Synthesis of CD3-labeled 11-cis-retinals and application to solid-state deuterium NMR spectroscopy of rhodopsin

Article abstract of DOI:10.1246/bcsj.80.2177

Efficient synthesis of 11-Z-retinals labeled with ²H at the C5, C9, or C13 methyl groups is described. The ²H-labeled retinals were used to regenerate the visual pigment rhodopsin for structural investigations. Solid-state ²H NMR data provided the orientation of retinal within the rhodopsin binding pocket as well as its conformation. Extension of the approach to other membrane receptors can yield knowledge of their mechanisms of activation as a guide for ligand-based drug design.

Full text of DOI:10.1246/bcsj.80.2177

ꢀ 2007 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 80, No. 11, 2177–2184 (2007) 2177
Synthesis of CD3-Labeled 11-cis-Retinals and Application
to Solid-State Deuterium NMR Spectroscopy of Rhodopsin#
y
yy
Katsunori Tanaka,1; Andrey V. Struts,2 Sonja Krane,1;
yyy
yyyy
2
Naoko Fujioka,1;
Gilmar F. J. Salgado,3;
and Koji Nakanishi1
´
Karina Martınez-Mayorga,
ꢀ2;3
Michael F. Brown,
1Department of Chemistry, Columbia University, New York, New York 10027, USA
2Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA
3Department of Biochemistry & Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA
Received January 9, 2007; E-mail: mfbrown@u.arizona.edu
2
Efficient synthesis of 11-Z-retinals labeled with H at the C5, C9, or C13 methyl groups is described. The 2H-
2
labeled retinals were used to regenerate the visual pigment rhodopsin for structural investigations. Solid-state H NMR
data provided the orientation of retinal within the rhodopsin binding pocket as well as its conformation. Extension of the
approach to other membrane receptors can yield knowledge of their mechanisms of activation as a guide for ligand-based
drug design.
Site-directed nuclear spin labeling in combination with
solid-state NMR spectroscopy can give unique information
regarding the conformation and orientation of ligands bound
to integral membrane proteins and G protein-coupled receptors
such as rhodopsin. Biomolecular applications of NMR typi-
cally rely on the availability of suitable isotopically labeled
compounds. Effectively, two strategies are possible: either
[9-CD3]-, and 11-Z-[13-CD3]-retinals (structures shown in
Fig. 1). Deuterium labeling enables us to obtain site-specific
orientational restraints to model the structure of retinal bound
to rhodopsin and other photoreceptors in combination with
bioorganic and biophysical data.
Results and Discussion
2
the molecules are uniformly labeled, e.g. with 13C, 15N, or H
Synthesis of 11-cis-Retinals Deuterated at C5, C9, and
C13 Methyl Groups. The stereoselective synthesis of deuter-
ated retinals, namely, 11-Z-[5-CD3]-retinal, 11-Z-[9-CD3]-
retinal, and 11-Z-[13-CD3]-retinal was carried out by the Ito’s
procedure, which utilizes the diene/tricarbonyliron complex
(Schemes 1–3).19 Other methods for the preparation of 2H-
labeled retinals have been described previously.26,27 For syn-
thesis of [5-CD3]- and [9-CD3]-retinals, the regioselectively
deuterated ꢀ-ionones 4 and 7 were used as the starting materials
and were prepared according to Scheme 1. Thus, 2,2-dimeth-
ylcyclohexanone (1) was reacted with CD3I where lithium di-
isopropylamide (LDA) was used as a base to provide 2 in 80%
yield, which was converted to the triflate 3 by treatment with
LDA and N-phenylbis(trifluoromethanesulfonimide) (NPhTf2)
in 65% yield. The triflate 3 was then subjected to the Heck re-
action with methylvinylketone in the presence of PdCl2(PPh3)2
in DMF, providing the [5-CD3]-ꢀ-ionone (4) in 50% yield.
