Crystallization Scale Preparation of a Stable GPCR
Signaling Complex between Constitutively Active
Rhodopsin and G-Protein
Shoji Maeda1, Dawei Sun1, Ankita Singhal1, Marcello Foggetta2, Georg Schmid2, Joerg Standfuss1,
Michael Hennig2, Roger J. P. Dawson2, Dmitry B. Veprintsev1*, Gebhard F. X. Schertler1*
1 Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen, Switzerland and Department of Biology, ETH Zurich, Zurich, Switzerland, 2 pRED Pharma Research
and Early Development, Small Molecule Research, Discovery Technologies, F. Hoffmann-La Roche Ltd, Basel, Switzerland
Abstract
The activation of the G-protein transducin (Gt) by rhodopsin (Rho) has been intensively studied for several decades. It is the
best understood example of GPCR activation mechanism and serves as a template for other GPCRs. The structure of the
Rho/G protein complex, which is transiently formed during the signaling reaction, is of particular interest. It can help
understanding the molecular details of how retinal isomerization leads to the G protein activation, as well as shed some
light on how GPCR recognizes its cognate G protein. The native Rho/Gt complex isolated from bovine retina suffers from
low stability and loss of the retinal ligand. Recently, we reported that constitutively active mutant of rhodopsin E113Q forms
a Rho/Gt complex that is stable in detergent solution. Here, we introduce methods for a large scale preparation of the
complex formed by the thermo-stabilized and constitutively active rhodopsin mutant N2C/M257Y/D282C(RhoM257Y) and
the native Gt purified from bovine retinas. We demonstrate that the light-activated rhodopsin in this complex contains a
covalently bound unprotonated retinal and therefore corresponds to the active metarhodopin II state; that the isolated
complex is active and dissociates upon addition of GTPcS; and that the stoichiometry corresponds to a 1:1 molar ratio of
rhodopsin to the heterotrimeric G-protein. And finally, we show that the rhodopsin also forms stable complex with Gi. This
complex has significantly higher thermostability than RhoM257Y/Gt complex and is resistant to a variety of detergents.
Overall, our data suggest that the RhoM257Y/Gi complex is an ideal target for future structural and mechanistic studies of
signaling in the visual system.
Citation: Maeda S, Sun D, Singhal A, Foggetta M, Schmid G, et al. (2014) Crystallization Scale Preparation of a Stable GPCR Signaling Complex between
Constitutively Active Rhodopsin and G-Protein. PLoS ONE 9(6): e98714. doi:10.1371/journal.pone.0098714
Editor: Pere Garriga, Universitat Politècnica de Catalunya, Spain
Received March 24, 2014; Accepted May 7, 2014; Published June 30, 2014
Copyright: ß 2014 Maeda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All data are included within the manuscript.
Funding: SM is supported by the Roche postdoctoral fellowship (RPF113, www.roche.com). This work was supported by the Swiss National Science Foundation
(www.snf.ch) grants 133810 (DBV, JS, GFXS), 135754 (DBV), 141898 (DBV, GFXS), 132815 (GFXS, JS), 141235 (JS) and NCCR Structural Biology (GFXS). Roche
provided funding in the form of salaries for the employees and co-authors (MF, GS, MH and RJPD). The Swiss National Science Foundation had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors of this manuscript declared the following competing interests: the authors, DBV and JS are paid employees of the Paul
Scherrer Institut, Villigen; Switzerland; GFXS of the ETH Zurich, Zurich, Switzerland and is a member of the board of directors of the Paul Scherrer Institut, Villigen,
Switzerland; MF, GS, MH and RJPD are paid employees of the F. Hoffmann-La Roche AG, Basel, Switzerland. This does not alter the authors’ adherence to all the
PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products to declare.
* Email: dmitry.veprintsev@psi.ch (DBV); gebhard.schertler@psi.ch (GFXS)
remarkably accelerated in recent years due to the innovative
protein engineering [5,6,7], novel crystallization techniques [8]
and crystallography methods [9,10], only a few structures are
crystallized in the active conformation [11,12,13,14,15] and only
one structure of an active GPCR/G-protein complex [16] has
been determined so far. Apart from rhodopsin, structures of active
GPCRs are obtained from heavily modified proteins with
truncated termini/loops, fusion domains and co-crystallized
antibodies. Further structures from additional receptors with
minimum modifications and in complex with other types of Gproteins, arrestins and kinases will therefore be needed for a
comprehensive understanding of the molecular mechanisms of
GPCR signaling.
