Archives of Biochemistry and Biophysics 450 (2006) 215222

www.elsevier.com/locate/yabbi

Assembly of single bacteriorhodopsin trimers in bilayer nanodiscs

Timothy H. Bayburt a,b, Yelena V. Grinkova a,b, Stephen G. Sligar a,b,c,
a
Department of Biochemistry, University of Illinois, 116 Morrill Hall, 505 S. Goodwin, Urbana, IL 61801, USA
b
Beckman Institute for Advanced Science and Technology, University of Illinois, 116 Morrill Hall, 505 S. Goodwin, Urbana, IL 61801, USA
c
Department of Chemistry, University of Illinois, 116 Morrill Hall, 505 S. Goodwin, Urbana, IL 61801, USA

Received 9 February 2006, and in revised form 9 March 2006

Available online 29 March 2006

Abstract

Nanodiscs, phospholipid bilayer assemblies of controlled size, were used to self-assemble bacteriorhodopsin (bR) into single trimers.
Self-assembly at optimal bR to Nanodisc and phospholipid stoichiometry yielded particles containing three bR molecules. Analysis of
solution small angle X-ray scattering indicated that bacteriorhodopsin is embedded in a discoidal phospholipid bilayer structure. Forma-
tion of trimers, as evidenced by visible circular dichroism of the retinal absorbance bands, is facilitated in Nanodiscs at a speciWc size
threshold, suggesting that a critical bilayer area or amount of lipid is necessary to maintain a native oligomeric state. The lipid to bR ratio
in the assembly process was also found to be an important factor in determining oligomerization state. These nanoscale bilayers oVer the
opportunity to understand and control the assembly of oligomeric integral membrane proteins critical to macromolecular recognition
and cellular signaling.
2006 Elsevier Inc. All rights reserved.

Keywords: Model bilayer; Membrane protein; Nanoparticle; Bicelle; Self-assembly; Oligomer

Membrane proteins are associated with 2030% of all
open reading frames from archaebacterial, bacterial, and
eukaryotic organisms and are perhaps among the most
challenging and potentially rewarding subjects of study [1].
Indeed, a majority of currently marketed therapeutics are
active against membrane protein targets, and the large
number of membrane associated orphan receptors and
enzymes of unknown function, as revealed by the human
genome sequencing eVorts, are thought to represent excit-
ing pharmacological opportunities. A central problem
encountered by biochemists and biophysicists is the solubi-
lization of membrane proteins in a functional state using
detergents (for a recent review, see [2]).
Detergentmembrane and detergentprotein interaction
depends on molecular properties and the physicochemical
make-up of the solution and detergentprotein interactions
are often destabilizing. Once solubilized, the protein
requires the continued presence of detergent above its criti-
*
Corresponding author. Fax: +1 217 265 4073.
E-mail address: s-sligar@uiuc.edu (S.G. Sligar).
cal micelle concentration to avoid aggregation during puri-
Wcation, manipulation, assay, and structural studies.
Maintenance of activity also often requires the presence of
phospholipid or lipid components [3]. Finally, membrane
protein oligomers exist whose association is concentration-
dependent and potentially critical for function.
The G-protein coupled receptors, seven-transmembrane
helix proteins with roles in cellular signal transduction, are
of great interest in human disease and as therapeutic tar-
gets. These receptors are believed to form hetero- and
homo-oligomers of functional consequence, and methods
for controlling and isolating monomeric and oligomeric
states would be a signiWcant contribution to understanding
the functional role of oligomerization [4,5]. Clearly, tech-
niques are needed to maintain stability and oligomerization
state in the absence of deleterious eVects of detergent.
We have recently introduced a new type of stable
phospholipid bilayers termed Nanodiscs, which have a
monodisperse size, into which membrane proteins can be
self-assembled [610]. Nanodiscs are composed of
phospholipid present in a bilayer domain with two copies