Alternatively, the ꢀ-ionone was treated with bromine and
sodium hydroxide in 1,4-dioxane to give the acid 5 in 88%
yield, which was converted to the Weinreb amide 6 by reaction
with methoxymethylamine hydrochloride in the presence of
DMAP and EDC in 89% yield. The amide 6 was successfully
treated with deuterated methylmagnesium iodide (CD3MgI) in
THF to yield [9-CD3]-ꢀ-ionone (7) in 66% yield.
isotopes through bacterial expression in the case of proteins,1–4
or rather organic synthesis is used to introduce isotopic labels
to probe specific regions of interest within the molecule.5,6
For solid-state NMR spectroscopy of biomolecules specific
labeling is often used.7–9 Deuterium substitution in the case
of 2H NMR is widely applicable to membrane lipids,10,11 mem-
brane proteins,7,12 and nucleic acids.13,14 Another important
application of 2H-isotope labeling involves neutron diffraction
of membrane lipids15 and membrane proteins.16,17 Organic
synthesis6,18–20 has been applied extensively to 13C and 2H
labeling of retinal for solid-state NMR applications to bacterio-
rhodopsin21–23 or rhodopsin.8,24,25 In view of the biological
importance of retinal, it is desirable that additional synthetic
methods for 2H-isotope labeling are described. We report
herein the efficient synthesis of deuterated retinals having
methyl 2H isotope labels, namely, 11-Z-[5-CD3]-, 11-Z-
y Present address: Department of Chemistry, Graduate School of
Science, Osaka University, 1-1 Machikaneyama, Toyonaka,
Osaka 560-0043
yy Present address: Publications Division, American Chemical
Society, Washington, DC 20036, USA
yyy Present address: Shionogi & Co., Ltd., Osaka
The [5-CD3]- and [9-CD3]-ꢀ-ionones (4 and 7), prepared
in Scheme 1, were respectively converted to the diene/tricar-
bonyliron complex 8 by treatment with triirondodecacarbonyl
´
´
´
yyyy Present address: Faculte de Pharmacie, Universite Rene
Descartes, Paris 75270, France
Published on the web November 13, 2007; doi:10.1246/bcsj.80.2177

2178 Bull. Chem. Soc. Jpn. Vol. 80, No. 11 (2007)
Synthesis of Deuterated 11-cis-Retinals
Fig. 1. 11-Z-[5-CD3]-, 11-Z-[9-CD3]-, and 11-Z-[13-CD3]-retinals.
Scheme 1. Synthesis of [5-CD3]- and [9-CD3]-ꢀ-ionones.
Scheme 2. Synthesis of 11-Z-[5-CD3]- and 11-Z-[9-CD3]-retinals.

K. Tanaka et al.
Bull. Chem. Soc. Jpn. Vol. 80, No. 11 (2007) 2179
Scheme 3. Synthesis of 11-Z-[13-CD3]-retinal.
(Fe3(CO)12) in hot benzene (Scheme 2). The iron complex 8
was alkylated with the lithium salt of acetonitrile in THF,
which was subsequently reduced by diisobutylaluminum
hydride (DIBAL) to provide the (E)-conjugated aldehyde 10
stereoselectively, with the concomitant shift of the tricarbonyl-
iron group to the C7–C8/C9–10 diene. Stereoselective trans-
formation to the Z-ester 11 was achieved by Peterson olefin-
ation with ethyl trimethylsilylacetate, by virtue of the steric
bulkiness of the tricarbonyliron moiety,19 which was then con-
verted to the Weinreb amide 12 by reaction with iso-propyl-
magnesium bromide and methoxymethylamine in THF. Alkyl-
ation of the amide 12 with methyl lithium in THF and E-selec-
tive Horner–Emmons reaction with diisopropyl cyanomethyl-
phosphonate and sodium hydride gave the nitrile 14.
Demetalation was realized upon treatment with copper(II)
dichloride in ethanol. Finally, DIBAL reduction of the nitrile
15 followed by purification by silica-gel chromatography
provided the 11-Z-[5-CD3]-retinal and 11-Z-[9-CD3]-retinal,
respectively.
11-Z-retinal with MHþ at m=z ¼ 285:1. (The molar mass of
protiated retinal is 284.4). However, for 11-Z-[9-CD3]-retinal,
the ESI mass spectrum gave four peaks at m=z ¼ 288:1, 287.2,
286.1, and 285.1/285.3. Hence, it is possible that some ex-
change of deuterium occurred to yield retinal isotopomers with
CD3, CHD2, CH2D, or CH3 groups.
Application to 2H NMR Spectroscopy of Rhodopsin.
