Getmanova, E. V.; Eilers,
M.; Khorana, H. G.; Smith, S. O. J. Mol. Biol. 2005, 347, 803–812.
(10) Verdegem, P. J. E.; Bovee-Geurts, P. H. M.; De Grip, W. J.;
Lugtenburg, J.; de Groot, H. J. M. Biochemistry 1999, 38, 11316–
11324.
(22) Spooner, P. J. R.; Sharples, J. M.; Verhoeven, M. A.; Lugtenburg, J.;
Glaubitz, C.; Watts, A. Biochemistry 2002, 41, 7549–7555.
(23) Spooner, P. J. R.; Sharples, J. M.; Goodall, S. C.; Seedorf, H.;
Verhoeven, M. A.; Lugtenburg, J.; Bovee-Geurts, P. H. M.; DeGrip,
W. J.; Watts, A. Biochemistry 2003, 42, 13371–13378.
(24) Salgado, G. F. J.; Struts, A. V.; Tanaka, K.; Fujioka, N.; Nakanishi,
K.; Brown, M. F. Biochemistry 2004, 43, 12819–12828.
(25) Salom, D.; Lodowski, D. T.; Stenkamp, R. E.; Le Trong, I.; Golczak,
M.; Jastrzebska, B.; Harris, T.; Ballesteros, J. A.; Palczewski, K. Proc.
Natl. Acad. Sci. U.S.A. 2006, 103, 16123–16128.
(11) Okada, T.; Sugihara, M.; Bondar, A. N.; Elstner, M.; Entel, P.; Buss,
V. J. Mol. Biol. 2004, 342, 571–583.
(12) Sugihara, M.; Hufen, J.; Buss, V. Biochemistry 2006, 45, 801–810.
(13) Schoenlein, R. W.; Peteanu, L. A.; Wang, Q.; Mathies, R. A.; Shank,
C. V. J. Phys. Chem. 1993, 97, 12087–12092.
(14) Tsutsui, K.; Imai, H.; Shichida, Y. Biochemistry 2007, 46, 6437–6445.
(15) Honig, B.; Dinur, U.; Nakanishi, K.; Baloghnair, V.; Gawinowicz,
M. A.; Arnaboldi, M.; Motto, M. G. J. Am. Chem. Soc. 1979, 101,
7084–7086.
(26) Luca, S.; White, J. F.; Sohal, A. K.; Filippov, D. V.; van Boom, J. H.;
Grisshammer, R.; Baldus, M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
10706–10711.
(16) Smith, S. O.; Courtin, J.; de Groot, H.; Gebhard, R.; Lugtenburg, J.
Biochemistry 1991, 30, 7409–7415.
(27) Ratnala, V. R. P.; Kiihne, S. R.; Buda, F.; Leurs, R.; de Groot, H. J. M.;
DeGrip, W. J. J. Am. Chem. Soc. 2007, 129, 867–872.
9
J. AM. CHEM. SOC. VOL. 131, NO. 42, 2009 15161

A R T I C L E S
Ahuja et al.
by standard methods.33,34 Rhodopsin pigments in DDM micelles
were regenerated with HPLC purified 11-cis 13C-labeled retinal by
illumination of concentrated samples in the presence of a 2:1 molar
ratio of retinal-to-protein.
2.4. Solid-State NMR Spectroscopy. Solid-state NMR experi-
ments were performed at a 1H frequency of either 360 or 600 MHz
on Bruker AVANCE spectrometers using 4 mm MAS probes
containing 1.6-4.0 mM rhodopsin. Variable amplitude cross-
polarization35 was used with a 2 ms contact time, and two-pulse
phase-modulated36 or SPINAL6437 was used for proton decoupling
during the acquisition periods with 85-90 kHz field strengths. The
MAS spinning rate was maintained between 8 and 12 kHz to within
(5 Hz. The sample temperature was maintained at 190 K for the
duration of the experiment.
T1 relaxation measurements for the 13C18 methyl group on the
ꢀ-ionone ring were performed on retinoic acid crystals (monoclinic
and triclinic forms) and retinal in rhodopsin at 190 K using a
standard cross-polarization inversion-recovery sequence.17
NMR measurements were first made on rhodopsin in the dark.
Subsequently, the samples were illuminated for 45-60 s at room
temperature using a 400 W lamp with a >495 nm cutoff long pass
filter. The rotor was then recapped and placed in the NMR probe
with the probe stator warmed to 5 °C. Under slow spinning (2 kHz),
the heater was turned off and the sample was frozen within 3 min
using N2 gas cooled to 190 K.
11-cis retinal PSB chromophore in rhodopsin and with retinal
model compounds suggests that the Schiff base moiety is in a
polar environment in Meta II. Interestingly, the largest chemical
shift differences between the all-trans retinal SB in Meta II and
in solution exist at the Schiff base and ꢀ-ionone ring ends of
the chromophore. The 15Nε resonance of the unprotonated SB
in Meta II is further upfield (shielded) and the 13C15 chemical
shift is further downfield (deshielded) than in all-trans SB model
compounds, reflecting a highly polarized CdN bond. Similarly,
the 13C5 resonance within the retinal ꢀ-ionone ring of Meta II
has an unusual upfield chemical shift despite our determination
that the C6-C7 bond is in the 6-s-cis conformation in both
rhodopsin and Meta II. The 13C NMR chemical shifts of the
central portion of the all-trans retinal SB chromophore in Meta
II are not dramatically different from those in the retinal model
systems. For example, the 13C chemical shifts of the C19 and
C20 methyl groups in the dark state of rhodopsin are unique
due to the conformation and environment of the protein within
a tight retinal-binding cavity. However, in Meta II they are
roughly the same as in all-trans SB model compounds in
solution. Together these results indicate that protein interactions
at the two ends of the all-trans retinal SB chromophore play a
role in the activation mechanism of rhodopsin.
2.5. Chemical Shift Referencing. All 13C high-resolution NMR
and solid-state MAS NMR spectra were externally referenced to
the 13C resonance of neat TMS at 0 ppm at room temperature. Using
TMS as the external reference, we calibrated the carbonyl resonance
of solid glycine at 176.46 ppm. Our chosen reference compound is
different from 1% TMS in CDCl3, which is the IUPAC recom-
mended standard.38 This choice of reference compound was based
on the fact that appreciable solvent shifts were observed for the
13C resonance of TMS in CDCl3 (+0.68 ppm) and CD3OH (-0.84
ppm). Zilm and co-workers39 have reported similar solvent shifts
earlier for TMS in deuterated chloroform and methanol. Addition-
ally, we compare our 13C chemical shifts of the retinal chromophore
in rhodopsin and Meta II with previous NMR studies conducted
on retinal model compounds40 where the 13C chemical shifts have
been referenced externally to neat TMS.
2. Methods and Materials
2.1. Expression and Purification of 13C-Labeled Rhodop-
sin. Opsin was expressed and isotope labeled using stable tetracycline-
inducible HEK293S cells30 containing the wild-type opsin gene31
as previously described.32 Rhodopsin was regenerated in harvested
cells by the addition of 14 mM 11-cis retinal in ethanol.