Rhodopsin is one of the most extensively studied members of
the GPCR family. It works as a photoreceptor pigment protein in
retinal rod cells where it senses light via covalently bound 11-cis-
Introduction
Intercellular signaling is essential for complex biological
processes in higher animals, such as differentiation, immune
response, metabolic regulation, and neural activity. The largest
group of proteins involved in these processes is the G-proteincoupled receptors (GPCRs) that transmit the signal across the
cellular membrane. Despite their functional and ligand diversity,
all GPCRs share a seven alpha-helical transmembrane architecture and presumably transduce the activation signal by a common
mechanism via a heterotrimeric guanine nucleotide-binding
protein (G-protein). GPCR malfunction is often associated with
pathological outcomes [1,2,3] and hence, these receptors constitute an important pharmaceutical target, with almost 30% of
currently prescribed drugs acting through the GPCR family of
proteins [4]. Although structure determination of GPCR has been
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Stable GPCR/G-Protein Complex for Structural Studies
retinal that in the dark acts as a potent inverse agonist and
suppresses activity of the receptor. Upon absorption of light, 11-cisretinal isomerizes to the full agonist all-trans-retinal which in turn
starts a series of conformational changes resulting in the formation
of metarhodopsin-II (MetaII), the fully active species that couples
with the heterotrimeric G-protein transducin (Gt). The MetaII
catalyzes exchange of GDP to GTP in the Gta subunit. Gat and
Gbct then dissociate and diffuse to transmit the signal to
downstream effectors [17].
Similar to ligand binding in other GPCRs, retinal isomerization
in rhodopsin disturbes the equilibrium between different states of
the receptor. In contrast to most GPCRs, rhodopsin however has
an exceptionally low level of basal activity i.e. G-protein signaling
in the absence of ligands [18]. The rate of spontaneous activation
events in primate rod cells has been estimated to be 5.2610211 per
second, corresponding to a half life of the inactive state of over 400
years [19]. Even the apoprotein opsin is 106-fold less active than
the active metarhodopsin II state with covalently bound agonist
all-trans retinal [20]. This exceptionally low level of basal activity
is required to create such a sensitive light detector as the rod cell
but can be easily disturbed even by single point mutations. Such
constitutively active mutations are known to shift the equilibrium
towards the active form and increase the basal activity of opsin
[21,22]
In previous studies we have combined constitutively active
mutants with a cysteine double mutant (N2C/D282C) that
increases thermal stability but does not change retinal binding,
G-protein activation, activation pathways or structure of the
protein [23,24,25]. Of particular interest is the combination with
the M257Y mutant (RhoM257Y), one of the strongest constitutively active mutants known in rhodopsin [26]. We have previously
solved the crystal structure of this mutant in complex with a
peptide resembling the C-terminus of the Ga protein subunit [11].
Our previous analysis of the structure suggested that the
constitutive activity of this mutant stems from a stabilization of
the open G-protein-binding pocket. Here we demonstrate how the
RhoM257Y can be used for the purification of large quantities of
complete rhodopsin/G-protein complexes for structural studies.
phosphate, during the light activation improved the efficiency of
the RhoM257Y/Gt complex formation by preventing re-binding
of GDP. After the improvement the protein was eluted as a
symmetric peak from the size exclusion column that contained all
components of the RhoM257Y/Gt complex (Figure 1, small inset).
A typical yield from 40 g of cell pellet and 50 retinas was 6–9 mg
of RhoM257Y/Gt complex with the purity suitable for highthroughput crystallization screening.
Spectroscopic characterization of the RhoM257Y/Gt
complex
The photoactive ligand bound to rhodopsin is an 11-cis-retinal
molecule covalently attached via a protonated Schiff base (SB)
with K296 in TM7. Upon absorption of a photon, 11-cis-retinal
isomerizes to all-trans-retinal and the proton is transferred from the
SB to the retinal counterion E113 as part of a series of
conformational and spectrophotometrically detectable changes
leading to formation of the G-protein binding conformation
MetaII [17,28,29,30,31]. The UV/VIS spectrum of the
RhoM257Y/Gt complex shows two major peaks, one at 380 nm
arising from unprotonated retinal (Figure 2) and one at 280 nm
derived from the protein moiety. The ratio between the two peaks
is 3.5 and agrees well with a single retinal per complex based on
the theoretical extinction coefficients and reports in the literature
[27,32,33]. When a purified sample of the complex was denatured
by addition of acid, the 380 nm peak position was shifted to a new
lmax at 440 nm characteristic of a protonated retinylidene Schiff
base, with little change in absorbance at 280 nm. This complete
shift of the 380 nm peak under acid conditions suggests that most
of the retinal is covalently bound to protein, and virtually no
retinal remained free in the sample, as expected for the native
Rho/Gt complex. In contrast to previously reported preparations
of detergent solubilized native Rho/Gt complex, the covalently
bound retinal in our mutated complex was stably trapped within
the protein with no observable spectral changes after one month in
detergent solution (Figure 2).
RhoM257Y/Gt complex activity and stability
Besides the availability in sufficient amounts, it is critical for a
successful crystallization to maintain the protein in a folded and
functional state. We therefore tested the purified RhoM257Y/Gt
complex for specific dissociation upon binding of the nonhydrolysable GTP analogue GTPcS. Indeed the profile obtained
by analytical size exclusion chromatography showed a near
complete dissociation of the complex upon specific binding of
GTPcS, indicative for a high activity in the purified RhoM257Y/
Gt complex (Figure 3, A). And purified RhoM257Y/Gt did not
lose its activity at 4uC for 27 days after purification (Figure 3, B).
Furthermore, RhoM257Y/Gt complex was resistant to wide range
of pH and high salt conditions (Figure 3, C,D).
