Double-quantum 13C nuclear magnetic resonance of bathorhodopsin, the first photointermediate in mammalian vision

Article abstract of DOI:10.1021/ja803801u

The ¹³C chemical shifts of the primary visual photointermediate bathorhodopsin have been observed by performing double-quantum magic-angle-spinning NMR at low temperature in the presence of illumination. Strong isomerization shifts have been ob

Full text of DOI:10.1021/ja803801u

Published on Web 07/22/2008
Double-Quantum 13C Nuclear Magnetic Resonance of Bathorhodopsin, the
First Photointermediate in Mammalian Vision
Maria Concistre`,† Axel Gansmu¨ller,† Neville McLean,† Ole G. Johannessen,† Ildefonso Mar´ın
Montesinos,† Petra H. M. Bovee-Geurts,§ Peter Verdegem,‡ Johan Lugtenburg,‡
Richard C. D. Brown,† Willem J. DeGrip,§ and Malcolm H. Levitt*,†
School of Chemistry, UniVersity of Southampton, SO17 1BJ Southampton, U.K., Leiden Institute of Chemistry,
Gorlaeus Laboratories, NL-2300 RA Leiden, The Netherlands, and Nijmegen Centre for Molecular Life Sciences,
Radboud UniVersity, NL-6500 HB Nijmegen, The Netherlands
Received May 21, 2008; E-mail: mhl@soton.ac.uk
Dim-light vision in mammals is initated when a photon is
absorbed by the retinylidene prosthetic group of the seven-helix
transmembrane protein rhodopsin, a G-protein coupled receptor
(GPCR) which is abundant in the rod cells of the retina at the back
of the eye. The photon absorption leads to an ultrafast (<100 fs)
and highly selective isomerization of the retinylidene conjugated
chain from the initial 11-Z configuration to a distorted all-E
configuration (Figure 1). The resultant photostate is called bathor-
hodopsin since its optical absorption is red-shifted relative to
rhodopsin.1,2 Bathorhodopsin is a highly energetic species that stores
2
of the absorbed photon energy.1 Under physiological
Figure 1. Sketch of the distorted all-E retinylidene chromophore in
about
/
3
bathorhodopsin, showing the numbering of the carbon sites and the
protonated Schiff base linkage to the K296 side chain. The structural data
are from the Protein Data Bank entry 2g87 (see ref 4).
conditions, bathorhodopsin is converted rapidly into a series of less
energetic photostates, culminating in the active state metarhodopsin-
II, which is capable of binding and activating many copies of the
G-protein transducin, a process that leads to a modulation of the
transmembrane potential within the cell and induction of an optical
nerve signal.2
ppm deshielding shift of the 13C isotropic chemical shift of site
C10 when bathorhodopsin is generated.
The new data were generated using 13C2-labeled isotopomers of
rhodopsin, prepared by total organic synthesis of 13C2-retinals,11
followed by regeneration of 13C2-rhodopsin using bleached opsin
pigments isolated from bovine retinas, and reconstitution with
natural-composition lipid membranes. Four 13C2-labeled rhodopsin
isotopomers were studied, namely, [9,10-13C2], [11,12-13C2], [12,13-
13C2], and [14,15-13C2]-retinylidene rhodopsin. Since the reconsti-
tuted samples are optically dense, the light penetration was improved
by grinding frozen rhodopsin samples in liquid nitrogen to form
particles of diameter ∼200 µm which were then mixed with ∼100
µm diameter glass beads. The rhodopsin/glass bead mixtures were
packed into 4 mm diameter thin-wall zirconia rotors for the magic-
angle-spinning NMR experiments. All procedures were performed
in dim red light to avoid premature isomerization.