Deuterium NMR in combination with site-directed 2H-nuclear
spin labeling provides structural information for ligands bound
to integral membrane proteins such as rhodopsin, and is com-
plementary to X-ray crystallographic studies. An advantage of
solid-state NMR is that single crystals are unnecessary, and
moreover dynamical information is available.10 We applied
2
solid-state H NMR spectroscopy to investigate the conforma-
tion and orientation of retinal within rhodopsin as a prototype
for G protein-coupled receptors (GPCRs). This knowledge is
broadly significant for GPCR biology and pharmacology in
general. More specifically, the nature of the primary photo-
chemical events involving rhodopsin is crucial for understand-
ing the process of visual signaling.28,29
Similarly, the 11-Z-[13-CD3]-retinal (Scheme 3) was syn-
thesized from the Weinreb amide 16, stereoselectively pre-
pared from the ꢀ-ionone via the same sequences of the reac-
tions shown in Scheme 1. In order to incorporate the CD3 moi-
ety at the C13 position, the amide 16 was alkylated with
CD3MgI in 59% yield. Subsequent Horner–Emmons reaction
(92%), demetalation (64%), and DIBAL reduction (58%) suc-
cessfully provided the desired 11-Z-[13-CD3]-retinal. All three
CD3-labeled 11-cis-retinals thus prepared were stereochemi-
cally pure, and required no further HPLC purification.
The synthetic 11-Z-[5-CD3]-, 11-Z-[9-CD3]-, and 11-Z-
[13-CD3]-retinals were stored in benzene solution at ꢁ70 ꢂC
in total darkness. They were characterized by their UV–visible
We conducted studies of the synthetic CD3-labeled retinals
covalently bound to rhodopsin in bilayer membranes using
solid-state NMR spectroscopy (Fig. 2). This procedure in-
volved the regeneration of rhodopsin with 11-Z-[5-CD3]-,
11-Z-[9-CD3]-, or 11-Z-[13-CD3]-retinal prepared by organic
synthesis. Rhodopsin was then purified and recombined
with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)
(1:50 molar ratio) by detergent dialysis.30 Membranes con-
taining 11-Z-[5-CD3]-retinylidene rhodopsin, 11-Z-[9-CD3]-
retinylidene rhodopsin, or 11-Z-[13-CD3]-retinylidene rho-
dopsin embedded in a POPC bilayer were macroscopically
aligned on planar glass substrates by isopotential ultracentrifu-
gation.25 Deuterium NMR lineshapes were then measured
for the C5, C9, or C13 deuterated methyl substituents of the
retinylidene ligand to elucidate the orientation, conformation,
and mobility of the chromophore of rhodopsin.
1
2
spectra, Fourier transform infrared spectra, and their H, H,
and 13C NMR spectra (see Experimental Section). The deute-
rium content of the synthetic deuterated retinal samples was
2
confirmed by H NMR spectroscopy, and each gave a single
peak in benzene solution. Mass spectrometry in acetone/aceto-
nitrile using positive electrospray ionization (ESI) further
confirmed the purity of the synthetic deuterated retinals. For
the 11-Z-[5-CD3]- and 11-Z-[13-CD3]-retinals, the (quasi)-
molecular MHþ ions were at m=z ¼ 288:1, consistent with a
single-deuterated methyl group, as compared with authentic
Solid-state 2H NMR spectra for rhodopsin containing retinal
2
specifically H-labeled at the C5, C9, or C13 methyl groups
in aligned membrane bilayers are displayed in Figs. 2a–2c,
2
respectively. Comparison of the H NMR spectra for the C9
methyl group in Fig. 2b to the corresponding spectra for the
C5 and C13 methyl groups in Figs. 2a and 2c indicates that

2180 Bull. Chem. Soc. Jpn. Vol. 80, No. 11 (2007)
Synthesis of Deuterated 11-cis-Retinals
Fig. 3. Structures for 11-cis-retinal in the dark state of rho-
2
dopsin calculated from orientational H NMR constraints
and 13C NMR distance restraints. Enantiomeric structures
(a) and (b) are related by a vertical symmetry plane. The
right-hand structure (b) is excluded based on circular
dichroism results (see text).