Results
Milligram scale preparation of the RhoM257Y/Gt complex
The activated rhodopsin/Gt complex was prepared by first
solubilizing HEK293S-GnTI2 cells expressing the N2C/M257Y/
D282C opsin in 1.25% DDM (w/v), centrifuging the material to
remove nuclei and insoluble fractions, and applying the supernatant fraction to a 1D4-antibody immunoaffinity matrix (Figure 1)
essentially as described previously [24,27]. The immobilized opsin
was then reconstituted with 11-cis-retinal to ground state
rhodopsin while still bound to the resin, free unbound retinal
was washed away and excess Gt (typically 1.5 times molar ratio of
rhodopsin) was added. Complex formation was induced by
isomerization of 11-cis-retinal to the full agonist all-trans-retinal
using a xenon lamp with a 495 nm long-pass filter to prevent
isomerization of unbound retinal. After extensive washing of the
resin, rhodopsin in complex with Gt was released from the
immunoaffinity matrix by incubating with the 1D4-elution peptide
resembling the C-terminus of rhodopsin (TETSQVAPA). The
eluted fraction was concentrated and further purified by sizeexclusion chromatography to remove free components. Initially
size-exclusion chromatography showed that our preparation
contained a relatively large amount of free rhodopsin, supposedly
due to dissociation of the complex by re-binding of GDP to Gat.
Adding apyrase, an enzyme that hydrolyzes GDP to GMP and
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Complex Stoichiometry
Bovine rhodopsin was observed to form arrays of dimers in the
native rod outer segment membrane [34,35] and some biochemical/biophysical data suggested the formation of heteropentameric
complex with Gt [36,37,38] at a stoichiometry of 2 rhodopsin to
1 Gt. Other data, however, suggested a stoichiometry of 1
rhodopsin to 1 Gt both in the membrane and detergent solution
[27,33,39,40]. To address the controversial stoichiometry of
rhodopsin/Gt complex, we employed analytical ultracentrifugation (AUC) to determine the molecular weight of the purified
complex more accurately than it is possible by size exclusion
chromatography. Fitting of the equilibrium sedimentation profile
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Stable GPCR/G-Protein Complex for Structural Studies
Figure 1. Preparation diagram of RhoM257Y/Gt. A typical 1D4 immuno-affinity purification and preparative size-exclusion chromatography of
RhoM257Y/Gt from recombinant OpsinM257Y and native Gt. SDS-PAGE analysis (small inset) shows clean bands for the Gat and Gbt subunits
whereas rhodopsin runs as less sharp band due to partly heterogeneous glycosylation. The Gct is not resolved in this gel system due to its small
molecular size.
doi:10.1371/journal.pone.0098714.g001
(Figure 4) yielded a molecular weight of 194+/21 kDa. However
determination of the oligomerisation state of the RhoM257Y/Gt
complex is complicated by the presence of detergents, which
contribute to the molecular weight and affect the buoyancy of the
particle. The size of the detergent micelle can be estimated from
the excess molecular weight (DM) corrected for the partial specific
volume difference. Using Mdet = DM(12Vp*r)/(12Vd*r), where
Vp and Vd are partial specific volumes of the protein and
detergent, respectively, and r is the density of the buffer, we
obtained a micelle size of 70 kDa. The expected molecular weight
of the protein component of the 1:1 complex is 124 kDa, the
molecular weight of the Rho alone is 39 kDa. Taken the detergent
component into account, as discussed above, this leaves the only
possibility that the complex we observed has 1: 1 stoichiometry.
have established a thermo-shift assay to compare the stability of
the RhoM257Y/Gt complex under various conditions. The
employed fluorescence assay is based on binding of the thiol
specific maleimide CPM to cysteines that become exposed during
unfolding of the protein [41]. Melting curves of the RhoM257Y/
Gt complex obtained with this assay showed a clear transition of
the fluorescence signal at 22uC (Figure 5B) and a later transition at
56uC. Among 8 cysteines the Gat subunit possesses, there are two
cysteine residues that are supposedly protected upon complex
formation, one located at the boundary between Gbct subunit and
the other one within the C-terminus. The latter one will be buried
deep within the G-protein binding pocket in the rhodopsin,
assuming the C-terminus of the Gat subunit binds similarly as in
the structure of the RhoM257Y with co-crystallized GaCT peptide
[11]. Upon complex dissociation, these cysteines will be exposed
and become available for reaction with the fluorescent dye. While
the first transition is thus likely due to dissociation of the complex,
the second transition likely stems from unfolding of the complex
components, as both active rhodopsin and the G-protein subunits
unfold between 50uC and 60uC. This interpretation of the melting
curves correlates well with the data from the analytical size
Thermostability improvement of the complex
Whereas the purified RhoM257Y/Gt complex exhibits full
activity and resistance to wide range of pH and high salt
concentrations, it readily dissociated at room temperature
(Figure 5A). To be able to measure stability more accurately and
in a higher throughput than by size exclusion chromatography we
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Stable GPCR/G-Protein Complex for Structural Studies
Figure 2. RhoM257Y/Gt complex formation evaluated by UV/
VIS absorption spectroscopy. The spectrum of the purified
RhoM257Y/Gt complex (blue) consists of two components, a peak at
280 nm from the protein component and a peak at 380 nm from
retinal. The ratio of the two peaks for the active MetaII state is 1.6 but
rises to 3.5 upon binding of the Gt protein. Based on the extinction
coefficients of all components the spectrum indicates one retinal per
RhoM257Y/Gt complex. Acid denaturation of the complex (red) shifts
the retinal peak to 440 nm corresponding to a protonated retinylidene
Schiff base. The retinal contained in our preparation is thus covalently
bound to the RhoM257Y/Gt complex. Importantly even after incubation
for 31 days at 4uC (cyan) the retinal is still covalently bound (orange)
indicating that we prevented hydrolysis of the SB and trapped the
active Rho/Gt complex under these conditions.