0003-9861/$ - see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2006.03.013
216 T.H. Bayburt et al. / Archives of Biochemistry and Biophysics 450 (2006) 215222
of an amphipathic -helical Membrane ScaVold Protein
(MSP)1 that wrap around the periphery of the bilayer. They
form reproducibly and in high yield through a directed self-
assembly process in which detergent is removed from initial
detergentphospholipid micelles, either by dialysis or treat-
ment with hydrophobic detergent-removal media [7,10].
The potential uses of Nanodiscs for stabilization, puriWca-
tion, manipulation, and structural studies of refractory
membrane proteins, enzymes and receptors are exciting
prospects.
We have recently shown that Nanodisc size can be con-
trolled by extending the length of the MSP [10] so that the
number of phospholipids can vary from 160 to 340 lipids
per particle, hence potentially allowing an increase in the
buried volume of a membrane protein target. Our original
work with membrane scaVold proteins used a construct
with 200 amino acids which formed the encircling amphi-
pathic helices and was termed MSP1. The extended MSP
constructs MSP1E1, MSP1E2, and MSP1E3 have 22, 44,
and 66 additional amino acid residues, respectively, com-
pared to MSP1 [10] corresponding to increases in the cir-
cumference of the 280 helical belt by 30, 70, and 100 .
In this work we used a model seven-transmembrane helix
membrane protein to investigate the eVects of increasing
the size of Nanodiscs on the oligomerization state of the
solubilized protein.
GPCRs are notoriously diYcult to obtain in large quan-
tities and therefore bacteriorhodopsin (bR), a seven-trans-
membrane proton pump from Halobacterium salinarum
purple membrane, has often been used as a model GPCR
with multiple membrane spanning helices. The use of bR as
a model membrane protein allowed assessment of the util-
ity of Nanodiscs for solubilization of membrane proteins in
amounts necessary for physical characterizations of the
Nanodiscs containing monomeric protein [8]. The native
form of bR is a trimer that is further organized in the pur-
ple membrane into a two-dimensional hexagonal crystal
[11] thereby presenting the possibility of addressing the
introduction of protein oligomers into Nanodiscs. Here, we
establish the utility of Nanodiscs for solubilizing large inte-
gral membrane protein complexes, and suggest a role for a
critical number of phospholipids necessary to preserve a
native oligomerization state in the Nanodisc bilayer struc-
ture.
Materials and methods
Materials
Purple membrane was isolated from H. salinarum JW-3
cultures as described [12]. Sucrose was removed by centrifu-
gation at 35,000 rpm in a Beckman Ti-45 rotor for 15 min
followed by resuspension in water. This process was
1
Abbreviations used: bR, bacteriorhodopsin; CD, circular dichroism;
DMPC, dimyristoyl phosphatidylcholine; MSP, Membrane ScaVold Pro-
tein; SAXS, small angle X-ray scattering.
Fig. 1. Schematic of MSP constructs used in this work. MSP1E1,
MSP1E2, and MSP1E3 have the indicated 22, 44, and 66 amino acid
sequence insertions, respectively, compared to MSP1. MSP1D1() lacks
the His tag, linker and residues 111 found not to participate in the helical
belt [10]. The Nanodisc structure consists of a discoidal phospholipid
bilayer domain encircled by two MSPs in a belt fashion.
repeated three times and the sample was aliquoted, lyophi-
lized, and stored at 20 C. MSP1, MSP1D1, MSP1E1,
MSP1E2, and MSP1E3 were grown and puriWed as
described previously [10]. The MSPs used in the experi-
ments had N-terminal polyhistidine tags, with the exception
of MSP1D1(). The N-terminal polyhistidine tag was
removed from MSP1D1 to make MSP1D1() as described
[10]. The MSP constructs are shown schematically in Fig. 1
and full amino acid sequences are available as Supplemen-
tary material. Concentrations were determined from the
absorbance at 280 nm using calculated extinction coeY-
cients [13]. Dimyristoyl phosphatidylcholine (DMPC) was
obtained from Avanti Polar Lipids (Alabaster, AL, USA),
dissolved in chloroform and quantitated by phosphate
analysis [14]. Bacteriorhodopsin was biotinylated with EZ-
Link-Sulfo-NHS-LC-LC-biotin (Pierce Biotechnology,
Inc., Rockford, IL, USA) as described [8]. All buVers con-
sisted of 10 mM TrisHCl, pH 7.4, 0.1 M NaCl, 0.01%
NaN3 unless stated otherwise. Water was puriWed with a
Milli-Q system (Millipore, Billerica, MA, USA). All other
materials were commercially available high-quality
reagents.
Self-assembly of nanodiscs with bacteriorhodopsin
Bacteriorhodopsin was initially solubilized with 4% w/v
Triton X-100 as described [15]. MSP stock solutions (200
400 M) and a DMPC/cholate mixture (200/400 mM in
buVer) were added to bR (typically 190 M) at mole
ratios of two MSP to three bR with varying amounts of
phospholipid to optimize self-assembly. After 1 h at room
temperature, detergent was removed by treatment for 34 h
at room temperature with 500 mg wet Biobeads SM-2
(Bio-Rad, Hercules, CA, USA) per ml of solution, with gen-
tle agitation to keep the beads suspended [16]. Beads were
removed by centrifugal Wltration. In some cases, samples
were found to be proteolytically degraded upon prolonged
storage at 4 C. Addition of a complete protease inhibitor
 T.H. Bayburt et al. / Archives of Biochemistry and Biophysics 450 (2006) 215222
cocktail without EDTA (Roche Applied Science, Indianap-