2
Fig. 2. Solid-state H NMR spectra of retinal ligand bound
to rhodopsin in aligned membranes at 76.8 MHz (11.7 T)
in the dark state. Data are shown for (a) 11-Z-[5-CD3]-
retinylidene rhodopsin, (b) 11-Z-[9-CD3]-retinylidene
rhodopsin, and (c) 11-Z-[13-CD3]-retinylidene rhodopsin,
i.e. with 11-cis-retinal deuterated at the C5, C9, or C13
methyl groups, respectively, at temperatures of ꢁ150,
ꢁ60, and ꢁ30 ꢂC. 2H NMR spectra were acquired for
rhodopsin/POPC recombinant membranes (1:50 molar
ratio) with the membrane normal aligned at ꢁ ¼ 0 and
90ꢂ relative to the main magnetic field B0. Samples nomi-
nally contained 5 mM HEPES buffer prepared with 2H-
Fig. 4. Structural solutions for trans retinal within the bind-
ing pocket of rhodopsin in the meta I state. Angular
restraints are provided by simulating the 2H NMR data
for the C5, C9, and C13 methyl groups together with linear
dichroism results in terms of a simple three-plane model.
Structures (a), (b), (g), (h) shown in green, and (c), (d),
(e), (f) in red, are obtained from each other by reflection
symmetry transformations. Only the top left structure
(a) and its mirror image (b) are consistent with electron
crystallographic data.
1
depleted H2O at pH 6.8.
the intensity as revealed by the signal-to-noise ratio is substan-
tially less, despite the greater number of scans. We ascribe this
to incomplete deuteration of the C9 methyl substituent (see
above), together with a relatively long spin-lattice relaxation
time (T1Z; not shown); both require longer spectral acquisitions
versus the other two positions. The solid-state 2H NMR spectra
reveal that the methyl substituents undergo rapid three-fold
rotation at all temperatures. The orientation-dependent line-
shapes are related to the angles that the three-fold symmetry
axes of the different methyl groups make to the membrane sur-
We derived two possible structures for retinal in the dark
2
state based on the angular restraints from H NMR (Fig. 3).34
However, circular dichroism studies of locked retinoids imply
that the twist is positive for the C11=C12–C13=C14 dihedral
angle;35 whereas it is negative for the C5=C6–C7=C8 angle.36
This leaves as the only plausible physical structure the one
shown in Fig. 3a. In the meta I state of bleached rhodopsin,
eight possible solutions are obtained from the orientational
2H NMR and distance 13C NMR restraints (Fig. 4).37 The solu-
tions in Figs. 4c–4h can be eliminated, since based on electron
2
face normal.31 In addition, the H NMR spectra in Fig. 2 are
relatively constant from ꢁ150 to ꢁ60 ꢂC, which demonstrates
the stability of the retinal conformation over a wide tempera-
ture range. This lack of temperature variation of the methyl
group orientations is important with regard to X-ray crystallo-
graphic studies of rhodopsin and its photointermediates, which
are typically conducted at cryogenic temperatures.32,33

K. Tanaka et al.
Bull. Chem. Soc. Jpn. Vol. 80, No. 11 (2007) 2181
by preparative TLC using silica-gel plates, 60 F-254, 0.25 mm
1
crystallography the position of the ꢀ-ionone ring in meta I re-
mains the same as in the ground state.38 Molecular modeling34
also reveals that the conformation in Fig. 4b is less likely due
to steric hindrance within the binding pocket of rhodopsin. As
a result, the structure in Fig. 4a is the most plausible for retinal
in the meta I state, having a negative dihedral angle of ꢁ32ꢂ
for the C6–C7 bond. This conclusion is consistent with
measurements of the C8-to-C17 and C8-to-C18 distances in
meta I.39
(E. Merck). H NMR spectra were acquired with a Bruker DMX
400 MHz spectrometer and were referred in parts per million
(ppm) to TMS (ꢂ), with coupling constants (J) in Hertz (Hz).
Proton decoupled 13C spectra were obtained with a Bruker DRX
600 spectrometer and chemical shifts were referenced to the
CDCl3 solvent peak at 77.0 ppm. Assignments were made by
comparison with published retinal 13C chemical shifts.48,49 The
13C peaks originating from 5-CD3, 9-CD3, or 13-CD3 groups
were not observed due to heteronuclear 13C–2H coupling confirm-
ing the low abundance of residual non-deuterated methyl groups.
High-resolution 2H NMR spectra were obtained with a Bruker
DRX 400 spectrometer and each of the compounds in benzene
gave a single peak assigned to the deuterated CD3 groups. ESI
mass spectra were measured in acetone/acetonitrile using positive
electrospray ionization with a JEOL HX110A high-resolution
mass spectrometer.