doi:10.1371/journal.pone.0098714.g002
Figure 4. RhoM257Y/Gt complex has 1:1 stoichiometry as
shown by the equilibrium sedimentation.
doi:10.1371/journal.pone.0098714.g004
screen we investigated the possibility to stabilize the complex
through binding of small molecules from the Silver bullet additive
screen (Hampton). This screen contains 96 different conditions
each containing 2–20 small molecules that are included for their
ability to stabilize intermolecular, hydrogen bonding, hydrophobic
and electrostatic interactions with proteins. Colored solutions were
excluded in our screen as would interfere with the fluorescencebased assay. Thermostability of the RhoM257Y/Gt complex in
the presence of the remaining 72 conditions (Figure 5 C) varied
with a maximal stabilizing effect of 5.8uC at a standard deviation
of 0.9uC obtained from 20 measurements in the absence of
additives. The best hit (E1) contained a digest of DNA and RNA
exclusion chromatography indicating a dissociation of the complex
between 20uC and 30uC.
In a next step we used the thermo-shift assay for the search of
conditions that could further stabilize the complex. In an initial
Figure 3. Analytical SEC of RhoM257Y/Gt. A,B: RhoM257Y/Gt complex retains its activity as shown by its dissociation upon incubating with
GTPgS 1day after and even 27days after purification. C,D: RhoM257Y/Gt complex is resistant to various pH range from 5.6 to 8.2 and up to 2M NaCl
salt concentration.
doi:10.1371/journal.pone.0098714.g003
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Stable GPCR/G-Protein Complex for Structural Studies
Figure 5. RhoM257Y/Gt complex dissociates at room temperature. A: analytical SEC. B: Fluorescent dye-assisted assay gives two transition phases
arising from dissociation and unfolding of the components. Raw data (red triangles) are fitted by biphasic Boltzmann sigmoidal equation. C: The plot shows
the stabilizing or destabilizing effect of 72 small molecule mixes from the Silver Bullet (Hampton) additive screen on the RhoM257Y/Gt complex.
doi:10.1371/journal.pone.0098714.g005
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Stable GPCR/G-Protein Complex for Structural Studies
[31]. More recently several groups have undertaken purification of
the complex in detergent solution [33,44]. These efforts have used
native rhodopsin isolated from bovine retina and employed
sucrose density gradient centrifugation or size exclusion chromatography to separate the activated complex from unbound
material. In an earlier study [27], we introduced two modifications
to achieve preparation and purification of an activated complex.
First, we used immunoaffinity chromatography with a rhodopsinspecific 1D4 antibody for purification of the complex. Second, we
used recombinant rhodopsin in order to take advantage of
constitutively active mutations in the protein that supposedly
enhances formation and stability of the activated complex. The
activated complex could be formed and purified readily beginning
with crude extracts from stably transfected HEK293 cells that
were applied directly to the 1D4-immunoaffinity resin. Immobilization of opsin on the matrix was followed by incubation with
retinal and Gt protein to form an active complex, which could be
isolated in purified form by specifically eluting from the matrix
with the 1D4 peptide. Although the isolated complex displayed the
expected functional characteristics, it was proved unsuitable for
routine large-scale purifications due to the low expression levels of
the E113Q mutant and an overall low stability of the complex. In
the present study we therefore utilized the RhoM257Y that leads
to similar levels of constitutive activity as the E113Q mutation
through a specific stabilization of the G-protein binding site [11].
Introduction of the enzyme apyrase dramatically increased the
efficiency of the complex formation by preventing re-binding of
GDP, which otherwise leads to the dissociation of the complex. An
additional size exclusion chromatography improved homogeneity
of the complex and furthermore allows for an easy exchange of
detergents in crystallization screening. After successive optimization yields of the RhoM257Y/Gt complex reached 6–9 mg,
sufficient amount for crystallization screens. The purified complex
contained stoichiometric amounts of the retinal ligand, covalently
bound by means of an unprotonated Schiff base for over a month.
Incubation with GTPcS caused dissociation of the complex
demonstrating that the isolated complex is functionally active.
