Differential dynamics in the G protein-coupled
receptor rhodopsin revealed by solution NMR
Judith Klein-Seetharaman*†, Naveena V. K. Yanamala*, Fathima Javeed*, Philip J. Reeves‡, Elena V. Getmanova‡§,
Michele C. Loewen‡¶, Harald Schwalbe†, and H. Gobind Khorana‡?
*Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;†Zentrum fur Biologische Magnetische Resonanz, Johann
Wolfgang Goethe-Universita ¨t Frankfurt, Marie-Curie-Strasse 11, D-60439 Frankfurt?Main, Germany; and‡Departments of Biology and Chemistry,
Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139
Contributed by H. Gobind Khorana, December 30, 2003
G protein-coupled receptors are cell-surface seven-helical mem-
brane proteins that undergo conformational changes on activa-
tion. The mammalian photoreceptor, rhodopsin, is the best-
studied member of this superfamily. Here, we provide the first
evidence that activation in rhodopsin may involve differential
dynamic properties of side-chain versus backbone atoms. High-
resolution NMR studies of ?-15N-labeled receptor revealed large
backbone motions in the inactive dark state. In contrast, indole
side-chain15N groups of tryptophans showed well resolved,
equally intense NMR signals, suggesting restriction to a single
cell-surface receptors, the G protein-coupled receptors
(GPCRs). These receptors perform extremely diverse func-
tions that include responses to light, odorant molecules, neu-
rotransmitters, hormones, and a variety of other signals.
GPCRs all contain seven transmembrane (TM) helices (Fig.
1A) as shown by structural studies of rhodopsin by cryoelectron
microscopy (1) and confirmed more recently in a 3D x-ray
crystal structure in the inactive state (2–4) (Fig. 1B). The
chromophore in rhodopsin, 11-cis-retinal, is covalently bound
to the protein via a protonated Schiff base to the ?-amino
group of Lys-296 located in TM7. Capture of a photon by
rhodopsin results in isomerization of retinal to the all-trans
form, which triggers a series of transient changes in the protein
culminating in the active conformation. The application of
NMR spectroscopy to the study of rhodopsin, the best studied
member of GPCRs, has begun only recently (5). This has been
made possible by the development of high level expression of
a variety of rhodopsin mutants from the corresponding genes
(6, 7). Previously, we reported on NMR spectroscopy of
19F-labeled rhodopsin mutants in detergent micelles. Distinct
chemical shifts in the dark and changes in them on activation
of rhodopsin were observed for19F labels placed at different
positions in the cytoplasmic domain (8). Further, nuclear
Overhauser effects were observed between certain pairs of19F
labels (9). More recently, study of ?-15N-lysine-labeled rho-
dopsin by high-resolution heteronuclear NMR spectroscopy
(10) showed that whereas motions were detected on the
nanosecond time scale in the single lysine located in the C
terminus (Fig. 1A), lysines present elsewhere in rhodopsin
showed motions in micro- to millisecond time scales. In
addition, more than the expected number of signals with
variable intensities was detected. This finding indicated mul-
tiple backbone conformations. Here, we have studied ?,?-15N-
tryptophan-labeled rhodopsin. In contrast with lysines in
rhodopsin that are located mostly in the cytoplasmic domain
(Fig. 1, orange), four of the five tryptophans are in the TM
domain, and one is in the extracellular domain (Fig. 1, green).
Here, we report that the indole side chains show the expected
number of clearly defined signals with relatively homogeneous
intensity; however, the backbone amide shows a larger than
hodopsin, the vertebrate dim-light photoreceptor, is the
prototypic member of the largest known superfamily of
expected number of signals with varying intensities. These
results indicate that whereas the indole side chains have
one specific conformation, there are conformational fluctua-
tions on the micro- to millisecond time scale in the backbone
amide groups, as found previously for ?-15N-lysine-labeled
Amino acids are numbered according to the general GPCR
numbering scheme proposed by (11) and by their position in the
rhodopsin sequence. All experimental methods have been de-
scribed (10) with the following exceptions.
NMR Spectroscopy.NMR spectra were obtained at a spectrometer
1H frequency of ?800 MHz by using a Bruker (Billerica, MA)
spectrometer. Data were acquired and analyzed by using Bruker
UXNMR V.2.1 software. Data processing and analysis was also
carried out by using FELIX V.98.0.
