Accurate measurements of 13C-13C J-couplings in the rhodopsin chromophore by double-quantum solid-state NMR spectroscopy

Article abstract of DOI:10.1021/ja0581604

A new double-quantum solid-state NMR pulse sequence is presented and used to measure one-bond ¹³C-¹³C J-couplings in a set of ¹³C₂-labeled rhodopsin isotopomers. The measured J-couplings reveal a perturbation of the electronic structure at the terminus of the conjugated chain but show no evidence for protein-induced electronic perturbation near the C11-C12 isomerization site. This work establishes NMR methodology for measuring accurate ¹J_CC values in noncrystalline macromolecules and shows that the measured J-couplings may reveal local electronic perturbations of mechanistic significance. Copyright

Full text of DOI:10.1021/ja0581604

Published on Web 03/03/2006
Accurate Measurements of 13C-13C J-Couplings in the Rhodopsin
Chromophore by Double-Quantum Solid-State NMR Spectroscopy
Wai Cheu Lai, Neville McLean, Axel Gansmu¨ller, Michiel A. Verhoeven, Gian Carlo Antonioli,
Marina Carravetta, Luminita Duma, Petra H. M. Bovee-Geurts, Ole G. Johannessen,
Huub J. M. de Groot, Johan Lugtenburg, Lyndon Emsley, Steven P. Brown, 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, P.O. Box 9502, NL-2300 RA Leiden, The Netherlands, Nijmegen Centre for Molecular Life
Sciences, Radboud UniVersity Nijmegen Medical Centre, NL-6500 HB Nijmegen, The Netherlands, Department of
Physics, UniVersity of Warwick, CoVentry CV4 7AL, U.K., Laboratoire de Chimie, UMR-5182 CNRS/ENS,
Laboratoire de Recherche ConVentionne´ du CEA (DSV 23V/DSM 0432), Ecole Normale Supe´rieure de Lyon,
69364 Lyon, France
Received December 1, 2005; E-mail: mhl@soton.ac.uk
Rhodopsin is a G-protein coupled receptor (GPCR) responsible
for dim light vision in vertebrates. Rhodopsin consists of a seven-
helix transmembrane protein bound by a protonated Schiff base
(PSB) linkage to the 11-Z-retinylidene chromophore, which is the
primary light receptor.1 On photoactivation, the retinylidene chro-
mophore is isomerized to the all-E-retinylidene configuration. This
process is exceptionally specific and fast, and it is not fully
understood how the protein environment stereoselectively steers
Figure 1. Double-quantum-filtered spin-echo pulse sequence for the
measurement of J-couplings in MAS 13C2-labeled biomolecules. The shaded
pulse sequence elements are given a four-step phase cycle. Note the absence
and accelerates the photoisomerization process. NMR estimations
of 13C-13C distances within the retinylidene chain suggested that
the PSB positive charge is partially delocalized and stabilized near
the isomerization site by polar protein side chains and an associated
water molecule.2 However, density functional calculations did not
support this hypothesis.3
1
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of an H decoupling field during the R202 recoupling elements.
To obtain an independent estimation of the bond conjugation in
the retinylidene chromophore, we have developed a double-
quantum-filtered spin-echo (DQF-SE) NMR method to measure
one-bond 13C-13C J-couplings for large biomolecules in the solid
1
state. The JCC values are sensitive to the bond order, and double
bonds display couplings around 15 Hz larger than single bonds.
Accurate measurements of 1JCC couplings should therefore provide
an unambiguous mapping of the bond conjugation within the
retinylidene chain. We have applied this method to six different
13C2-labeled rhodopsin isotopomers, namely [9,10-13C2], [10,11-
13C2], [11,12-13C2], [12,13-13C2], [13,14-13C2], and [14,15-13C2]-
retinylidene rhodopsin.