Analytical ultracentrifugation analysis of the isolated complex
demonstrated the component stoichiometry of RhoM257Y and
the Gt protein to be at a 1:1 molar ratio. While this result disagrees
with a recent electron microscopic and spectroscopic characterization of a native Rho/Gt complex [38,45], it agrees well with
previous reports for the complex with native rhodopsin purified by
size-exclusion chromatography in DDM solution [27,33] and that
of the b2AR/Gs complex [16]. Our data also corroborate the
stoichiometry observed for the rhodopsin/Gt complex in nanodiscs [39]. Based on stoichimetry, ligand-bound state, and
nucleotide binding activity we conclude that the purified
RhoM257Y/Gt complex consists of the active species MetaII
bound to the empty-pocket state of the Gt in a 1:1 ratio.
One factor that has long prevented the crystallization of
membrane proteins and particularly GPCRs is their low stability
in detergent solution. Our analysis demonstrates that the complex
consisting of the thermostabilized, constitutively active
RhoM257Y and the native Gt can be stored for over a few weeks
at 4uC in detergent solution, long enough for a successful
crystallization. Therefore, our studies suggest the purified
RhoM257Y/Gt complex is suitable for crystallization by vapor
diffusion at 4uC. However, dissociation of the RhoM257Y/Gt
complex at temperatures above 20uC limits the chances for success
in crystallization in lipidic cubic phases (LCP). This technique so
far delivered most GPCR structures. In order to further stabilize
the complex, we implemented a fluorescence-based high-throughput thermal stability assay that requires protein in the low
with DnaseI and RnaseA, while the second best hit (F1) contained
sodium pyrophosphate and sodium triphosphate, two nucleotide
analogues. In both cases the observed stabilizing effect therefore is
likely conferred from binding to the nucleotide-binding pocket of
the Ga subunit. Because the binding of GDP or GTP leads to the
dissociation of the complex, it is somewhat counter-intuitive that
other nucleotides or phosphates stabilize it. However, the products
of DNA and RNA digestion are complex mixtures of di-, tri- and
oligo- monophosphate nucleotides and probably cannot induce
the same conformational changes in Ga as GDP or GTP. On the
other hand, they may stabilize the local structure of the binding
pocket without causing dissociation of the complex.
Preparation and characterization of RhoM257Y/Gi
complex
Bovine rhodopsin couples with Gt protein in the native
photoreceptor, but it has been shown to activate also Gi type Gprotein in vitro [42]. In the trial to obtain a stable rhodopsin/Gprotein complex, we prepared RhoM257Y/Gi instead of
RhoM257Y/Gt and investigated the thermostability using fluorescence-detection size-exclusion chromatography-based thermostability assay (FSEC-TS) [43]. Unlike Gat, Gai1 can be highly
expressed recombinantly in bacterial cells and purified in large
quantity. After reconstitution of Gi heterotrimer by combining
with Gbct separated from native Gt heterotrimer, Gi was mixed
with the ground state RhoM257Y followed by photo-isomerization
to form RhoM257Y/Gi in the same way as RhoM257Y/Gt
(Figure 6).
The apparent affinities of Gt and Gi to photo-activated
rhodopsin were measured by an enzymatic reaction based assay
[33]. The initial rate of G-protein activation was determined by
monitoring the rise of intrinsic tryptophan fluorescence. Plotting
the initial rate constants against the titrated G-protein concentrations in the reaction gave a Michaelis-Menten type hyperbolic
function and Km value obtained after curve fitting represents
apparent affinity. While Km of Gt is .800 nM (Figure 7A), that of
Gi is ,10 nM (Figure 7B), significantly higher apparent affinity.
Consistent with the higher affinity of Gi, purified RhoM257Y/Gi
gave the dissociation temperature (Td) of 45uC in FSEC-TS and
40.5uC in CPM thermo-stability assay, respectively (Figure 8A, B).
RhoM257Y/Gi holds the activity to specifically bind GTP
analogues and dissociate, which was demonstrated by analytical
SEC after incubating the complex with various nucleotides
(Figure 8C).
Resistance to detergents is another critical factor for the
crystallization of membrane proteins as it allows the screening of
a larger crystallization space. We therefore tested detergent
resistance of the purified RhoM257Y/Gi complex by diluting it
into a range of detergents. After incubation for 30 minutes the
diluted complexes were analyzed for their structural integrity by
fluorescence-detection
size
exclusion
chromatography
(Figure 8D,E,F,G). While dissociated in relatively harsh detergents, the complex survived in a number of detergents and was
further investigated for the Td in selected ones. As expected,
RhoM257Y/Gi showed Td ranging from 27uC in nonyl-glucoside
to 45uC in LMNG (Figure 8H).
Discussion
The earliest attempts at the isolation of a rhodopsin/Gt
complex in soluble form were based on immobilizing rhodopsin
on a ConA-Sepharose column. While this approach did not allow
purification of the complex, it could be shown that Gt bound to
the column and could be specifically eluted by addition of GTPcS
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Figure 6. Preparation diagram of RhoM257Y/Gi complex. Heterotrimeric Gi protein is prepared by combining recombinant Gai1 and native
Gbct separated from heterotrimeric Gt protein. RhoM257Y/Gi is formed on the 1D4-sepharose matrix and further purified by a size exclusion
chromatography in the same way as RhoM257Y/Gt.
doi:10.1371/journal.pone.0098714.g006
receptors and Gi in other cell types and the high affinity is not
required. Moreover, the off-rate of activated Gt from the
rhodopsin may have to be high (resulting in the decreased affinity)
in order to improve the overall speed of the visual signaling
cascade. From the practical perspective, Gt and Gi are highly
homologous and the RhoM257Y/Gi complex can be transferred
into a wide range of detergents including different members of the
maltoside and cymal classes that are among the most commonly
used for the structure determination of membrane proteins.