Rhodopsin was solubilized in 1.0% n-dodecyl-ꢀ-D-maltoside
(DDM) in phosphate buffered saline at pH 6.0 and purified by
affinity chromatography using the 1D4 antibody (National Cell
Culture Center, Minneapolis, MN).31 A peptide corresponding to
the 9 C-terminal amino acids of rhodopsin in 0.02% DDM and
sodium phosphate buffer at pH 6.0 was used as the antibody epitope
to elute rhodopsin.
The 15N solid-state MAS NMR spectra collected on rhodopsin
and Meta II were referenced to the 15N resonance of 5.6 M aqueous
NH4Cl at 0.0 ppm at room temperature by using 15N-labeled glycine
as an external reference and the reported value of glycine at 8.1
ppm relative to 5.6 M aqueous NH4Cl.41
2.2. Synthesis of Retinal Model Compounds. All-trans-N-
retinylidene-n-butylimine was prepared by reacting all-trans retinal
powder (Sigma-Aldrich) in methanol with an excess of butylamine
over 3.0 Å molecular sieves as described by Harbison et al.17 We
obtained the natural abundance 13C chemical shifts for all-trans-
N-retinylidene-n-butylimine in polar (CD3OD) and nonpolar
(CDCl3) solvents at 25 °C. These NMR spectra were obtained on
3. Results and Discussion
1
a Bruker Avance NMR spectrometer operating at a H frequency
3.1. Characterization of Meta II Trapped at Low Temper-
ature. Meta II is the only intermediate in the rhodopsin
photoreaction cascade with an unprotonated Schiff base nitrogen.
We first confirmed that in our experiments the all-trans retinal
of 700 MHz.
All-trans retinoic acid crystals in monoclinic and triclinic form
were prepared as described by Harbison et al.17 The 13C chemical
shifts for the retinoic acid crystals were obtained using a 4 mm
MAS probe at a 1H frequency of 600 MHz (see below) and
referenced externally to the carbonyl 13C chemical shift for
powdered glycine at 176.46 ppm.
(33) Lugtenburg, J. Pure Appl. Chem. 1985, 57, 753–762.
(34) Crocker, E.; Patel, A. B.; Eilers, M.; Jayaraman, S.; Getmanova, E.;
Reeves, P. J.; Ziliox, M.; Khorana, H. G.; Sheves, M.; Smith, S. O.
J. Biomol. NMR 2004, 29, 11–20.
2.3. Synthesis of 13C-Labeled Retinals and Regeneration
of Rhodopsin. Retinals with specific 13C-labels were synthesized
(35) Metz, G.; Wu, X.; Smith, S. O. J. Magn. Reson., Ser. A 1994, 110,
219–227.
(28) Ahuja, S.; Crocker, E.; Eilers, M.; Hornak, V.; Hirshfeld, A.; Ziliox,
M.; Syrett, N.; Reeves, P. J.; Khorana, H. G.; Sheves, M.; Smith, S. O.
J. Biol. Chem. 2009, 284, 10190–10201.
(36) Bennett, A. E.; Rienstra, C. M.; Auger, M.; Lakshmi, K. V.; Griffin,
R. G. J. Chem. Phys. 1995, 103, 6951–6958.
(37) Fung, B. M.; Khitrin, A. K.; Ermolaev, K. J. Magn. Reson. 2000,
142, 97–101.
(29) Ahuja, S.; Hornak, V.; Yan, E. C. Y.; Syrett, N.; Goncalves, J. A.;
Hirshfeld, A.; Ziliox, M.; Sakmar, T. P.; Sheves, M.; Reeves, P. J.;
Smith, S. O.; Eilers, M. Nat. Struct. Mol. Biol. 2009, 16, 168–175.
(30) Reeves, P. J.; Kim, J. M.; Khorana, H. G. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 13413–13418.
(38) Harris, R. K.; Becker, E. D.; De Menezes, S. M. C.; Granger, P.;
Hoffman, R. E.; Zilm, K. W. Solid State Nucl. Magn. Reson. 2008,
33, 41–56.
(39) Morcombe, C. R.; Zilm, K. W. J. Magn. Reson. 2003, 162, 479–486.
(40) Harbison, G. S.; Herzfeld, J.; Griffin, R. G. Biochemistry 1983, 22,
1–4.
(31) Reeves, P. J.; Thurmond, R. L.; Khorana, H. G. Proc. Natl. Acad.
Sci. U.S.A. 1996, 93, 11487–11492.
(32) Eilers, M.; Reeves, P. J.; Ying, W. W.; Khorana, H. G.; Smith, S. O.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 487–492.
(41) Wang, J. X.; Balazs, Y. S.; Thompson, L. K. Biochemistry 1997, 36,
1699–1703.
9
15162 J. AM. CHEM. SOC. VOL. 131, NO. 42, 2009

Solid-State 13C NMR of Retinal in Metarhodopsin II
A R T I C L E S
chromophore in Meta II trapped in DDM detergent is bound to
Lys296 through an unprotonated SB linkage, and that only a
single intermediate is present in the NMR sample. 15Nε-Lysine
was incorporated into rhodopsin, and the 15Nε chemical shifts
were obtained of the 11-cis retinal PSB in rhodopsin and the
all-trans retinal SB in Meta II using solid-state MAS NMR. In
Figure 1C, the 15Nε resonance for the PSB nitrogen in rhodopsin
solubilized in DDM is observed at 155.4 ppm, similar to the
chemical shift for rhodopsin reconstituted in DOPC lipids.32 In
Meta II (Figure 1D), the 15Nε resonance shifts to 282.8 ppm.
The resonances at ∼8.7 ppm correspond to the other 10 free
lysines in the protein. The natural abundance 15N signal from
the amide nitrogens is observed as a broad resonance at 95.3
ppm, and the intense peak at ∼47 ppm results from 15N-labeling
of arginine in the same sample.
The large change in the 15N chemical shift of the SB in Meta
II is attributed to deprotonation of the PSB upon activation. In
bacteriorhodopsin, two resolved M intermediates are observed
with 15Nε chemical shifts of 288.6 ppm (Mn) and 296.4 ppm
(Mo).42 In contrast, the all-trans-N-retinylidene-n-butylimine SB
is observed at 315.3 ppm.40 The upfield chemical shifts of the
M states relative to the SB model compound, as well as the
upfield shift of the Mn-state of bacteriorhodopsin relative to the
Mo-state, were attributed to stronger basicity.42 The 15Nε
chemical shift in Meta II is unusual in being even further upfield
than in the Mn-state of bacteriorhodopsin. One of the major
differences between the rhodopsin and bacteriorhodopsin pho-
toreactions is that the Schiff base linkage in rhodopsin is
hydrolyzed in the transition of Meta II to opsin, while in
bacteriorhodopsin the Schiff base nitrogen is reprotonated
between the M and N photocycle intermediates. As we will
discuss below, the chemical shift of the Schiff base nitrogen in
Meta II may reflect the polarization of the CdN bond and
facilitate hydrolysis, thereby shifting the receptor to the inactive
opsin conformation. Hence, the Schiff base 15N chemical shift
may reflect a key difference between the linear photoreaction
in the visual receptors and the cyclic proton-pumping reaction
in bacteriorhodopsin.