It has recently been shown that homonuclear J-couplings may
be estimated accurately by spin-echo modulations in solids, even
though the size of these couplings is often less than the line width.4,5
To measure one-bond 13C-13C J-couplings in rhodopsin, we have
combined the spin-echo technique with symmetry-based double-
Figure 2. MAS 13C NMR spectra of [9,10-13C2]-retinylidene rhodopsin,
at a spinning frequency of 10.00 kHz, a field of 9.4 T, and a temperature
of 170 K. (a) Cross-polarization spectrum. (b) Double-quantum-filtered
spectrum showing the peaks from the 13C labels. (c) Experimental double-
quantum-filtered signal intensities as a function of the echo interval τ and
a fitted echo modulation curve (solid line).
quantum dipolar recoupling6 to suppress the natural abundance 13
C
background, which otherwise obscures the signals of interest. This
new NMR technique allows the measurement of J-couplings, and
hence bond orders, with high accuracy in large, noncrystalline
biomolecules.
of symmetry-based R2029 recoupling6 to excite DQ 13C coherences
in the 13C2-labeled species and to reconvert these coherences into
observable magnetization. Four-step phase cycling is used to
suppress signals from isolated 13C nuclei that cannot support DQ
coherence, thereby removing the natural abundance 13C background
The DQF-SE pulse sequence is shown in Figure 1 and consists
of a preparation block, a DQ filtration block, and a spin-echo block.
Magic-angle spinning (MAS) is used to improve the sensitivity and
1
9
the resolution. The preparation module consists of ramped H-
signal, as shown in Figure 2b. The R202 sequence consists of a
13C cross-polarization followed by a strong 90° pulse (90° out-of-
phase with the spin-locking field) to enhance the longitudinal 13C
magnetization. The DQ filtration module consists of two intervals
repeating pulse pattern 908127026190-81270-261 where the flip angles
and phases are specified in degrees, and the nutation frequency
under the rf field is 10 times the MAS frequency.6 This sequence
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10.1021/ja0581604 CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
1
The JCC values for rhodopsin and the PSB model compound
agree in all positions within experimental error. The J-couplings
therefore provide no indication that the protein environment
significantly perturbs the electronic structure of the conjugated
system, apart from the localized effects of the PSB at the chain
terminus. These data therefore support the conclusions of Okada
et al.3 who argued, on the basis of X-ray diffraction and density
functional calculations, that the bond alternation in the retinylidene
chromophore is insensitive to the protein environment.
The fact that the dipole-dipole coupling2 and chemical shift12
measurements both indicate a disruption of conjugation penetrating
deep into the conjugated chain, while a corresponding perturbation
of the J-couplings is not observed, suggests that a single “bond
order” parameter is too simplistic to describe the interplay of
electronic structure, chemical shifts, internuclear distances, and
J-couplings in a conjugated system. Quantum computations are
planned to clarify this relationship.
In summary, we have developed and applied a solid-state NMR
experiment that allows accurate and robust measurements of one-
bond J-couplings in large noncrystalline macromolecules, such as
membrane proteins. The magnitudes of J-couplings monitor very
local features of the electronic bonding structure. In the case of
rhodopsin, our measurements support the thesis that the protein
environment does not perturb the chromophore electronic structure
significantly in the vicinity of the isomerization site.
1
Figure 3. Filled circles with error bars: JCC values for the six rhodopsin
isotopomers and their confidence limits. The inset shows the numbering
scheme of the 11-Z-retinylidene chromophore. Diamonds and solid line:
13C-13C J-couplings for all-E N-tert-butyl retinylidene imine triflate in
solution. Open squares and dashed line: 13C-13C J-couplings for all-E
retinal in solution.
provides efficient DQ recoupling at a moderate MAS frequency
without requiring a H decoupling field.7
1
The DQ-filtered longitudinal magnetization is transformed into
transverse magnetization by a second strong 90° pulse. A τ/2 -
180 - τ/2 spin-echo is performed in the presence of a 1H
decoupling field, and the 13C NMR signal is detected. The rotor-
synchronized spin-echo interval τ is varied in a series of experi-
ments, and the oscillatory modulation of the 13C signal is recorded.