Although additives and substitution of the G-protein stabilize
the rhodopsin/G-protein complex, the overall instability and,
more particularly, the flexibility of the alpha-helical domain of the
Ga protein might be still high to hamper the crystallization
[46,47]. Consequently, further efforts would be needed on the
stabilization of the complex through conformation-specific antibodies or nanobodies [16,48,49,50], or engineering G-protein in
order to increase the chances of successful crystallization.
microgram range and delivers an accuracy of 1uC to measure the
influence of chemical additives to complex stability. Among the 72
conditions screened from the Silver Bullet crystallization additive
screen (Hampton Research), 14 conditions (containing 2–20 small
molecules each) increased the thermal stability by more than two
standard deviations with a maximal stabilizing effect of 5.8uC.
These initial hits contained nucleotide analogues that stabilize the
Gt protein possibly by specific binding to the nucleotide binding
pocket, as well as small molecules like benzamidine, glycerol,
sugars or individual amino acids that stabilize the complex by less
specific mechanisms. Interestingly similar nucleotide analogues
have been used as additives in the recent crystallization of the
b2AR-Gs protein complex [16].
We also found that Gi, another G-protein subtype, forms higher
affinity and far more thermostable complex with rhodopsin. The
reason for the higher affinity of Gi to rhodopsin is not immediately
obvious. One possibility is that the concentration of both
rhodopsin and Gt in rod cells is high in comparison to Gi-coupled
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Purification of the Gt protein and separation of Gbct
subunit
The Gt protein was purified from bovine retina essentially as
described previously [52]. Frozen dark adapted bovine retinas,
purchased from W L Lawson Company (Omaha, NE), were
exposed to the room light at 4uC for overnight to form rhodopsin/
Gt in the rod photoreceptor cell outer segments (ROS). Following
isotonic and hypotonic washes, 40 mM GTP was added to release
Gt from the purified ROS membranes. Gt was then separated
from ROS by centrifugation, filtered through a 0.22 mm
membrane (Steriflip from Millipore Corp., Billerica, MA),
concentrated, and dialyzed against 10 mM Tris, pH 7.4, containing 2 mM MgCl2, 1 mM DTT, and 50% glycerol for storage at 2
20uC. Gbct was separated from Gt heterotrimer by Blue
Sepharose 6 Fast Flow (GE Healthcare) as described previously
[53], flash frozen and stored at 220uC until use.
Expression and purification of recombinant Gai1
Human G protein alpha-subunit (Gai1) was cloned into the
pJ411 vector (DNA 2.0), incorporating an N-terminal 10-histidine
tag followed by a TEV cleavage site. The sequenced plasmid was
transformed into E.coli BL21 (DE3) strain. The bacterial cells were
grown at 37uC. When OD600 reached 0.6, the protein expression
was induced by addition of 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) and the cells were further incubated for 20 hours
at 20uC. After centrifugal harvesting, the cell pellets were
resuspended in buffer A (25 mM Tris-HCl, pH 7.4, 0.5 M NaCl,
50 mM imidazole, 10% glycerol) and disrupted by sonication. The
supernatant was loaded into 5 ml His-Trap FF crude column (GE
Healthcare). The unbound protein was washed with buffer A and
eluted with buffer B (25 mM Tris-HCl pH 7.4, 0.5 M imidazole,
0.5 M NaCl and 10% glycerol). After the cleavage of the histidine
tag, the cleaved Gai1 protein was further purified by a size
exclusion chromatography (HiLoad Superdex 200, GE Healthcare).
Figure 7. Michaelis-Menten characterization of the Gt and Gi
activation by Rhodopsin using Trp fluorescence assay. The
Michaelis-Menten constant (Km) of Gt to the photoactive rhodopsin is
860 nM in DDM detergent solution (A), while that of Gi is 8.6 nM (B),
nearly 100 times higher than that of Gt.