Expression and Purification of Rhodopsin Containing Isotope-Labeled
Amino Acids in HEK 293S Cell Lines. The preparation of HEK 293S
stable cell lines containing the opsin gene for WT was described
(6). Stable cell lines were grown in media with composition
according to DMEM formulation, prepared from individual
components. Thus, all solutions were prepared as 100? concen-
trated stock solutions, except glucose, NaCl, glutamine, and
?,?-15N-tryptophan, which were added as solids. The glutamine
concentration was initially that of one-half of the DMEM
formulation, and the same amount was then added on day 5 or
6, together with 6 ml of 20% (wt?vol) glucose and 4 ml of 8%
(wt/vol) NaHCO3?5 mM sodium butyrate additions as described
(7). To remove unlabeled amino acids from FBS, it was dialyzed
three times against 10 liters of buffer A at 4°C with a tubing cut
off of 1 kDa as described (12). Immunoaffinity purification of
rhodopsin and preparation of samples for NMR spectroscopy
was as described (10).
Prediction of Chemical Shift Values Based on the Rhodopsin Crystal
programs SHIFTS(13)and PROSHIFT(14).1Hchemicalshiftswere
calculated by using the programs PROSHIFT (14) and SHIFTCALC
(15). All programs were applied to both chains in the two
available crystal structures with Protein Data Bank (PDB) ID
codes 1F88 (resolution 2.8 Å) and 1L9H (resolution 2.6 Å), and
the eight values were averaged for each of the five tryptophans
in both structures (2, 4).
15N chemical shifts were calculated by using the
Abbreviations: GPCR, G protein-coupled receptor; HSQC, heteronuclear single quantum
correlation; TM, transmembrane; TROSY, transverse relaxation optimized spectroscopy.
§Present address: Phylos, Inc., 128 Spring Street, Lexington, MA 02421.
¶Present address: Plant Biotechnology Institute, National Research Council of Canada, 110
Gymnasium Place, Saskatoon, SK, Canada S7N 0W9.
?To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2004 by The National Academy of Sciences of the USA
March 9, 2004 ?
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Conventional and Transverse Relaxation Optimized Spectroscopy
(TROSY)-Type 2D Heteronuclear Single Quantum Correlation (HSQC)
Spectra of ???-15N-Tryptophan-Labeled Rhodopsin. A 2D1H,15N-
HSQC spectrum of ?- and ?-15N-tryptophan-labeled rhodopsin
was recorded at 800 MHz (Fig. 2A). In the backbone region,
signals with variable intensities were observed instead of the five
expected. This heterogeneity in the backbone region is unlikely
to be due to deamination and scrambling of the15N label into the
? position of other amino acids as we have shown previously by
using mass spectrometry (10). The amino signals indicated by
arrow in Fig. 2A presumably originated from the natural abun-
dance signal from amino groups in rhodopsin because these were
also observed in ?-15N-lysine-labeled rhodopsin, and there was
no evidence for scrambling of15N to side-chain amino groups
either (10). TROSY resulted in decreases in signal intensities
overall, causing lowering of signal intensity below detection limit
for some of the signals (Fig. 2B). On the other hand, analysis of
the indole side-chain region, from ?10 ppm to 12 ppm, showed
the expected five signals. Their intensities were similar, except
for a stronger intensity for the central signal at ?10.5 ppm. This
signal most likely corresponds to Trp-35(1.30) (Fig. 1), the single
tryptophan in the extracellular domain in rhodopsin. The effect
of TROSY in this region of the HSQC spectrum (Fig. 2B) was
a smaller decrease in signal intensity than that observed for the
Comparison of Tryptophan Temperature Factors in the Rhodopsin
Crystal Structure. The temperature factors of side-chain and
backbone nitrogen atoms for all of the five tryptophan residues
were extracted from the rhodopsin crystal structure [PDB ID
code 1F88 (2)] and are shown in Fig. 3A. The four TM
tryptophan residues show similar temperature factors, ?35, for
both nitrogen atoms in each residue. On the other hand,
Trp-35(1.30) in the extracellular domain shows temperature
factors significantly elevated with values for both nitrogens of
?60. Thus, within any given tryptophan, the temperature factors
of the two nitrogens are essentially identical.
Solvent Accessibilities and Steric Restrictions of Nitrogens Vary With
the Position of Tryptophan Residues but Not Within ? or ?-Positions
in the Same Tryptophan.The conformational restriction of indole
side chains relative to the fluctuations in the amide groups is
opposite to that commonly observed in proteins, especially on
surfaces. Unless restricted by tertiary contacts, side chains can
have more freedom than the backbone. Thus, protein NMR
spectra usually indicate higher degrees of conformational
averaging in the side chains than in the backbone. Therefore,
we inspected the local environment of the tryptophan residues
tryptophan residues are indicated in green in comparison to the positions
of lysine residues (orange).