Figure 2. Representative one-dimensional 13C MAS NMR spectra of the dark
state of rhodopsin (black) and the active Meta II intermediate (red) containing
13C-labeled retinal chromophores. Regions of the 13C spectra from ∼110-180
ppm are shown for rhodopsin and Meta II containing retinal 13C-labeled at the
C9 (A), C13 (B), C5 (C), and C8 (D) positions. The C5 resonance overlaps
the natural abundance signal from the aromatics in the protein.
Table 1. 13C Chemical Shifts of the Retinal Carbons in Wild-type
Rhodopsin and Comparison with the Retinal PSB Model
Compound, N-(11-cis-Retinylidene)-n-propyliminium
Trifluoroacetate,a in CDCl3
carbon
Rho (ppm)
PSBa (ppm)
∆Rho (ppm)
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
131.0
137.1
132.8
139.3
148.9
128.0
141.6
132.2
168.5
122.1
165.7
30.6
132.1
137.2
132.3
137.2
147.8
126.4
138.7
128.7
165.8
120.5
163.3
28.9
-1.1
-0.1
0.5
2.1
1.1
1.6
2.9
3.5
2.7
1.6
2.4
1.7
-2.8
-0.5
2.1
26.1
21.6
14.7
16.4
28.9
22.1
12.6
18.8
-2.4
a Data from Shriver et al.45
The 13C chemical shifts of the retinal carbons along the
polyene chain also provide a way to characterize the ability to
trap a homogeneous Meta II intermediate at low temperature.
In this case, rhodopsin solubilized in DDM was regenerated
with 11-cis retinal selectively 13C labeled within the ꢀ-ionone
ring and along the conjugated polyene chain. One-dimensional
1H-13C cross-polarization (CP) MAS NMR experiments were
performed to obtain the chemical shifts of the retinal carbons
in rhodopsin and Meta II. Figure 2 presents regions of the 13C
CP-MAS spectra for rhodopsin regenerated with retinal 13C
labeled at the C5, C8, C9, and C13 carbons along the polyene
chain. The 13C MAS spectra of rhodopsin (black) have been
overlaid with the MAS spectra of Meta II (red). The quaternary
odd numbered carbons (C5, C9, and C13) with attached methyl
groups exhibit the largest chemical shift changes of the retinal
carbons among those shown in Figure 2.
168.5 ppm in rhodopsin overlaps the broad natural abundance
13C signal from the protein carbonyls centered at ∼175 ppm. A
20 ppm change in the 13C13 chemical shift to 148.2 ppm is
observed in Meta II. In contrast, the C8 resonance does not
shift significantly between rhodopsin and Meta II.
Integration of the 13C resonances shows that we can convert
>85% of rhodopsin to Meta II. The retinal 13C resonances are
narrow (1-2 ppm) in both rhodopsin and Meta II, exhibiting
no signs of splitting or differential broadening. The chemical
shifts are reproducible to within 0.3 ppm. For comparison, the
histamine ligand bound to the H1 receptor exhibits broad 13C
resonances (4-8 ppm), suggesting conformational heterogene-
ity.27
Following the thermal decay of Meta II to opsin and free
retinal, the 13C retinal resonances broaden considerably and are
not observed. The origin of the broadening most likely results
from heterogeneous interactions of the retinal with the protein
and detergent after hydrolysis of the Schiff base linkage.
Together these observations indicate that the conformational
heterogeneity of the retinal in rhodopsin and Meta II is similar
and limited.
The retinal C5 and C9 resonances are observed in rhodopsin
at 131.0 and 148.9 ppm, respectively, and shift to 126.0 and
139.6 ppm in the Meta II intermediate. The broad resonance at
127.0 ppm is due to the natural abundance 13C signal from the
aromatic carbons in the protein. The 13C resonance of C13 at
Table 1 lists the 13C chemical shifts for carbons C5-C20 of
the 11-cis retinal PSB in rhodopsin solubilized in DDM
detergent. The 13C chemical shifts are in agreement within
(42) Hu, J. G.; Sun, B. Q.; Bizounok, M.; Hatcher, M. E.; Lansing, J. C.;
Raap, J.; Verdegem, P. J. E.; Lugtenburg, J.; Griffin, R. G.; Herzfeld,
J. Biochemistry 1998, 37, 8088–8096.
9
J. AM. CHEM. SOC. VOL. 131, NO. 42, 2009 15163

A R T I C L E S
Ahuja et al.
Table 2. 13C Chemical Shift of the Retinal C12 Carbon in
Wild-type Rhodopsin and the E181Q Mutant of Rhodopsin and
Meta II
compound
wild type (ppm)
E181Q mutant (ppm)
rhodopsin
Meta II
132.2
137.9
129.8
137.4
chemical shift in Figure 3A is that observed for C12 because
the partial charges on the retinal carbons typically alternate
between partial negative on the even number carbons and partial
positive on the odd numbered carbons. We previously attributed
the large downfield chemical shift at C12 to a specific interaction
with a negative charge in the retinal binding site.47 This
observation was confirmed by the crystal structure of rhodop-
sin,48 which revealed that Glu181 on EL2 is oriented toward
the retinal with the side chain carboxyl group ∼4 Å from C12.
Glu181 is one of the residues that distinguishes the high-
sensitivity rod-cell receptors associated with vision under low
light conditions from the human green and red cone pigments
where the corresponding residue is a histidine, which forms part
of a chloride ion binding site.
To test whether the unusual chemical shift at C12 is due to
a charged Glu181 carboxyl group, we obtained NMR spectra
of the E181Q rhodopsin mutant regenerated with 11-cis retinal
13C-labeled at C12. The E181Q mutation results in a reduction
of the anomalous ∆Rho shift from +3.5 to +1.1 ppm (Table
2). The reduction in ∆Rho confirms that Glu181 is responsible
for the unusual shift at C12, which implies that Glu181 is also
responsible for the accumulation of excess positive charge along
the polyene chain and suggests that Glu181 is charged in
rhodopsin. The large change in the C12 chemical shift is
consistent with FTIR data that Glu181 is deprotonated in
rhodopsin,49 but is in contrast to the lack of a change in the
absorption spectra between wild-type rhodopsin and the E181Q
mutant.50 The lack of a change of the λmax may reflect the fact
that the residue at position 181 is interacting with the middle
of the retinal polyene chain and could potentially affect the
energy levels of both the ground and the excited electronic states
of the retinal in a similar fashion, leaving the λmax unperturbed.
Hall et al.51 have shown computationally that the S1 f S2
energy level splitting is almost identical for model systems
differing in the protonation state of Glu181. The E181Q mutant
does not appear to affect the ground-state C11dC12-C13 twists
in rhodopsin because the hydrogen out-of-plane and fingerprint
vibrations in resonance Raman spectra do not change as
compared to the E181Q mutant.52
Figure 3. Comparison of retinal 13C chemical shifts in rhodopsin with an
11-cis retinal PSB model compound and with Meta II. (A) Differences
∆(Rho) are plotted between the retinal chemical shifts for C5-C20 carbons
in rhodopsin and in the N-(11-cis-retinylidene)-n-propyliminium trifluoro-
acetate model compound in solution (red).43 The measurement of the 13C12
chemical shift in the E181Q mutant of rhodopsin is also shown (green bar).