doi:10.1371/journal.pone.0098714.g007
Purification of RhoM257Y/Gt and RhoM257Y/Gi complex
Purification of RhoM257Y/Gt complex was performed essentially in the same way as described previously [27] using N2C/
D282C/M257Y mutant bovine opsin (OpsinM257Y) instead of
N2C/D282C/E113Q mutant. Briefly, the whole HEK293SGnTI2 cells, stably expressing OpsinM257Y, are solubilized in
b-dodecyl-D-n-maltoside (DDM). After separating the supernatant, OpsinM257Y was first immobilized onto the 1D4-antibody
immunoaffinity sepharose and reconstituted with 11-cis retinal to
form the ground state RhoM257Y. The ground state RhoM257Y
was mixed with purified Gt and irradiated for 10 to 15 minutes
through 495 nm long-pass filter, converting the inverse agonist 11cis retinal to the full agonist all-trans retinal and forming
RhoM257Y/Gt complex on the sepharose resin. The resulting
RhoM257Y/Gt complex was detergent-exchanged from 0.02%
DDM to 0.02% lauryl-maltose neopentyl glycol(LMNG) and
eluted from the resin by incubating with 1D4-elution peptide
(TETSQVAPA). The eluent was further purified by size-exclusion
chromatography. Adding 25 mU/ml Apyrase (New England
Biolabs) during the light activation improved the efficiency of
the RhoM257Y/Gt complex formation by preventing re-binding
of the GDP that was released from Gat after the binding of Gt to
the active RhoM257Y [16]. Heterotrimeric Gi protein was formed
by mixing purified Gai1 and Gbct subunits at equimolar ratio for
30 min on ice, and used for the formation of RhoM257Y/Gi
complex in the same way as RhoM257Y/Gt complex.
Materials and Methods
Expression of constitutively active rhodopsin at
bioreactor scale
The N2C,M257Y,D282C rhodopsin mutant (RhoM257Y) was
expressed in stably transfected HEK293S-GnT12 cells [51]
constructed as described previously [11]. In contrast to our
previous reports we expressed the RhoM257Y in fully instrumented 20 L stirred-tank bioreactors (Sartorius, Germany) under
controlled conditions (120 rpm, pH 7.2, pO2 30% air saturation)
to be able to produce sufficient protein for crystallization
screening. Typically, 5 days after inoculation from shake flask
cultures the cell density reached 4–5610E6 viable cells/mL (PEM
medium (Life Technologies, USA) with 5% FBS, 4 mM
glutamine, G418 and blasticidin as selection markers). Protein
expression was induced by adding tetracycline in 500 mL of PEM
medium (final concentration of 2 mg/mL tetracycline). 800 mL
concentrated feeding solution (Roche, proprietary composition)
was added to avoid nutrient limitations. 48 h post-induction the
culture was supplemented with sodium butyrate (final concentration of 3 mM butyrate) and with additional feeding solution
(400 mL). Cells were harvested 72 h post-induction by centrifugation at 3,000 g for 10 minutes at 4uC (1 L beakers, Beckman).
Cell pellets (700–900 g in total) were washed once in PBS and
frozen in 50 mL Falcon tubes until purification of the complex.
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Stable GPCR/G-Protein Complex for Structural Studies
Figure 8. RhoM257Y/Gi is active and shows higher stability than RhoM257Y/Gt. A: fluorescent dye (CPM) assisted thermal denaturation
assay and B: FSEC-TS assay. C: The Gi in RhoM257Y/Gi complex adopts active conformation showing dissociation upon incubating with GTPgS or
GDP and AlF4. D,E,F,G: RhoM257Y/Gi complex is resistant to wide variety of detergents. H: Thermostability analysis of RhoM257Y/Gi by FSEC-TS in
selected detergents.
doi:10.1371/journal.pone.0098714.g008
repeated with increasing concentrations of Gi or Gt. The initial G
protein activation rate was determined by fitting the fluorescence
intensity to an exponential association curve y = y0+a[12exp(2
kr9t)] using Origin 8.5, where kr9 is the apparent rate constant and t
is the time in seconds. Apparent rate constant (kr9) of the initial
fluorescence increase was plotted against G protein concentrations. The data were fitted by Michaelis-Menten equation:
kr9 = (Vmax?[G])/(Km+[G]).
Acid denaturation assay
UV/Vis absorption spectra were measured by using a
UV_2401PC spectrophotometer (Shimadzu). Spectra of the intact
complex were measured by diluting 0.2 ul of purified
RhoM257Y/Gt complex (60 mg/ml) into 100 ul of buffer
(100 mM NaCl, 10 mM Hepes pH 7.5 and 0.02% LMNG).
Spectra of the acid denaturated complex were measured after
adding 2 ul of 25%H2SO4 and 3.5 ul of 20% Sodium dodecyl
sulfate to make the pH around 1.9 to denature the protein and
protonate retinilydene Schiff bases.
CPM fluorescence assisted Thermo-stability assay
Thermal dissociation was monitored by the formation of the
thiol specific malemaide fluorochrome CPM (N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]malemeide) adduct [41] attached to the protected cystein residue between Gat or Gai1, and
Gbct.
For RhoM257Y/Gt, thermo-stability assays were performed
using Eclipse fluorimeter (Varian) equipped with a multisample
holder. 2 ml of purified RhoM257Y/Gt (1 mg/ml) was diluted into
98 ml ice cold buffer (100 mM NaCl, 10 mM Hepes pH 7.5 and
0.02% LMNG). Immediately before the measurement, CPM
(3 mg/ml in DMSO) was diluted 1:30 into buffer and 10 ml of the
diluted CPM was added to the reaction mix. Cuvettes were placed
into the fluorimeter and fluorescence intensity (lex: 387 nm,
lem:464 nm) was monitored while ramping temperature from 4uC
to 90uC at the rate of 2uC/min. The resulting curve was fitted
using a sigmoidal Boltzmann equation to obtain Td50 values.