olis, IN, USA) to the self-assembly mixture and stored sam-
ples prevents degradation.
Size exclusion and monoavidin aYnity chromatography
Self-assembled mixtures were concentrated, Wltered
through 0.22 m Wlters and injected onto a Superdex 200
HR 10/30 column (Amersham Biosciences, Piscataway, NJ,
USA) run at 0.5 ml/min at room temperature with collec-
tion of 1 min fractions. Peak elution was monitored at 280
and 560 nm. The column was calibrated as described [10].
AYnity puriWcation of Nanodiscs containing biotinylated
bR was performed using a 5 ml soft-release monoavidin
column (Promega, Madison, WI, USA) according to the
manufacturers protocol. Nanodiscs containing biotin-
modiWed bR were eluted with 2.5 mM biotin in 0.1 M phos-
phate buVer, pH 7.0. The recovery of biotinbR nanodiscs
from the monoavidin resin is typically in the range of 50
70% mostly due to incomplete elution of the sample.
Protein quantitation and determination of lipid stoichiometry
Phosphate analysis of aYnity-puriWed bR Nanodisc
samples was performed after extensive dialysis to remove
phosphate buVer [14]. The amount of lipid per MSP in the
absence of bR was determined from the amount of lipid
phosphate and the concentration of MSP in the sample
based on the absorbance at 280 nm. The amount of lipid
per bR was determined using an extinction coeYcient
560 D 56,600 1200 M1 cm1 measured using retinal titra-
tion of bleached Nanodisc samples [17]. The concentration
of MSP was calculated by subtracting contributions of bR
to absorbance at 280 nm using values of 59,600 M1 cm1
based on the amino acid composition of bR and a mea-
sured value of 42,000 1200 M1 cm1 for contribution by
bound retinal obtained from the retinal titration. MSP to
bR ratios were also estimated after separation on 415 or
20% SDSPAGE and staining with Coomassie blue R-250.
Use of the MSP1D1() construct was necessary because
MSP1 does not separate eYciently from bR on SDS
PAGE. MSP1D1() and MSP1 form Nanodiscs of the
same size and lipid stoichiometry [10] and can be consid-
ered interchangeable. Gels were scanned and quantitated
using Scion Image for Windows (Scion Corporation, Fred-
erick, MD, USA). Calibration of staining intensities were
performed with standards consisting of mixtures bR and
MSP at known ratios. The ratios of bR to MSP in Nano-
disc samples were determined from least squares Wts to the
calibration curves.
Spectroscopy
Circular dichroism spectra were measured with a Jasco
J-720 spectrapolarimeter at 20 C at a sample OD560 of
approximately one. Samples contained non-biotinylated
bR and were puriWed by gel Wltration. UVvisible spectra
217
were measured with a Varian Cary 300 or Hewlett Packard
8453 spectrophotometer at room temperature.
Solution small angle X-ray scattering
Small angle X-ray scattering measurements were per-
formed at the Advanced Photon Source (Argonne National
Laboratory, Argonne, IL). The bare MSP1E3 and bR tri-
mer MSP1E3 samples were puriWed by gel Wltration. Sam-
ples were sealed in 1.5 mm glass capillaries (Charles Supper
Co, Natick, MA, USA) and placed in a sample holder with
a sample to detector distance of 1.5 m. Intensities were mea-
sured at ambient temperature using a wavelength of
0.826 . A silver behenate standard was used for calibration
of the detector array. Data were processed with the pro-
gram FIT2D [18,19] and scattering intensities of sample
consisting of buVer were subtracted. The momentum trans-
fer is deWned as s D 4sin()/, where  D 0.826 and  is
half the scattering angle. Scattering curves were Wt with
GNOM [20].
Model structures were made using an MSP1E3 sequence
without polyhistidine tag and residues 111 (see Fig. 1), as
these N-terminal amino acids do not contribute to the
perimeter of Nanodiscs [10]. The phi and psi angles were
adjusted to form a 3 to 11 helical pitch so that hydrophobic
residues line one side of the amphipathic helix [21]. A
monolayer consisting of 148 molecules of DMPC in a hex-
agonal array and molecular area of 59 2 was placed in the
center of a circular MSP1E3 molecule and juxtaposed with
itself to form a Nanodisc structure. The value for the
molecular area was chosen based on the experimentally
determined number of DMPC molecules (see Results) and
the estimated area of lipid in MSP1E3 Nanodiscs [10]. Bac-
teriorhodopsin trimer (PDB ID 1C3W) [22] was positioned
at the center of the Nanodisc structure and models were
constructed in which the trimer was translated from the
center of the Nanodisc. The trimer displaced 90 molecules
of DMPC on average. Calculated scattering curves were
generated with the program CRYSOL [23] and the curves
were summed using weighting factors based on the frac-
tional area encompassed by concentric circular bounding
intervals for each model.
Results
Optimization of bR reconstitution mixtures
The MSP constructs used are depicted schematically in
Fig. 1 and consist of an N-terminal polyhistidine tag, a
linker/protease recognition sequence and a sequence, encod-
ing the amphipathic -helical MSP. The extended MSP con-
structs were obtained by inserting a repeat of amino acids
5677, 5699, and 56121 of the parent MSP1 sequence after
residue 55, to make MSP1E1, MSP1E2, and MSP1E3,
respectively [10]. Nanodiscs result from the self-assembly of
two membrane scaVold proteins and a Wxed number of phos-
pholipids (Table 1 and [7,10]). The number of phospholipids
218 T.H. Bayburt et al. / Archives of Biochemistry and Biophysics 450 (2006) 215222