(B) Differences ∆(Rho-MII) are plotted between the retinal chemical shifts
for C5-C20 plotted in rhodopsin and Meta II. All chemical shifts are
externally referenced to neat TMS.
experimental error (0.3-0.4 ppm) with those reported earlier
for rhodopsin reconstituted in lipids by de Groot and co-
workers.43 The similar chemical shifts (and absorption spectra)
indicate that the conformation of the 11-cis retinal PSB in
rhodopsin solubilized in DDM is the same as for rhodopsin
reconstituted into a lipid environment. In a comprehensive
comparison of spin-labeled rhodopsin, Hubbell and co-workers44
found that the most notable difference between the DDM and
lipid environments is that the receptor has increased flexibility
in detergent, which facilitates the conversion of rhodopsin to
Meta II.
3.2. Charge Delocalization along the Retinal Polyene Chain
in Rhodopsin. Table 1 compares the 13C chemical shifts of
rhodopsin with those of the 11-cis retinal PSB model compound
in CDCl3 having a trifluoroacetate counterion (N-(11-cis-
retinylidene)propyliminium trifluoroacetate).45 The differences
in chemical shifts (∆Rho) listed in Table 1 are shown as a
histogram in Figure 3A. ∆Rho represents the effect of the
protein on the conformation and environment of the 11-cis
retinal PSB. As noted previously,46 the positive ∆Rho values
observed for the C8-C15 carbons along the retinylidene chain
are an unusual feature of the histogram. The most unusual
In Meta II spectra of the E181Q mutant, the 13C12 chemical
shift does not exhibit a significant change from its chemical
shift in wild-type Meta II (Table 2). While the influence of
protein charges on retinal chemical shifts should be much
(47) Han, M.; DeDecker, B. S.; Smith, S. O. Biophys. J. 1993, 65, 899–
906.
(48) Palczewski, K.; Kumasaka, T.; Hori, T.; Behnke, C. A.; Motoshima,
H.; Fox, B. A.; Le Trong, I.; Teller, D. C.; Okada, T.; Stenkamp,
R. E.; Yamamoto, M.; Miyano, M. Science 2000, 289, 739–745.
(49) Lu¨deke, S.; Beck, R.; Yan, E. C. Y.; Sakmar, T. P.; Siebert, F.; Vogel,
R. J. Mol. Biol. 2005, 353, 345–356.
(43) Creemers, A. F. L.; Kiihne, S.; Bovee-Geurts, P. H. M.; DeGrip, W. J.;
Lugtenburg, J.; de Groot, H. J. M. Proc. Natl. Acad. Sci. U.S.A. 2002,
99, 9101–9106.
(50) Yan, E. C. Y.; Kazmi, M. A.; De, S.; Chang, B. S. W.; Seibert, C.;
Marin, E. P.; Mathies, R. A.; Sakmar, T. P. Biochemistry 2002, 41,
3620–3627.
(44) Kusnetzow, A. K.; Altenbach, C.; Hubbell, W. L. Biochemistry 2006,
45, 5538–5550.
(45) Shriver, J. W.; Mateescu, G. D.; Abrahamson, E. W. Biochemistry
1979, 18, 4785–4792.
(51) Hall, K. F.; Vreven, T.; Frisch, M. J.; Bearpark, M. J. J. Mol. Biol.
2008, 383, 106–121.
(46) Smith, S. O.; Palings, I.; Miley, M. E.; Courtin, J.; de Groot, H.;
Lugtenburg, J.; Mathies, R. A.; Griffin, R. G. Biochemistry 1990, 29,
8158–8164.
(52) Yan, E. C. Y.; Kazmi, M. A.; Ganim, Z.; Hou, J. M.; Pan, D. H.;
Chang, B. S. W.; Sakmar, T. P.; Mathies, R. A. Proc. Natl. Acad.
Sci. U.S.A. 2003, 100, 9262–9267.
9
15164 J. AM. CHEM. SOC. VOL. 131, NO. 42, 2009

Solid-State 13C NMR of Retinal in Metarhodopsin II
A R T I C L E S
Table 3. 13C Chemical Shifts of the all-trans Retinal SB in Meta II
and Comparison with the Model Compound
N-All-trans-retinylidene-tert-butylimine in a Nonpolar (MNP) and in a
Polar Solvent (MP)
Meta II
(ppm)
SBa CDCl3
(ppm)
MNP CDCl3
(ppm)
MP CD3OH
(ppm)
∆MIINP
(ppm)
∆MIIP
(ppm)
carbon
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
126.0
139.5
127.6
139.6
139.6
131.4
127.8
137.9
148.2
126.9
163.1
28.6
129.9
137.4
127.4
137.3
138.0
129.8
127.8
135.8
144.4
129.0
159.7
28.8
130.4
138.5
128.5
138.2
138.6
130.7
128.5
136.8
144.6
130.2
160.0
29.7
129.3
138.2
128.2
138.2
138.2
130.5
128.2
136.1
146.9
129.3
161.5
28.7
-4.3
1.0
-0.8
1.5
1.0
0.8
-0.6
1.2
3.7
-3.2
3.2
-1.0
3.8
-1.4
0.4
-3.3
1.3
-0.6
1.4
1.4
1.0
-0.4
1.9
1.4
-2.4
1.7
0.0
4.8
-0.2
1.9
Figure 4. Comparison of retinal 13C chemical shifts between Meta II and
all-trans retinal model compounds. Differences are shown between Meta
II and the N-all-trans-retinylidene-tert-butylimine model compounds in a
nonpolar solvent (CDCl3) (∆MII, gray,45 ∆MIINP, orange) and in a polar
solvent (∆MIIP, CD3OD, blue). The smaller differences between retinal
13C13, 13C14, and 13C15 in Meta II and the butylimine model compound in
a polar solvent suggest that the binding cavity is more polar near the SB
end of the retinal in Meta II. All chemical shifts are externally referenced
to neat TMS.
33.4
20.9
13.8
13.7
28.8
21.9
12.9
13.1
29.7
22.4
13.5
13.7
28.7
21.2
12.0
12.2
0.1
1.5
a Data from Shriver et al.45
weaker for the unprotonated retinal SB as compared to the PSB,
the absence of an influence on the C12 chemical shift would
be consistent with lack of interaction between C12 and Glu181
in Meta II. Nevertheless, mutation of Glu181 does have a
pronounced effect on the visible absorption maximum of Meta
II.50 The λmax shifts from 375 nm in the E181F mutant of Meta
II to 392 nm in E181Y mutant. Yan et al.50 concluded that the
Glu181-retinal interactions may in fact be more dramatic in
Meta II than in the dark state of rhodopsin. In this regard, it is
important to note that FTIR studies do not find a difference
band associated with Glu181, indicating that the protonation
state of the carboxyl side chain does not change relative to
rhodopsin.49 That is, the influence of Glu181 on the C12
chemical shift or on the λmax is not due to a change in protonation
state. A shift of Glu181 away from C12 would be consistent
with the lack of a change in the C12 chemical shift when
comparing wild-type Meta II and Meta II in the E181Q mutant.