Since bathorhodopsin is stable at temperatures below 125 K,1
its structure and properties may be studied by illuminating rhodopsin
at low temperature and using techniques such as X-ray diffraction
and nuclear magnetic resonance. An early solid-state NMR study
attempted to measure 13C chemical shifts in the retinylidene side
chain of 13C-labeled bathorhodopsin and thereby obtain information
on the electronic structure.3 However, only minor chemical shift
changes were observed relative to rhodopsin.3
A more recent 2.7Å X-ray structure of bathorhodopsin has
verified the distorted all-E configuration of the chromophore and
also identified various structural changes in the immediate protein
environment, including a small displaceent of the negatively charged
Glu-113 counterion relative to the positively charged nitrogen atom
of the protonated Schiff base (PSB) linking the chromophore to
the lysine-296 side chain of the opsin protein.4 The electronic
structure of bathorhodopsin has been modeled using density
functional theory,5,6 QM/MM,7–9 and molecular dynamics10 cal-
culations, but in general, the level of agreement between the reported
NMR chemical shifts and the calculated values has been quite poor.
13C NMR spectra of the labeled chromophores were recorded
using the double-quantum filtered pulse sequence reported in ref
12. Symmetry-based double-quantum recoupling using pulse se-
9
quences with the symmetry R202 , combined with phase-cycling
to remove signals that do not pass through double-quantum
coherence, achieves a clean suppression of the natural 13C
background signals from the protein and the lipid. The spectra in
Figure 2 show clearly resolved signals from the 13C labels of the
chromophore.
Bathorhodopsin was generated by illuminating the rhodopsin
samples in situ in a custom-built NMR probe at a sample
temperatures between 110 and 125 K. The light was generated by
two 250 W halogen lamps passed through 420 ( 5 nm interference
filters (Edmund optics, UK) and led into the sample region by 14
optical fibers. The illumination wavelength was chosen so as to
improve the light penetration into the sample and to minimize the
undesirable photoisomerization of bathorhodopsin into the 9-Z
In this communication, we report on new measurements of the
13C chemical shifts in the retinylidene chromophore of bathor-
hodopsin, performed with modern double-quantum recoupling
techniques and with great care paid to the calibration of the real
sample temperature and to the illumination methodology. In contrast
to the 1991 results,3 the new values indicate a considerable
perturbation in bathorhodopsin. For example, we observe a +9.4
† University of Southampton.
‡ Leiden Institute of Chemistry.
§ Nijmegen Centre for Molecular Life Sciences.
9
10490 J. AM. CHEM. SOC. 2008, 130, 10490–10491
10.1021/ja803801u CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Isotropic 13C Chemical Shifts of Rhodopsin (δrho) and
Bathorhodopsin (δbatho) As Found in This Worka
13C site
δ
rho/ppm
δ
batho/ppm
∆δ/ppm
9
10
11
12
13
14
15
148.9
127.9
141.4
131.8
167.4
122.3
165.4
149.6
137.3
144.4
132.8
171.2
117.5
164.4
+ 0.7
+ 9.4
+ 3.0
+ 1.0
+ 3.8
-4.8
-1.0
a The last column shows the isomerization shift, ∆δ ) δbatho - δrho
.
All chemical shifts have a confidence limit of (0.5 ppm and are
referenced indirectly to TMS using the rhodopsin shift data in ref 16.
The isotropic 13C chemical shifts of the C9 to C15 sites in
bathorhodopsin are summarized in Table 1. The +9.4 ppm
isomerization shift of C10 is particularly striking. The isomerization
shifts tend to be in the deshielding direction toward the center of
the chain but in the shielding direction at the end of the chain.
Comparisons with previous NMR data and with numerical shift
calculations are presented in the Supporting Information.
We are currently in the process of interpreting the bathorhodopsin
chemical shifts. The isomerization shifts of the odd-numbered
carbons might be interpreted in terms of the small displacement of
the negatively charged counterion from the vicinity of the positively
charged protonated Schiff base linkage, while the isomerization
shifts of the even-numbered carbons are due to the torsional twists.
Figure 2. Double-quantum filtered 13C NMR spectra of (a) [9,10-13C2],
(b) [11,12-13C2], (c) [12,13-13C2], (d) [14,15-13C2] rhodopsin before (above)
and after (below) 10 h of illumination with 420 ( 5 nm light. All spectra
are the Fourier transforms of ∼12 000 NMR signals acquired at a
temperature <120 K with magic-angle spinning at 7.00 ( 0.05 kHz. The
positions of the bathorhodopsin signals are indicated by asterisks.