Table 1
Composition and size of bare and bR incorporated Nanodiscs
Bare Nanodiscs bR Nanodiscs
ScaVold protein Dia. (nm) Lipids Dia. (nm) bR per Nanodisca Lipids per bR
MSP1 9.1 154 4 10.7 4.8 1.6 25 2
MSP1D1() 9.2 11.2 (2.6 0.3)
MSP1E1 10.0 205 20 11.1 2.7 0.4 (3.3 0.4) 24 3
MSP1E2 10.7 244 6 12.0 2.9 0.4 (2.6 0.5) 55 4
MSP1E3 12.1 295 4 12.6 2.6 0.4 (3.0 0.4) 69 7
a
calculated from absorbance spectrum, values using SDSPAGE quantitation in parentheses.

Fig. 2. Optimization of Nanodisc self-assembly with bR. Size exclusion chromatograms were obtained with MSP1 (A), MSP1E1 (B), MSP1E2 (C) and
MSP1E3 (D) assembled in the presence of the indicated mole ratio of DMPC to MSP. bR and MSP were held at mole ratios of 1.51. Following self-
assembly, samples were injected onto a Superdex 200 HR 10/30 column (Amersham Biotech). Void volume, v; Nanodisc peak, I; larger aggregates, II;
smaller species are marked with asterisks ().