An increase in the distance between Glu181 and the retinal C12
carbon might result from retinal isomerization, translation of
the retinal within the binding site, or displacement of EL2 from
the retinal binding site upon rhodopsin activation.28 Different
mutants of Glu181 may influence the position of the retinal and/
or EL2 and thereby modulate the electrostatic interactions that
regulate the λmax of the retinal SB possibly through alteration
of the hydrogen-bonding network to the Schiff base nitrogen.53
3.3. Retinal 13C Chemical Shifts Suggest a Polar Retinal
Binding Site in Metarhodopsin II. The isotropic 13C chemical
shifts of the retinal chromophore are sensitive to their environ-
ment and have been used in the past to map the polarity of the
retinal binding site in rhodopsin.43,46 Table 3 lists the 13C
chemical shifts of the C5-C20 carbons of the all-trans retinal
in Meta II. The plot of the chemical shift differences in Figure
3B between rhodopsin and Meta II indicates a strong alternation
of chemical shift from C5 to C15 due to a difference in electron
delocalization along the polyene chain.47 The odd numbered
carbon resonances are shifted upfield and the even numbered
carbon resonances are shifted downfield in Meta II, with the
most pronounced shifts being associated with the odd numbered
carbons. This pattern reflects the dominant influence that the
protonation state of the Schiff base has on electron delocaliza-
tion. The retinal C16-C20 carbons do not exhibit significant
shifts, indicating that they are not affected by charge delocal-
ization along the retinal polyene chain. The major exception is
C17, discussed below.
Table 3 and Figure 4 (orange) present the chemical shift
differences (∆MIINP) between Meta II and the all-trans-N-
retinylidene-n-butylimine model compound in a nonpolar solvent
(CDCl3). Because the retinal SB is unprotonated in both the
Meta II and the all-trans retinal butylimine model compound,
one would expect smaller differences than for ∆Rho. However,
significant differences are seen at retinal carbons C5, C13, C14,
C15, and C17. To account for the effect of the choice of solvent
on the model compound spectra, we compared the chemical
shifts of Meta II with an all-trans-N-retinylidene-n-butylimine
model compound in a polar solvent (CD3OD) (Figure 4, blue).
The downfield shifts for the retinal resonances along the polyene
chain (C13, C14, and C15) are reduced, suggesting that the
binding cavity near the SB end of the retinal is polar in Meta
II. This observation is consistent with experiments showing that
the retinal binding cavity becomes accessible to water (and
hydroxylamine) in Meta II.54,55 The observation that the C15
carbon is strongly deshielded (large partial positive charge) and
the SB nitrogen is strongly shielded (large partial negative
charge, see discussion above) shows that the CdN bond is
highly polarized in Meta II.
The accessibility to water and hydroxylamine is likely
associated with motion of EL2 away from the retinal SB in
Meta II.28 Weaker interactions between the retinal and EL2 in
Meta II are also reflected in chemical shift changes of the retinal
carbons. First, the lack of chemical shift changes at the C12
position in Meta II as observed for the E181Q mutation is
consistent with the loss of a strong Glu181 interaction with the
retinal chain. Second, the chemical shifts of the C19 and C20
(54) Farrens, D. L.; Khorana, H. G. J. Biol. Chem. 1995, 270, 5073–5076.
(55) Wald, G.; Brown, P. K. J. Gen. Physiol. 1953, 37, 189–200.
(73) Scheerer, P.; Park, J. H.; Hildebrand, P. W.; Kim, Y. J.; Krauss, N.;
Choe, H. W.; Hofmann, K. P.; Ernst, O. P. Nature 2008, 455, 497–
502.
(74) Park, J. H.; Scheerer, P.; Hofmann, K. P.; Choe, H. W.; Ernst, O. P.
Nature 2008, 454, 183–187.
(53) Gat, Y.; Sheves, M. Photochem. Photobiol. 1994, 59, 371–378.
9
J. AM. CHEM. SOC. VOL. 131, NO. 42, 2009 15165

A R T I C L E S
Ahuja et al.
Table 4. Comparison of the 13C Chemical Shifts of the Retinal C5
and C8 Carbons along with the T1 Relaxation Measurement for
the C18 Methyl Group in Wild-type Rhodopsin, Meta II, and
Retinal Model Compounds
methyl groups in rhodopsin are unique (Table 1, Figure 3) due
to the conformation and environment of the protein within a
tight retinal-binding cavity. However, in Meta II they are roughly
the same as in all-trans SB model compounds in solution,
suggesting a reduced interaction with the protein upon
isomerization.
The retinal 13C14 chemical shift is sensitive to isomerization
about the CdN bond of the SB.56 In rhodopsin, the PSB linkage
between the retinal and Lys296 on H7 is characterized by a
15-anti CdN geometry.48,57-59 When the CdN bond is in a
syn configuration, steric interaction between the proton attached
to C14 and the protons on the ε-carbon of Lys296 leads to an
upfield shift of the isotropic resonances at both positions,
referred to as a γ-effect.56,60 In our case, the ∼3.0 ppm upfield
shift of the 13C14 resonance is not associated with a corre-
sponding upfield shift in the Lys296(Cε) resonance, which is
observed at 60.8 ppm in Meta II.28 Together, the 13C14 and
Lys296(Cε) chemical shifts are in agreement with FTIR studies
that the geometry of the CdN double bond remains 15-anti in
Meta II.61
Despite the polar nature of the binding site in Meta II, it is
known that the unprotonated Schiff base in Meta II is not
titratable.62,63 FTIR studies have shown that proton uptake in
Meta II results from protonation of Glu134; in wild-type
rhodopsin reconstituted into membranes, there is a single
titration with an apparent pKa of 6.3 that is abolished in the
E134Q mutant.64,65 Deprotonation of the Schiff base in the Meta
I-II transition results from an internal proton transfer to Glu113.