2
Investigations of rhodopsin with a bound H-labeled ligand
2
open the door to application of H NMR to study the confor-
mation, orientation, and dynamics of retinal in the dark state,25
where the ligand is 11-cis, as well as the meta I state with an
all-trans polyene chain (see Appendix).11,37 The angular-
dependent 2H NMR lineshapes for deuterated retinal yield
the orientation of the labeled C–CD3 methyl group axes rela-
tive to the membrane normal, which cannot be obtained with
11-Z-[5-CD3]-Retinal: 1H NMR (400 MHz, CDCl3) ꢂH 1.07
(s, 6H), 1.43–1.46 (m, 2H), 1.52–1.59 (m, 2H), 1.74 (s, 3H), 1.76
(d, 3H, J ¼ 1:2 Hz), 1.91 (ddd, 2H, J ¼ 6:4, 6.4, 1.6 Hz), 5.59 (d,
1H, J ¼ 12:0 Hz), 6.10 (d, 1H, J ¼ 7:6 Hz), 6.21 (d, 1H, J ¼ 16:0
Hz), 6.34 (d, 1H, J ¼ 16:0 Hz), 6.38 (dd, 1H, J ¼ 12:0, 12.0 Hz),
6.58 (d, 1H, J ¼ 12:0 Hz), 9.90 (d, 1H, J ¼ 7:8 Hz); 13C NMR
(150.9 MHz, CDCl3) ꢂC 13.0 (9-CH3), 13.1 (13-CH3), 19.1 (C3),
29.0 (1,1-CH3), 33.1 (C4), 34.2 (C1), 39.7 (C2), 125.5 (C10),
129.0 (C7 or C12), 129.4 (C12 or C7), 129.7 (C14), 132.5 (C11),
134.5 (C10, from all-trans retinal), 137.0 (C6 or C8), 137.6 (C8 or
C6), 141.2 (C9), 154.7 (C13), 191.1 (C15); ESI-MS (positive) m=z
288.1 [ðM þ HÞþ].
2
other biophysical techniques.10 Thus, H NMR provides angu-
lar information for the retinylidene bond orientations that can
be combined with constraints about the geometry of the mole-
cule, and with the results of other biophysical,39–43 bioorgan-
ic,35,36 and biochemical44 methods. In particular, 2H NMR data
can be used to refine the results of X-ray and electron diffrac-
tion studies of rhodopsin in the dark state32,45–47 and its photo-
intermediates.32,33,38 Moreover, apart from giving information
2
complementary to other techniques, using H NMR rhodopsin
can be studied in a native-like membrane bilayer environment,
in contrast to X-ray diffraction studies of single crystals32,45,47
or electron diffraction of 2-D crystals.38 Last, we note that we
obtain information about the retinal ligand in the meta I state,
which is currently unavailable from X-ray crystallography.
11-Z-[9-CD3]-Retinal: 1H NMR (400 MHz, CDCl3) ꢂH 1.10
(s, 6H), 1.42–1.45 (m, 2H), 1.54–1.59 (m, 2H), 1.67 (s, 3H), 1.77
(s, 3H), 1.91 (dd, 2H, J ¼ 6:2, 6.2 Hz), 5.58 (d, 1H, J ¼ 11:8 Hz),
6.09 (d, 1H, J ¼ 7:8 Hz), 6.20 (d, 1H, J ¼ 16:0 Hz), 6.32 (d, 1H,
J ¼ 16:0 Hz), 6.37 (dd, 1H, J ¼ 11:9, 11.9 Hz), 6.57 (d, 1H, J ¼
12:4 Hz), 9.90 (d, 1H, J ¼ 7:8 Hz); 13C NMR (150.9 MHz, CDCl3)
ꢂC 13.1 (13-CH3), 19.1 (C3), 21.8 (5-CH3), 29.0 (1,1-CH3), 33.1
(C4), 34.2 (C1), 39.7 (C2), 125.5 (C10), 129.0 (C7 or C12), 129.4
(C12 or C7), 129.7 (C14), 130.5 (C5), 132.5 (C11), 134.5 (C10,
all-trans retinal), 137.0 (C6 or C8), 137.6 (C8 or C6), 154.7
(C13), 191.1 (C15); ESI-MS (positive) m=z 288.1 [ðM þ HÞþ].