Secondary (A) and three-dimensional (B) structure model of bovine
at 800 MHz. (A) Conventional HSQC spectra of 0.4 mM rhodopsin in dodecyl
maltoside at 37°C. (B) TROSY-HSQC spectra. Tryptophan backbone regions of
the spectra are labeled ‘‘?,’’ and side-chain regions are labeled ‘‘?.’’ Back-
ground signals from amino groups are indicated by arrow and label ‘‘NH2.’’
www.pnas.org?cgi?doi?10.1073?pnas.0308713101 Klein-Seetharaman et al.
in the crystal structure model for differences in the immediate
steric packing of the indole side chains and the backbone.
Connolly surfaces were calculated for these residues and the
surface accessibility is plotted in Fig. 3B. The surfaces of the
rhodopsin molecule surrounding the tryptophans are shown in
Fig. 4. Large differences in the degree of steric restrictions for
each tryptophan are found. Trp-35(1.30) (Fig. 4C), and to a
lesser extent Trp-175(4.65) (Fig. 4B), and the side-chain
nitrogen of Trp-161(4.50) (Fig. 4B) are solvent-accessible.
However, the nitrogen atoms of Trp-126(3.41) and all atoms of
Trp-265(6.48) (Fig. 4 A and B) are completely buried and
solvent-inaccessible. Despite the above differences between
tryptophans, each individual tryptophan shows a very similar
environment of the backbone amide group and the indole side
Prediction of Chemical Shifts Based on the Rhodopsin Crystal Struc-
tures. Finally, we tested whether a subset of the larger than
expected number of signals of backbone amide groups in
HSQC spectra could be attributed to a conformation similar
to the crystal structure model. The chemical shift values for
both nitrogen and hydrogen for backbone amide groups were
calculated by using the programs SHIFTS, PROSHIFTS, and
SHIFTCALC (see Methods), both for the two chains with PDB ID
codes 1F88 and 1L9H. The averages, SDs, and minima?
maxima of predicted chemical shifts at each of the five
tryptophan positions are listed in Table 1. Very little overlap
between predicted and experimentally observed chemical
shifts was observed.
The motions in the backbone amide groups of the polypeptide
chain indicated by the HSQC spectra of ?,?-15N-tryptophan-
labeled rhodopsin at 37°C in detergent solution is in agreement
with the previous results with ?-15N-lysine-labeled rhodopsin
based on two observations (1). More than the expected
number of signals was observed, indicating the presence of
multiple conformations. We tested whether a subset of the
signals reflects the rhodopsin crystal structure conformation
by predicting the chemical shifts by using the crystal structure
as a template. However, there was little overlap between
predicted and experimentally observed chemical shift values.
This may be due to changes in experimental chemical shifts as
a result of the micelle environment that is lacking in the
reference chemical shifts calculated by using soluble proteins.
Furthermore, large variation in predicted chemical shifts were
observed for the two different crystal structure models, and for
each of the chains within the rhodopsin dimers, suggesting that
the low resolution of the crystal structures may further limit
the accuracy of chemical shift predictions (2). Exchange
broadening causes low intensity of the majority of signals in
conventional HSQC spectra, and using the TROSY scheme
resulted in a decrease of sensitivity. This observation would be
expected for a system with exchange contributions to the T2
relaxation. It would be desirable to quantify these exchange
contributions both experimentally and through modeling.
However, the low sensitivity of the signals due to the combined
effects of high-molecular-weight exchange contributions and
signal intensity distribution over multiple signals prevents
application of NMR spectroscopic approaches to measure
relaxation time directly. Estimation of the exchange contribu-
tions through modeling of the relaxation sources in the local
environments of the tryptophans based on the crystal structure
is unreliable because of the above described differences be-
tween the crystal structure and the aqueous micellar system.
Future perdeuteration will likely enhance the quality of
TROSY spectra (16) and may thus make the quantification of
the exchange contributions experimentally measurable, but
perdeuteration is currently not feasible by using our mamma-
lian expression system. The conformational exchange ob-
served here for rhodopsin may prove general for TM proteins,
as evidence for microsecond–millisecond exchanges was also
tryptophan residues in the crystal structure. (A) Temperature factors. The
factors were obtained from the published PDB ID code 1F88 (2). (B) Surface-
surface calculations by INSIGHT II software.
Properties of backbone (?) and side-chain (?) nitrogen atoms of
phan residues. Helices are colored as in Fig. 1B. (A) Overall structure. (B)
Expansion of region around Trp-126(3.41), Trp-161(4.60), and Trp-175(4.65).