We used carefully adjusted SPINAL-648 proton decoupler modula-
tion during the echo interval to prolong the echo decays.9 An
experimental spin-echo modulation curve for [9,10-13C2]-reti-
nylidene rhodopsin is shown in Figure 2c.
Acknowledgment. This research was supported by BBSRC
(UK), EPSRC (UK), NWO (NL), CMSB (NL), EU Grants BIO4-
CT97-2101 and LSHG-CT-2004-504601, and Varian Instruments.
Supporting Information Available: Numerical values of the
J-couplings, full citation for ref 2, details of retinal synthesis and sample
preparation, choice of NMR methodology, experimental NMR param-
eters, pulse sequence code, solid-state and solution-state NMR spectra,
spin-echo modulation curves, and confidence limit analysis. This
material is available free of charge via the Internet at http://pubs.acs.org.
Duma et al.5 showed that the spin-echoes of isolated spin-pair
systems in MAS solids obey the equation
0
J
s(τ) ) p exp(-τ/T2 ) + (1 - p) cos(πJτ) exp(-τ/T2 )
References
(1) Ernst, O. P.; Hofmann, K. P.; in Palczewski, K. Photoreceptors and Light
Signalling; Batschauer, A., Ed.; Comprehensive Series in Photochemistry
& Photobiology; Royal Society of Chemistry: Cambridge, 2003; Vol. 3,
pp 77-123.
The J-coupling is estimated by fitting this equation to the
0
J
experimental data, adjusting the parameters T2 , T2 , p, and J. The
confidence limits on J are derived by taking into account the
experimental noise (see Supporting Information). The J-coupling
estimates for the six rhodopsin isotopomers, and their confidence
limits, are displayed graphically as a function of chain position in
Figure 3. This figure also shows solution-state J-couplings measured
for a PSB model compound (all-E N-tert-butyl retinylidene imine
triflate10), and all-E-retinal, which has an aldehyde group instead
of the protonated PSB moiety.11 The J-couplings of the PSB model
compound were measured by solution-state double-quantum NMR
(see Supporting Information).
(2) Carravetta, M.; et al. J. Am. Chem. Soc. 2004, 126, 3948-3953.
(3) Okada, T.; Sugihara, M.; Bondar, A.-N.; Elstner, M.; Entel, P.; Buss, V.
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M. H. ChemPhysChem 2004, 5, 815-833.
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97-101.
The measured J-couplings follow a zigzag pattern and correlate
well with the alternation of single and double C-C bonds in the
conjugated retinylidene chain. Some perturbations of the zigzag
(9) (a) De Pae¨pe, G.; Lesage, A.; Emsley, L. J. Chem. Phys. 2003, 119, 4833-
4841. (b) De Pae¨pe, G.; Giraud, N.; Lesage, A.; Hodgkinson, P.;
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D. Can. J. Chem. 1996, 74, 591.
1
are clearly visible. At the end of the chain, the JCC value for the
14-15 single bond is enhanced in both rhodopsin and the PSB
model compound, relative to retinal. This enhancement may be
attributed to the positively charged PSB neighbor, which creates a
conjugation defect. The penetration of the PSB positive charge into
the end of the chain is also reflected in the chemical shift values12
and the 13C-13C distances.2
(11) Groesbeek, M.; Rood, G. A.; Lugtenburg, J. Recl. TraV. Chim. Pays-Bas
1992, 111, 149-154.
(12) Creemers, A. F. L.; Kiihne, S. R.; Bovee-Geurts, P. H. M.; DeGrip, W.
J.; Lugtenburg, J.; de Groot, H. J. M. Proc. Natl. Acad. Sci. U.S.A. 2002,
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