Conclusion
Our findings illustrate how specific 2H-isotopic labeling can
2
be used in conjunction with solid-state H NMR spectroscopy
to investigate ligands bound to GPCRs and other membrane
proteins. As a specific example, we have focused on the visual
receptor rhodopsin. Additional GPCRs and ligand-gated ion
channels are attractive pharmaceutical targets. Organic synthe-
11-Z-[13-CD3]-Retinal:
1H NMR (400 MHz, CDCl3) ꢂH
2
2
sis of H-labeled retinoids together with solid-state H NMR
spectroscopy gives unique information about the local environ-
ment of ligands in GPCRs such as rhodopsin. Experimental
2H NMR data and theoretical lineshape analysis yield the con-
formation of the retinal ligand, and show how it is oriented
within the binding cavity of rhodopsin in the membrane-bound
state. This knowledge is pertinent to investigating the molecu-
lar basis for the pharmacology of agonists and antagonists
in terms of receptor activation. Extension of this approach to
other receptors can facilitate ligand-based drug discovery
and the design of new pharmaceutical agents.
1.07 (s, 6H), 1.43–1.46 (m, 2H), 1.53–1.59 (m, 2H), 1.68 (s, 3H),
1.74 (s, 3H), 1.91 (dd, 2H, J ¼ 6:1, 6.1 Hz), 5.59 (d, 1H, J ¼ 11:8
Hz), 6.10 (d, 1H, J ¼ 7:7 Hz), 6.21 (d, 1H, J ¼ 16:0 Hz), 6.33 (d,
1H, J ¼ 15:7 Hz), 6.38 (dd, 1H, J ¼ 11:9, 11.9 Hz), 6.57 (d, 1H,
J ¼ 12:4 Hz), 9.90 (d, 1H, J ¼ 7:8 Hz); 13C NMR (150.9 MHz,
CDCl3) ꢂC 13.0 (9-CH3), 19.1 (C3), 21.8 (5-CH3), 29.0 (1,1-CH3),
33.1 (C4), 34.2 (C1), 39.7 (C2), 125.5 (C10), 129.0 (C7 or C12),
129.4 (C12 or C7), 129.7 (C14), 130.5 (C5), 132.5 (C11), 134.5
(C10, all-trans retinal), 137.0 (C6 or 8), 137.6 (C8 or C6), 141.2
(C9), 191.1 (C15); ESI-MS (positive) m=z 288.1 [ðM þ HÞþ].
Preparation of 2H Retinylidene Rhodopsin in POPC
Membranes. All procedures involving rhodopsin were carried
out under dim red light or in total darkness at 4 ꢂC unless other-
wise specified. Rhodopsin-containing membranes were isolated
from frozen bovine retinas (W. L. Lawson Co., Lincoln, NE) as
described.30 Suspensions of rod outer segment (ROS) membranes
in 10 mM HEPES buffer at pH 6.8 containing 100 mM hydroxyl-
amine were bleached for 30 min at 4 ꢂC, using yellow light. Well-
homogenized suspensions were prepared and gently handled using
a Pasteur pipette. The opsin-containing membranes thus obtained
were centrifuged for 30 min at 20000 rpm in a Sorvall T865 rotor
Experimental
Materials and Methods. Synthesis of 11-Z-[5-CD3]-, 11-Z-
[9-CD3]-, and 11-Z-[13-CD3]-retinals followed reported proce-
dures.19 Anhydrous dichloromethane, diethylether, benzene, and
acetonitrile were dried and distilled. Unless otherwise noted,
materials were obtained from a commercial supplier and were
used without further purification. All reactions were performed
in pre-dried glassware under Ar. Purification was performed either
by column chromatography using ICN silica gel (32–63 mesh) or

2182 Bull. Chem. Soc. Jpn. Vol. 80, No. 11 (2007)
Synthesis of Deuterated 11-cis-Retinals
(40 660 ꢃ g) at 4 ꢂC, after which the pellet was resuspended in
10 mM HEPES buffer without hydroxylamine, repeating the
procedure 4ꢃ. Benzene solutions containing the synthetic 11-Z-
[5-CD3]-retinal, 11-Z-[9-CD3]-retinal, or 11-Z-[13-CD3]-retinal
were evaporated using a light stream of N2, after which retinal
was re-dissolved in EtOH (maximum of 1% v/v with respect to
buffer). Synthetic retinal was immediately added to opsin-contain-
ing membranes in 1.5 molar excess, and regeneration was carried
out for 1.5 h at 37 ꢂC. Rhodopsin was dissolved in dodecyltrimeth-
ylammonium bromide (DTAB) detergent and was purified on a
hydroxyapatite column. The regenerated rhodopsin was then re-
combined with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC) in a 1:50 protein/lipid molar ratio by detergent dialysis.30
Solid-State 2H NMR Spectroscopy. Membranes containing
11-Z-[5-CD3]-retinylidene rhodopsin, 11-Z-[9-CD3]-retinylidene
rhodopsin, or 11-Z-[13-CD3]-retinylidene rhodopsin and POPC