(C) Molecule turned around z axis and expanded to show region around
Views of surface-accessible area of the surroundings of the trypto-
Klein-Seetharaman et al.
March 9, 2004 ?
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observed for OmpA (17, 18), bacteriorhodopsin (19, 20), and
diacylglycerol kinase (21) in micelles.
In contrast to the flexibility of backbone groups, the indole
side chains in tryptophan residues appear to be restricted to
one conformation. The difference does not appear to result
from increased steric freedom of side chains that would lead to
rapid averaging of conformations in contrast to the amide
groups. This conclusion comes from the study of each
tryptophan in the crystal structure model: both indole-
side-chain and backbone groups show similar accessibility and
steric environments for individual tryptophans. Larger differ-
ences are only observed between tryptophans. Even though
NMR and x-ray crystallographic analyses measure different
types of dynamics, steric properties of the crystal structure
model suggest that the differences in side-chain and backbone
groups observed in the HSQC spectra are not due to overall
increased mobility of side chains because of lack of steric
At present, the reason for the observation of heterogeneity in
signal number and intensity is not clear. One possibility is the
presence of heterogeneous glycosylation causing fluctuations in
the backbone conformations of rhodopsin, a possibility that can
now be tested with the recent development of a rhodopsin
expression system in which glycosylation can be suppressed (22).
Backbone flexibility may also be modulated by the specific
detergent micelle structure present. The dynamic properties of
the dark state may have importance for the process of light-
activation of rhodopsin from the inactive state. The first event in
this process, after light-induced retinal isomerization, is a per-
changes in the cytoplasmic domain (reviewed in refs. 5, 23, and
24). The conformational flexibility in the backbone of the
polypeptide chain demonstrated here for tryptophans and pre-
viously for lysines (10) may be important for allowing the
light-induced conformational changes to occur at the microsec-
ond time scale of the light-activated Meta II state formation. In
the absence of light, or in the absence of ligand for GPCRs in
general, specific contacts lock the receptors in an inactive
conformation and the release of these constraints is a key event
in the activation process of rhodopsin (25, 26), the ?2-adrenergic
(27–30), ?1B-adrenergic (31, 32), muscarinic acetylcholine (33),
gonadotropin releasing hormone receptor (34), ? opioid (35),
dopamine D2 (36), cannabinoid (37), histamine H2 (38), sero-
tonin 5HT2A, and C5a (39) receptors, and probably GPCRs in
general (reviewed in ref. 40). These constraints cluster in con-
served domains, including a cluster of aromatic amino acids
surrounding the ligand binding pockets (25, 27, 41–44). In
rhodopsin, the highly conserved Trp-265(6.48) is in direct con-
tact with the retinal, as demonstrated by crosslinking (25) and
x-ray crystallography (2). On light-activation, retinal no longer
crosslinks to Trp-265(6.48) but instead to Ala-169(4.58) (45), a
residue located ?10 Ångstroms away from the ionone ring in the
dark state crystal structure (2). This would enhance the confor-
mational flexibility of Trp-265(6.48), allowing a rearrangement
of its indole side chain, shown to be important for activation of
rhodopsin (46, 47) and the ?2-adrenergic receptor (27). Another
highly conserved tryptophan, Trp-161(4.50), may contribute to
this process (45, 46). The results described in this paper provide
experimental evidence for differential dynamics on the amino
acid level in a membrane protein. This suggests that only some
strong constraints exist, whereas the majority of the molecule
experiences conformational flexibility within the same domains,
and even within the same amino acid. In particular, we showed
that tryptophan side chains are more restricted in conformation
than their backbone. Thus, it appears that the indole side-chain
contacts in part contribute to restricting the conformation in a
‘‘locked’’ dark state, without fully restricting motional fluctua-
tions in the overall molecule including the helical bundle itself.
The inference is that it would not be necessary to break and form
thousands of specific contacts within nanoseconds after retinal
isomerization in rhodopsin (or ligand binding in other GPCRs).
Rather, a few specific contacts restricting the inactive state need
to break on activation, and these changes are transmitted
through the entire membrane protein because of its dynamic
This work was supported by the National Institutes of Health (H.G.K.),
the National Science Foundation (H.G.K. and J.K.-S.), the Deutsche
Forschungsgemeinschaft (H.S.), the Howard Hughes Medical Institute
(J.K.-S.), and the Humboldt Foundation?Zukunftsinvestitionspro-
gramm der Bundesregierung Deutschland (J.K.-S.). This is paper no. 54
in the Structure and Function in Rhodopsin series.
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