The high upfield chemical shift of the 15N SB nitrogen and the
downfield shift of the 13C15 carbon indicate that the SB is highly
polarizedinMetaII.NMRstudiesshowthatintherhodopsin-Meta
II transition the C20 methyl group rotates toward the extracel-
lular surface, suggesting that the NH proton rotates toward the
hydrophobic receptor interior.28,29 In Meta II, such a rotation
would orient the free electron pair on the unprotonated SB
nitrogen toward Cys264/Ser298, while the C15H proton would
be oriented toward Glu113. The orientation of the C15H proton
would agree with the fact that the Schiff base is hydrolyzed in
the decay of Meta II to opsin and that the Meta II half-life
(which reflects the rate of hydrolysis) is increased from 12.5
min in wild-type rhodopsin to 153 min in the E113Q mutant.50
Hydrolysis likely involves nucleophilic attack at the retinal C15
carbon, a reaction that would be facilitated by a strongly
polarized CdN bond. Interestingly, the SB is not hydrolyzed
temperature
(K)
compound
13C5 (ppm)
13C8 (ppm)
13C18 (T1) (s)
6-s-transb
296
296
190
134.6-144.8 131.6-133.4 25.6-31.7
126.7-129.4 138.2-140.6 0.41-3.69
6-s-cisb
6-s-trans retinoic
acid (monoclinic)
6-s-cis retinoic
acid (triclinic)
rhodopsin
136.5
131.3
12.3
190
129.6
139.7
0.14
190
190
131.0
126.0
139.3
139.6
0.34
0.56
Meta II
b Data from Harbison et al.17
in squid rhodopsin where the “counterion” at the position
equivalent to 113 is a tyrosine. These results may also provide
an explanation for the conservation of Glu113 in the vertebrate
UV-absorbing pigments where the SB is unprotonated and
apparently not in need of a counterion. In these pigments,
Glu113 (or its equivalent) may have multiple roles,66 including
the regulation of the Meta II lifetime and SB hydrolysis. This
suggestion is supported by measurements in the mouse UV-
absorbing pigment where the Meta II half-life was increased
from ∼30 s in the wild-type pigment to 6 min in the E108Q
mutant.67
3.4. 6-s-cis Conformation of the ꢀ-Ionone Ring in
Rhodopsin and Metarhodopsin II. T1 relaxation time measure-
ments of the C18 methyl group have previously been used to
assess the conformation of the C6-C7 bond of the retinal
chromophore in bacteriorhodopsin.17 The C18 T1 relaxation
times are much longer (of the order of tens of seconds) for a
planar 6-s-trans conformation than for the skewed 6-s-cis
conformation (of the order of milliseconds). The difference is
mainly due to the strong steric interaction between the retinal
C18 methyl group and the proton on C7 in the 6-s-trans
geometry. Table 4 summarizes the T1 relaxation times for the
C18 methyl group in rhodopsin, as well as in 6-s-cis and 6-s-
trans retinal model compounds. T1 relaxation measurements of
6-s-cis and 6-s-trans retinoic acid obtained at the same external
1
field strength (600 MHz, H field) and temperature (190 K) as
rhodopsin fall outside of the range of the 6-s-cis and 6-s-trans
model compounds reported by Harbison et al.,17 but still differ
by roughly an order of magnitude and are characteristic of the
C6-C7 geometry. Figure 5 presents the T1 relaxation time
measurements of the C18 methyl group in rhodopsin (black)
and Meta II (red) at 190 K. In rhodopsin, the T1 relaxation time
is 343 ms, which is close to the value of 6-s-cis retinoic acid
and well outside the range of the 6-s-trans model compounds.
In Meta II, the T1 relaxation time is 561 ms, within the range
of 6-cis model compounds.
(56) Harbison, G. S.; Smith, S. O.; Pardoen, J. A.; Winkel, C.; Lugtenburg,
J.; Herzfeld, J.; Mathies, R.; Griffin, R. G. Proc. Natl. Acad. Sci. U.S.A.
1984, 81, 1706–1709.
(57) Bagley, K. A.; Balogh-Nair, V.; Croteau, A. A.; Dollinger, G.; Ebrey,
T. G.; Eisenstein, L.; Hong, M. K.; Nakanishi, K.; Vittitow, J.
Biochemistry 1985, 24, 6055–6071.
(58) Palings, I.; Pardoen, J. A.; van den Berg, E.; Winkel, C.; Lugtenburg,
J.; Mathies, R. A. Biochemistry 1987, 26, 2544–2556.
(59) Okada, T.; Fujiyoshi, Y.; Silow, M.; Navarro, J.; Landau, E. M.;
Shichida, Y. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5982–5987.
(60) Farrar, M. R.; Lakshmi, K. V.; Smith, S. O.; Brown, R. S.; Raap, J.;
Lugtenburg, J.; Griffin, R. G.; Herzfeld, J. Biophys. J. 1993, 65, 310–
315.
The 13C chemical shifts of the retinal C5 and C8 resonances
are also sensitive to the conformation of the ꢀ-ionone ring about
the C6-C7 single bond. Analysis of the chemical shift tensors
of retinal model compounds has shown that the C5 resonance
is not influenced by steric interactions, but by charge delocal-
ization along the polyene chain.17 The C5dC6 double bond is
within the ionone ring, and conjugation with the rest of the chain
is reduced in the 6-s-cis conformation. Comparison of the
(61) Vogel, R.; Siebert, F.; Mathias, G.; Tavan, P.; Fan, G. B.; Sheves, M.
Biochemistry 2003, 42, 9863–9874.
(62) Ja¨ger, F.; Fahmy, K.; Sakmar, T. P.; Siebert, F. Biochemistry 1994,
33, 10878–10882.
(63) Knierim, B.; Hofmann, K. P.; Ernst, O. P.; Hubbell, W. L. Proc. Natl.
Acad. Sci. U.S.A. 2007, 104, 20290–20295.
(64) Vogel, R.; Mahalingam, M.; Luedke, S.; Huber, T.; Siebert, F.; Sakmar,
T. P. J. Mol. Biol. 2008, 380, 648–655.
(66) Tsutsui, K.; Imai, H.; Shichida, Y. Biochemistry 2008, 47, 10829–
10833.
(65) Vogel, R.; Sakmar, T. P.; Sheves, M.; Siebert, F. Photochem.
Photobiol. 2007, 83, 286–292.
(67) Kusnetzow, A. K.; Dukkipati, A.; Babu, K. R.; Ramos, L.; Knox, B. E.;
Birge, R. R. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 941–946.
9
15166 J. AM. CHEM. SOC. VOL. 131, NO. 42, 2009

Solid-State 13C NMR of Retinal in Metarhodopsin II
A R T I C L E S
The 13C T1 relaxation data on the retinal chromophore clearly
show that we have predominantly a 6-s-cis conformer in both
rhodopsin and Meta II. However, these data do not allow us to
differentiate between negatively or positively twisted 6-s-cis
retinal enantiomers. Lau et al.68 have analyzed the conformation
of retinal in rhodopsin on the basis of molecular dynamic
simulations of rhodopsin. Their study indicates that the polyene
chain of the retinal is locked in a single, twisted conformation
due to multiple steric interactions with the surrounding protein.
However, they observe structural heterogeneity for the retinal
ꢀ-ionone ring in agreement with deuterium NMR measure-
ments.24 Their analysis yields a model of rhodopsin that can
accommodate both negatively and positively twisted 6-s-cis
retinal enantiomers; the deuterium NMR line shape of the C18
methyl group is best fit with a mixture of enantiomers where
the negatively twisted conformer is more abundant. Recently,
we described 2D dipolar-assisted rotational resonance (DARR)
NMR experiments on the position of the C18 methyl group
relative to Gly121 and to Tyr26828 that agree with the
conclusion that a negatively twisted 6-s-cis chromophore
predominates in rhodopsin. With a negative twist, the C18
methyl group is predicted to be in close proximity to Gly121,
while a positive twist places the C18 methyl group near Tyr268.