(1:50) were aligned on planar glass substrates by isopotential
ultracentrifugation.25,50 Recombinant membranes with rhodopsin
nominally in 5 mM HEPES buffer at pH 6.8 were subjected to
slow evaporation under a nitrogen atmosphere at 4 ꢂC. The glass
plates containing the recombinant membranes as a dry film were
hydrated in a sealed container, containing a saturated salt solution
(NaBr in 2H-depleted water) with a relative humidity ꢄ63% at
4 ꢂC. Thereafter, 25 glass plates were stacked and inserted in a
cut-off 8 ꢃ 22 mm NMR tube sealed with a Teflon plug. Recom-
binant POPC membrane samples used for 2H NMR typically
contained 22–25 mg of rhodopsin. The 2H NMR spectra were
acquired using a Bruker AMX 500 spectrometer with a narrow
bore magnet (11.7 T). A custom-built high-power 2H NMR
probe was utilized, which was optimized for the rhodopsin-con-
taining samples. A compꢂosite-pulse quadrupolar-echo sequence
(135ꢁꢂ x90xꢂ45ꢂꢁx–ꢃ–135yꢂ90ꢁy45ꢂy–ꢃ–acquisition) was employed
Fig. 5. Projection of the retinal molecule onto (a) the crys-
ꢀ
tallographic ð101Þ plane and (b) the (010) plane. The
retinal long axis is indicated by the broken line and the
transition dipole moment by the solid line with double
arrows (see text).
tively (cf. Fig. 4 of text). In both states, the transition dipole mo-
ment makes an angle of 16ꢂ to the membrane surface. However,
we also need to know the orientation of the main transition dipole
moment with respect to the retinal molecular axis. In the dark state
this angle is found to be 16ꢂ, where the molecular axis for 11-cis
retinal is chosen as the line connecting carbons C6 and C12.40
Now for the meta I state, referring to Fig. 5 the transition dipole
moment orientation of trans retinal with respect to the molecular
axis is determined by the angle ꢄ, which is calculated from linear
dichroism data53 in combination with the corresponding X-ray
crystal structure.54 Figure 5a shows the projection of the retinal
2
and was phase-cycled to acquire the H NMR signals. The delay
ꢃ varied from 40 to 70 ms and the recycle delay for the pulse
sequence was typically 0.1–1.5 s. The membrane tilt angle was
adjusted manually and was measured with a protractor.
Research was supported by U. S. National Institutes of
Health grants GM 36564 (to K N.) and EY 12049 (to M. F. B.).
We thank Constantin Job, Avigdor Leftin, and Neil Jacobsen
for assistance with the NMR experiments and helpful discus-
sions. K.T. is grateful to the JSPS for the award of a Post-
doctoral Fellowship for Research Abroad. G. F. J. S. received
a predoctoral fellowship award from the BIO5 Institute of the
University of Arizona. M. F. B. thanks the JSPS for the award
of a Research Fellowship at the Institute for Protein Research.
ꢀ
molecule within the crystal unit cell onto the ð101Þ plane, where
the long axis of the retinal molecule is defined as a line connecting
the C6 and O atoms. The double-headed arrow represents the main
transition dipole moment (m). The polyene chain plane is chosen
as passing through the molecular axis and minimizes the distances
of odd-numbered carbons including the C9 and C13 methyl
groups from the plane. The transition dipole moment was assumed
to lie in this plane and to form an angle ꢄ with the long axis of the
molecule.
Referring now to Fig. 5a, for a unit vector d collinear with the
transition moment we have:
Appendix
Modeling Retinal Conformation Bound to Receptor. The
possible orientations and structures of retinal based on NMR spec-
troscopy and linear dichroism data were obtained as follows. As a
specific example we consider the metarhodopsin I state with an
all-trans polyene chain. To calculate the orientation of retinal in
the binding pocket of rhodopsin, we introduce the angle of the
d ¼ k cos ꢄ þ i sin ꢄ:
Here, i and k are orthogonal unit vectors spanning the plane of the
polyene chain, and the vector k is collinear with the long molecu-
lar axis. Moreover, an axis b is defined that is perpendicular to
ð1Þ
?