Acknowledgment. This research was supported by BBSRC
(UK), EPSRC (UK), NWO (NL), CMSB (NL), and EC E-MeP
(NL). We thank M. Carravetta, P. Jansson, J. James, H. J. M. de
Groot, I. Heinmaa, D. Sebastiani, and K. Komatsu for technical
and experimental help, data, discussions, and samples.
isomer isorhodopsin.13 These conditions were optimized by nu-
merical finite-element simulations of the light penetration through
the optically dense sample, as described elsewhere.
Supporting Information Available: Full refs 15 and 16, sample
preparation, NMR equipment, temperature calibration data, chemical
shift comparisons, and experimental procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
The sample temperature is difficult to determine in magic-angle-
spinning experiments, due to frictional heating and warming caused
by the rotor bearing and drive nitrogen gas streams. As described
in the Supporting Information, the sample temperature was
calibrated during the illumination and acquisition of the NMR data
using the 207Pb chemical shift of lead nitrate,14 using the narrow
proton resonance of the endohedral dihydrogen-fullerene complex
H2@C60 as an independent chemical shift reference.15 The sample
temperature was found to exceed the temperature of the exiting
nitrogen gas by up to 40 K. The new results are clearly more reliable
than those obtained in 19913 using a vulnerable experimental and
data subtraction procedure.
As shown in Figure 2, double-quantum filtered NMR spectra
obtained after illumination show a clear splitting of at least one of
the 13C peaks, indicating the generation of bathorhodopsin. The
∼35% bathorhodopsin yield is probably limited by the partial
penetration of light into the optically dense particles and by
secondary photoisomerization of bathorhodopsin.
References
(1) Palczewski, K. Annu. ReV. Biochem. 2006, 75, 743–767.
(2) Rodieck, R. W. The First Step in Seeing; Sinauer Associates: Sunderland,
MA, 1998.
(3) Smith, S. O.; Courtin, J.; de Groot, H. J. M.; Gebhard, R.; Lugtenburg, J.
Biochemistry 1991, 30, 7409–7415.
(4) Nakamichi, H.; Okada, T. Angew. Chem., Int. Ed. 2006, 45, 4270–4273.
(5) Schreiber, M.; Buss, V. Int. J. Quantum Chem. 2003, 95, 882–889.
(6) Yan, E. C. Y.; Ganim, Z.; Kazmi, M. A.; Chang, B S. W.; Sakmar, T P.;
Mathies, R. A. Biochemistry 2004, 43, 10867–10876.
(7) Gascon, J. A.; Sproviero, E. M.; Batista, V. S. Acc. Chem. Res. 2006, 39,
184–193.
(8) Schreiber, M.; Sugihara, M.; Okada, T.; Buss, V. Angew. Chem., Int. Ed.
2006, 45, 4274–4277.
(9) Ro¨hrig, U. F.; Sebastiani, D. J. Phys. Chem. B 2008, 112, 1267–1274.
(10) Ro¨hrig, U. F.; Guidoni, L.; Laio, A.; Frank, I.; Rothlisberger, U. J. Am.
Chem. Soc. 2004, 126, 15328–15329.
(11) Groesbeek, M.; Lugtenburg, J. J. Photochem. Photobiol. 1996, 56, 903–
908.
(12) Mar´ın Montesinos, I.; Brouwer, D. H.; Antonioli, G.; Lai, W. C.;
Brinkmann, A.; Levitt, M. H. J. Magn. Reson. 2005, 117, 307–317.
(13) Birge, R. R.; Einterz, C. M.; Knapp, H. M.; Murray, L. P. Biophys. J.
1988, 53, 367–385.
(14) Neue, G.; Dybowski, C. Solid State Nucl. Magn. Reson. 1997, 4, 333–
336.
(15) Carravetta, M.; et al. Phys. Chem. Chem. Phys. 2007, 9, 4879–4894.
(16) Verhoeven, M. A.; et al. Biochemistry 2001, 40, 3282–3288.
The new 13C peaks that appeared after illumination were replaced
by broader signals at positions closer to the rhodopsin peaks when
the temperature of the illuminated sample was allowed to warm
above 125 K for several hours. This observation supports their
assignment as being due to bathorhodopsin.
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