We observed 13C· · ·13C correlations between the C18 methyl
group and Gly121 consistent with a ∼4 Å distance, but failed
to observe contacts between the C18 methyl group and Tyr268.
We can estimate on the basis of the signal-to-noise ratios in
our DARR NMR experiments that we would have been able to
observe a C18· · ·Tyr268 contact within ∼5 Å if the population
of the positively twisted 6-s-cis conformer were above 40%. In
the section below, we discuss additional NMR experiments that
support the conclusion that the ꢀ-ionone ring has predominantly
a single conformation with a negatively twisted 6-s-cis bond.
Finally, it is known that retinal analogues in which the
ꢀ-ionone ring is replaced with two ethyl groups are not able to
fully activate rhodopsin.69 In membranes, rhodopsin activity is
reduced to ∼20% of wild-type levels as assayed by GTPγS
binding to transducin. As a result, steric interactions between
the retinal ꢀ-ionone ring and helix H5 in Meta II are absent in
retinal analogues lacking the ionone ring. Without these
interactions, which may be responsible for the unusual C5
chemical shift and associated 6-s-cis ring conformation, changes
in the structure or position of H5 that are necessary for receptor
activation may not occur.
Figure 5. T1 relaxation curves for the retinal C18 methyl group in wild-
type rhodopsin (black) and Meta II (red). The T1 measurements were made
at a sample temperature of 190 K and external 1H field strength of 600
MHz.
chemical shifts of crystalline retinal derivatives shows that the
C5 chemical shift for 6-s-cis model compounds is ∼7-9 ppm
upfield of the 6-s-trans model compounds (Table 4). In contrast,
the C8 chemical shift appears to be predominantly influenced
by steric interactions.17 In the 6-s-trans conformation, the C8
proton is located between the C16 and C17 methyl groups, and
the C8 chemical shift for 6-s-trans model compounds is ∼5-8
ppm upfield as compared to the 6-s-cis model compounds. In
dark rhodopsin, we previously observed the C5 resonance at
131.0 ppm and the C8 resonance at 139.3 ppm, indicating a
6-s-cis conformation of the ꢀ-ionone ring.46
In the Meta II intermediate, the 13C5 and 13C8 chemical shifts
are observed at 126.0 and 139.6 ppm, respectively, in agreement
with a 6-s-cis conformation of the ring. The retinal C5 chemical
shift is shifted markedly upfield as compared to SB model
compound in both polar and nonpolar solvents (see Figure 4).
Although charge delocalization is reduced in unprotonated SB
chromophores, as compared to their protonated forms, the
upfield shift suggests that the ring is appreciably more nonplanar
with respect to the retinal polyene chain in Meta II than in the
retinal model compounds, and charge delocalization does not
extend into the C5dC6 bond of the ꢀ-ionone ring.
Interestingly, one might have anticipated a larger red-shift
in the λmax of Meta II on the basis of two observations. First,
we have shown that the 15N resonance for the Schiff base
nitrogen in Meta II is shifted further upfield than that observed
for the Mn and Mo states of bacteriorhodopsin. The upfield shift
is in the direction of protonation and is associated with stronger
basicity/hydrogen bonding.42 Second, Gat and Sheves53 have
shown using a series of retinal model compounds that the red-
shifted absorption maximum in the M state of bacteriorhodopsin
is correlated with the environment of the SB rather than the
planarity of the ꢀ-ionone ring. In DDM, the λmax of Meta II is
at 382 nm, as observed for the all-trans retinal SB in a
hydrophobic solvent, suggesting that the Schiff base nitrogen
is not involved in strong hydrogen-bonding interactions.53 One
way to possibly reconcile the unusual chemical shifts of the
retinal C5 carbon and the SB nitrogen with the 382 nm λmax of
Meta II is to conclude that there are appreciable twists about
the C6-C7 bond, which disrupts the conjugated π-system, and
that polarization of the CdN bond results from the SB nitrogen
being oriented toward a hydrophobic environment, while the
C15H proton is oriented toward a very polar environment.
3.5. Ring Conformation and Assignment of the 13C16 and
13C17 Chemical Shifts. According to IUPAC nomenclature, the
retinal C16 methyl group in Figure 1 is oriented into the plane
of the page, whereas the C17 methyl group is oriented out of
the plane of the page. In solution, the 13C16 and 13C17
resonances are at the same chemical shift of ∼28.9 ppm in both
11-cis PSB and all-trans SB retinal model compounds.43 In
rhodopsin, the 13C16 and 13C17 resonances are observed at 30.6
and 26.1 ppm, respectively (Table 1). The difference in chemical
shift between the retinal in solution and bound to the rhodopsin
is attributed to the tight binding site within the receptor and the
inability of different ring conformations to rapidly interconvert.
Ring inversion can change the position of the methyl groups
from an axial orientation to an equatorial orientation. In an axial
orientation, the protons of the methyl group are in steric contact
(68) Lau, P. W.; Grossfield, A.; Feller, S. E.; Pitman, M. C.; Brown, M. F.
J. Mol. Biol. 2007, 372, 906–917.
(69) Vogel, R.; Siebert, F.; Lu¨deke, S.; Hirshfeld, A.; Sheves, M.
Biochemistry 2005, 44, 11684–11699.
9
J. AM. CHEM. SOC. VOL. 131, NO. 42, 2009 15167

A R T I C L E S
Ahuja et al.
Figure 6. Two views of the retinal-binding site in Meta II highlighting the polar and aromatic residues interacting with the retinal chromophore (orange).
The position of the retinal (red) in the dark state of rhodopsin (pdb code: 1U1911) is shown for comparison. (A) View showing the position of EL2 relative
to the retinal chromophore. (B) View from the extracellular surface; EL2 is not shown for clarity. On the basis of the observed retinal chemical shifts and
T1 relaxation measurements, we conclude that the ꢀ-ionone ring has a 6-s-cis conformation about the C6-C7 single bond in rhodopsin and Meta II. The
location of the retinal in Meta II has been defined by 2D DARR NMR measurements between the retinal chromophore and surrounding amino acids in the
retinal binding site.28 The Cε-methyl resonance of Met207 exhibits DARR NMR crosspeaks with His211, Cys167, and the retinal carbons (C6, C7, C16, and
C18). These contacts allow us to position the ꢀ-ionone ring between Met207 and Phe208/Phe212 (Panel B) with the retinal C16 and C18 methyl groups on
the H3/H4 side of the retinal binding site. The Met207-C18 contact suggests a negative twist for the C6-C7 bond.
with the proton of the C3 carbon of the ꢀ-ionone ring. This
interaction produces an upfield chemical shift of the methyl
group22,43 (another example of a γ effect). Assuming that the
difference in the C16, C17 chemical shifts is due to a γ effect,
we assign the resonance at 26.1 ppm to the methyl group in the
axial orientation. An open question has been whether the 26.1
ppm resonance corresponds to the C16 or the C17 methyl group.
the ꢀ-ionone ring. As a consequence, in the rhodopsin to Meta
II transition, the C16 methyl resonance changes from 30.6 to
28.6 ppm, and the C17 methyl resonance changes from 26.1 to
33.4 ppm. These assignments, in turn, indicate that there is an
inversion in the ring conformation. Buss and co-workers70,71
have calculated that the energy barrier for ring inversion is
between 5-6 kcal/mol. Moreover, these results show that in
both rhodopsin and Meta II, the C16 and C17 chemical shifts
are unique and that averaging by rotation of the ionone ring
does not occur in the retinal binding site of the protein.