Comparison of thermo-stability in the presence of additives was
performed by supplementing the reaction mix with 10 ml of each
condition from the Silver Bullet additive screen. For each set of
G protein Activation Assay
The G protein activation was measured by monitoring the
change in intrinsic tryptophan fluorescence in the Ga subunit
upon exchange of GDP to GTPcS. All measurements were
performed by using Varian Cary Eclipse fluorescence spectrophotometer with settings of lex = 295 nm and lem = 340 nm. The
assays were carried out with 1 or 30 nM native rhodopsin (purified
from bovine retina), 0.01% DDM, and buffer C (50 mM Bis-Tris,
pH 7.3, 130 mM NaCl, 1 mM MgCl2, 1 mM DTT) in a final
volume 1 ml (1062 mm cuvette with stirring bar) at 20 degree.
The hetetrotrimeric Gi protein was reconstituted by mixing
equimolar ratio of the recombinant Gai1 and native Gbct
(purified from bovine retina) on ice for 30 min. The native Gt
was prepared from bovine retina. After forming the protein
complex of R*?G by irradiation to the orange light (.495 nm), the
basic fluorescence of R*?G was monitored for 5 min followed by
the addition of 10 mM GTPcS. The fluorescence intensity was
continuously recorded for 1 hr. The entire set of experiments was
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Stable GPCR/G-Protein Complex for Structural Studies
comparisons a control without additives was included and used to
calculate DTd values. The standard deviation from 20 measurements of the RhoM257Y/Gt complex was 0.94uC.
For RhoM257Y/Gi, thermo-stability assay was performed
using Rotor GeneQ (Qiagen). 5 ug of purified RhoM257Y/Gi
was diluted into 120 ul ice cold buffer (100 mM NaCl, 10 mM
Hepes pH 7.5 and 0.01%LMNG). 10 ul of the freshly prepared
40:1 dilution of CPM into measuring buffer was added immediately before the measurement. We prepare CPM stock at 3 mg/ml
in DMSO. Fluorescence intensity (lex: 365 nm, lem: 460 nm) was
monitored while ramping temperature from 25uC to 90uC at the
rate of 4uC/min. The resulting curves were analyzed by the Rotor
GeneQ package software.
NaCl, 10 mM Hepes pH 7.5, and each detergent followed by
FSEC.
For FSEC-TS, 1 ug of RhoM257Y/Gi in 100 ul of the buffer
was incubated at 4uC to 60uC for 30 minutes, ice cooled for
5 minutes, and then centrifuged at 9,000 g for 5 minutes. The
peak heights were normalized and then fit to a sigmoidal doseresponse curve to obtain Td values.
Analytical ultracentrifugation
Equilibrium sedimentation experiments were done in a Beckman Optima-XLI instrument at 4uC, using An-60Ti rotor and 6sector centrepieces. Data were collected at 11k, 15k and 20k rpm
and analyzed using the UltraSpin software (D. Veprintsev). Partial
specific volume was calculated from the protein sequence to be
0.737 using Sedenterp software (J. Philo). Buffer conditions were
20 mM KPi, 100 mM NaCl, 0.02% MNG-3. Partial specific
volume of LMNG was assumed to be similar to DDM (0.82 ml/
gr).
Analytical size-exclusion chromatography
For RhoM257Y/Gt, 18 ug of purified complex in 100 ul of
buffer composed of 100 mM NaCl, 10 mM Tris pH 7.5, and
0.02% LMNG or other salt or buffer conditions mentioned in the
results was loaded onto superdex 200 PC 3.2/30 (GE Healthcare)
equilibrated with 100 mM NaCl, 10 mM Tris pH 7.4, 20%
glycerol, and 0.02% LMNG and run at 0.05 ml/min. The elution
profile was monitored by the absorption of l: 280 nm. For
RhoM257Y/Gi, 1 ul of complex (1 mg/ml) was diluted into
100 ul of buffer composed of 100 mM NaCl, 10 mM Hepes
pH 7.5, and 0.01% was loaded onto superdex 200 packed in a
Tricorn 10/200 column (GE Healthcare) equilibrated with
100 mN NaCl, 10 mM Hepes pH 7.5, 0.01% LMNG. The
elution profile was monitored by protein-intrinsic fluorescence
with lex: 280 nm, lem: 340 nm. For detergent resistance test, 1 ul
of RhoM257Y/Gi (1 mg/ml) purified in DDM was diluted and
incubated for 30 minutes in 100 ul of buffer composed of 100 mM
Acknowledgments
We gratefully acknowledge Martin Siegrist (F. Hoffmann-La Roche AG)
for valuable assistance with fermentation of mammalian cells and Sandro
Mendieta (Paul Scherrer Institut) for valuable assistance with protein
purification.
Author Contributions
Conceived and designed the experiments: SM DS MH JS RJPD DBV
GFXS. Performed the experiments: SM DS AS MF GS JS DBV. Analyzed
the data: SM DS GS JS RJPD DBV GFXS. Contributed to the writing of
the manuscript: SM DS GS JS RJPD DBV GFXS.
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