ꢀ
the monoclinic b-axis and placed in the ð101Þ plane. Next, in ac-
2
C9 methyl group to the bilayer normal as obtained by H NMR.
cord with Fig. 5b we can write:
In addition we include the orientation of the main transition dipole
moment obtained from linear dichroism studies of rhodopsin in
either the dark state51 or the meta I state.52 These two axes define
the orientation of the central B plane of the retinylidene moiety
within the rhodopsin binding pocket. The orientations of the A
and C planes are defined by the C5 and C13 methyl axes, respec-
b
¼ x cos ꢅ þ z sin ꢅ;
ð2Þ
?
where b is a unit vector. Here ꢅ is the angle the b -axis
?
?
makes with the x-axis of the Cartesian system, and ðx; y; zÞ in
are unit vectors referred to the orthogonal crystallographic axes
ꢀ
ða; b; c Þ. The angle ꢅ can be calculated from the parameters of

K. Tanaka et al.
Bull. Chem. Soc. Jpn. Vol. 80, No. 11 (2007) 2183
ꢂ
˚
the unit cell (a ¼ 15:270, b ¼ 8:264, c ¼ 14:942 A, ꢀ ¼ 104:73 ),
10 M. F. Brown, in Biological Membranes. A Molecular
Perspective from Computation and Experiment, ed. by K. Merz,
giving:
ꢀ
ꢁ
Jr., B. Roux, Birkhauser, Basel, 1996, p. 175.
¨
c sin ꢀ
ꢅ ¼ tanꢁ1
:
ð3Þ
11 M. F. Brown, S. Lope-Piedrafita, G. V. Martinez, H. I.
Petrache, Modern Magnetic Resonance, ed. by G. A. Webb,
Springer, Heidelberg, 2006, p. 245.
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a ꢁ c cos ꢀ
The acute angle ꢁ is between the projection of the transition mo-
ꢀ
ment m of the retinal molecule onto the ð101Þ plane, i.e. the plane
of measurement, and the b axis, cf. Fig. 4a. It is determined from
?
linear dichroism as:
pffiffiffi
ꢁ ¼ tanꢁ1ðmb=mb?Þ ¼ tanꢁ1 P;
ð4Þ
where P ꢅ m2b=mb2? is the polarization ratio for the two directions
b and b . Combining Eqs. 1, 2, and 4 and using mb ¼ md y and
?
ꢆ
mb? ¼ md b leads to:
?
ꢆ
ðk cos ꢄ þ i sin ꢄÞ y
ꢆ
tan ꢁ ¼
:
ð5Þ
ðk cos ꢄ þ i sin ꢄÞ ðx cos ꢅ þ z sin ꢅÞ
Hence, we arrive at the following result:
ꢆ
ꢀ
ꢁ
ky ꢁ kx tan ꢁ cos ꢅ ꢁ kz tan ꢁ sin ꢅ
ix tan ꢁ cos ꢅ þ iz tan ꢁ sin ꢅ ꢁ iy
ꢄ ¼ tanꢁ1
:
ð6Þ
The angle ꢁ has been reported53 as 24.1ꢂ, from which one can de-
rive that ꢄ ¼ 4:4ꢂ. Knowing the orientation of the main transition
dipole moment with respect to (i) the retinal long axis (angle ꢄ)
and (ii) the membrane normal, together with (iii) the orientation
of the C9 methyl group, one can determine the inclination of the
retinal long axis to the membrane normal. In this way, for the dark
and meta I states we obtain values of 62 and 71ꢂ, respectively.
The possible solutions (cf. Fig. 4 of text) for the trans polyene
chain of retinal in meta I can then be calculated based on angular
restraints obtained from 2H NMR for the C5 and C13 methyl
groups37 and distance constraints from 13C rotational resonance
NMR.39,41 In Fig. 4, structures (c) through (h) are excluded on ac-
count of the different position of the ꢀ-ionone ring with respect to
retinal in the dark state.34 These structures as well as structure (b)
do not fit into the rhodopsin binding pocket in the dark state and
have numerous steric clashes, leaving structure (a) as the physical
solution.
´
22 V. Copie, A. E. McDermott, K. Beshah, J. C. Williams,
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