To determine which methyl group (C16 or C17) is at 26.1
ppm, we rely on recent 13C NMR studies that define the position
of the ꢀ-ionone ring in rhodopsin and in Meta II on the basis
of distance measurements between 13C groups on the retinal
and amino acids lining the retinal binding site.28 For example,
2D DARR NMR spectra have been obtained using rhodopsin
containing 13C-labels at the carbonyl carbons of Met207 and
His211 on H5, and regenerated with retinal 13C-labeled at both
the C16 and the C17 methyl groups. The strongest crosspeak,
corresponding to the shortest internuclear distance, is observed
to the retinal methyl group resonating at 30.6 ppm. According
to the rhodopsin crystal structures (pdb codes: 1U19 and
1GZM), the C16 methyl group is closer to the carbonyls of
Met207 (4.9 Å) and His211 (4.9 Å) on H5 than to the C17
methyl group, which is 7.0 Å away from the carbonyl of Met207
and 7.2 Å away from the carbonyl of His211.11 On this basis,
we can assign the resonance at 26.1 ppm to the C17 methyl
group in an axial orientation and the resonance at 30.6 ppm to
C16 methyl group in an equatorial orientation.
4. Conclusions
Crystal structures of the dark state of rhodopsin have provided
a number of clues for how the 11-cis retinal PSB chromophore
functions as an inverse agonist for activation.11,48,72 The lack
of a corresponding crystal structure of the active Meta II
intermediate has limited progress on understanding the mech-
anism by which the all-trans retinal SB functions as a full
agonist for activation. The recent crystal structure of opsin at
low pH,73,74 which contains several features of an activated
GPCR, lacks the covalently bound all-trans SB chromophore
that likely holds the extracellular side of the receptor in its active
conformation.28 Here, solid-state NMR measurements of the 13
C
chemical shifts and relaxation times of the all-trans SB
chromophore in Meta II provide the most detailed description
to date on the conformation and environment of a GPCR agonist.
The observation that the retinal C18 methyl group has a short
T1 relaxation time in both the dark state of rhodopsin and the
Meta II intermediate demonstrates that the C6-C7 single bond
has a twisted 6-s-cis conformation, which does not change upon
receptor activation. The 6-s-cis conformation contrasts with the
planar s-trans conformation of the retinal chromophore in
bacteriorhodopsin.17 The unusually high upfield chemical shift
of the retinal 13C5 resonance may be associated with steric
interactions of the ꢀ-ionone ring with helix H5 resulting in a
significant twist of the C6-C7 bond. Steric interactions involv-
On conversion to Meta II, the retinal moves toward H5 and
is positioned between Met207/His211 and Phe208/Phe212. The
C16 and C17 methyl group resonances are observed at 28.6
and 33.4 ppm, respectively. These assignments are based on
the following observations. First, a relatively strong DARR
NMR crosspeak is observed between the methyl resonance at
28.6 ppm and both the carbonyl carbons of Met207 and His211
on H5.28 Second, 2D DARR NMR data show that the side chain
of Met207 in Meta II is positioned between the ꢀ-ionone ring
and Cys167 on H4. Figure 6B shows that these constraints are
only consistent with assignment of the resonance at 28.6 ppm
to the C16 methyl group.
(70) Terstegen, F.; Buss, V. Chem. Phys. 1997, 225, 163–171.
(71) Terstegen, F.; Carter, E. A.; Buss, V. Int. J. Quantum Chem. 1999,
75, 141–145.
The upfield shift in the 28.6 ppm resonance in Meta II is
attributed to an axial orientation of the C16 methyl group and
the γ-effect produced by interaction with the protons on C3 on
(72) Li, J.; Edwards, P. C.; Burghammer, M.; Villa, C.; Schertler, G. F. X.
J. Mol. Biol. 2004, 343, 1409–38.
9
15168 J. AM. CHEM. SOC. VOL. 131, NO. 42, 2009

Solid-State 13C NMR of Retinal in Metarhodopsin II
A R T I C L E S
ing the ꢀ-ionone portion of the retinal are essential for rhodopsin
activation.69 In this regard, the ꢀ-ionone ring appears to be well
positioned to hold H5 in an active conformation. Recently, we
have shown that retinal isomerization is coupled to the motion
of EL2 and H5.29 Rotation of H5 is important for the insertion
of Tyr223 into the region of the ionic lock between H3 and
H6.29,73,74
Finally, it is possible to activate several opsin mutants above
basal (or constitutive) levels in membranes by the addition of
all-trans retinal as a diffusible ligand.75 However, the activity
never reaches the full light-induced activity of Meta II. This
observationsupportsthenotionthattherearespecificretinal-protein
contacts that form in the active site upon photoisomerization
that might stabilize unique “agonist” structural features (i.e.,
structural changes on the extracellular side of the receptor are
allosterically coupled to changes in the intracellular loops). In
fact, the agonist binding pocket optimal for receptor activity is
probably only achieved in the presence of the bound G protein,
analogous to the “high affinity” agonist state in ligand-activated
GPCRs.
The comparison between ∆MII values for the retinal C5-C20
carbons relative to model compounds in polar and nonpolar
solvents suggests that the SB linkage of the all-trans retinal
chromophore in Meta II is in a polar environment despite the
fact the SB cannot be titrated. These results are in agreement
with previous studies that the C20 methyl group rotates roughly
180° toward the extracellular surface28 and EL2 is displaced
slightly from the retinal binding site.29 The displacement of EL2
allows water to enter on the extracellular side of binding site.
In this regard, it is known that the Schiff base is more accessible
to hydroxylamine in Meta II.5,6 The orientation of the C20
methyl group suggests that the C15H proton is directed toward
Glu113 and the Schiff base nitrogen is oriented toward the
hydrophobic receptor interior. We propose that polarization of
the CdN bond, reflected in the NMR chemical shifts reported
here, results from this orientation of the retinal SB in Meta II
and that this facilitates Schiff base hydrolysis by dramatically
increasing the partial positive charge on the C15 carbon in the
presence of water.
Acknowledgment. This work was supported by a research grant
to S.O.S. from the NIH (GM-41412), by NIH-NSF instrumentation
grants (S10 RR13889 and DBI-9977553), and by the US-Israel
Binational Science Foundation to M.S. We gratefully acknowledge
the W. M. Keck Foundation for support of the NMR facilities in
the Center of Structural Biology at Stony Brook. M.S. acknowledges
support from the Kimmelman center for Biomolecular Structure
and Assembly.
JA9034768
(75) Han, M.; Smith, S. O.; Sakmar, T. P. Biochemistry 1998, 37, 8253–
8261.
9
J. AM. CHEM. SOC. VOL. 131, NO. 42, 2009 15169