per Nanodisc is related to the length of the MSP and the lipid
cross-sectional area [10]. Because of the Wxed geometry of
Nanodiscs, high yields of Nanodiscs require self-assembly at
a Wxed lipid to MSP ratio. Inclusion of bR in the assembly
mixture should result in lower optimal ratios of lipid to MSP
due to the displacement of lipid. As a starting point,
bRMSP mixtures at a ratio of 3:2 were used to provide an
average of three bR molecules per Nanodisc. To empirically
deWne the correct lipid ratio for bR trimer assembly into
Nanodiscs, self-assembly was performed with varying
amounts of DMPC and assessed by gel Wltration chromatog-
raphy. Representative chromatograms are shown in Fig. 2.
The optimal ratio was chosen as the ratio at which the main
peak I of solubilized bR is the major component with a mini-
mum amount of large species II and species eluting in the
void volume (v). At less than optimal ratios of DMPC to
MSP1E2 and MSP1E3, species of smaller size appear. This
behavior is also seen in the reconstitution of MSP and
phospholipid to form Nanodiscs with no incorporated pro-
tein [7]. The optimal ratios of DMPC to MSP1, MSP1E1,
MSP1E2, and MSP1E3 determined in this manner were
roughly 10:1, 10:1, 55:1, and 80:1, respectively, with yields of
bRnanodiscs of about 60%.
Size and stoichiometries of bR-containing nanodiscs
Re-injections of the pooled main peaks are shown in
Fig. 3A. The sizes based on calibration of the column with a
set of standard proteins are given in Table 1 and are slightly
larger than Nanodiscs formed in the absence of bR. Devia-
tions in the empirically determined calibration plot can
result in systematic errors and so, for example, MSP1E3
assemblies elute shortly after ferritin (12.2 nm) in Fig. 3A
but the size based on calibration data appears somewhat
larger. The small extramembrane loops of bR are expected
to alter the diVusion coeYcient of the Nanodisc by a small
amount. Modeling of the hydrodynamic radius with molec-
ular models using the program HYDROPRO [24] yields
approximately the same Stokes radii for Nanodiscs with
and without a bR trimer.
The stoichiometries of bR, MSP, and lipid were deter-
mined by phosphate analysis, quantitation by SDSPAGE
and absorbance after puriWcation by size exclusion and
monoavidin aYnity chromatography to isolate the main
bR population. The errors in bR to MSP ratios based on
absorbance are very sensitive to errors in the measured val-
ues of molar extinction for bR, which are in turn sensitive
 T.H. Bayburt et al. / Archives of Biochemistry and Biophysics 450 (2006) 215222 219

Fig. 3. Nanodiscs formed at the optimized ratio of lipid. (A) MSP1 (triangles), MSP1E1 (diamonds), MSP1E2 (squares), and MSP1E3 (circles) were
assembled at the optimal ratio of DMPC and bR and subjected to a size exclusion puriWcation step. The chromatographic traces are reinjections of the
pooled elution peak. (B) Visible circular dichroism of MSP1, MSP1E1, MSP1E2, and MSP1E3 bR Nanodiscs showing the presence of a bilobed trimer
spectrum for MSP1E2 and MSP1E3 Nanodiscs.

to the bR microenvironment. Therefore measurements
were also undertaken using an analysis of stained
SDSPAGE gels. The numbers of bR per Nanodisc are
given in Table 1 and are approximately three for all MSP
constructs.
Circular dichroism of bR containing nanodiscs
Circular dichroism is often used to probe for the pres-
ence of trimeric bacteriorhodopsin since bR in purple mem-
brane exhibits a positive and a negative peak in the visible
circular dichroism spectrum, while monomeric forms of bR
exhibit a single positive peak. Thus, CD spectra are often
used to measure assembly and disassembly of the bR lattice
in purple membrane and in liposomes composed of syn-
thetic phospholipid [25,26]. Spectra of bR solubilized by
MSPs at the optimal lipid ratios are shown in Fig. 3B.
MSP1E2 and MSP1E3 Nanodiscs have a negative peak
near 600 nm, indicating the existence of a trimer. Multiple
bR molecules are present in MSP1 and MSP1E1 Nanodiscs
(Table 1), but dont always appear to form an ordered tri-
meric structure. Heating of a MSP1 or MSP1D1() sample
to 35 C for ten minutes results in appearance of a trimer-
like CD spectrum with concomitant particle fusion as mea-
sured by gel Wltration (data not shown). The CD spectra
and gel Wltration elution proWle of the MSP1E2 and
MSP1E3 Nanodiscs, however, do not change when incu-
bated at the elevated temperature.
To assess the amount of trimer versus monomer, titra-
tion of bR into the assembly with MSP1E3 was performed.
The amount of DMPC in each reconstitution was adjusted
to account for displacement of lipids by the bR, based on
the results of Table 1. Circular dichroism spectra are shown
in Fig. 4A with titration proWles at wavelengths of 520 and
593 nm given in Fig. 4B. The characteristic trimer CD titra-
tion appears to saturate at approximately four bR mole-
cules per Nanodisc. Qualitatively, the positive and negative
amplitudes for the exciton splitting appear approximately
symmetric, suggesting that most if not all bR is present as a
trimer.

Fig. 4. (A) CD spectra of samples formed at ratios of 5, 4, 3, 2, 1, 0.5, and 0.2 bR per Nanodisc (DMPC to bR mole ratios of 8, 25, 53, 110, 280, 620, and
1640). (B) Titration proWle of spectral amplitudes at 520 nm (circles) and 593 nm (squares).
220 T.H. Bayburt et al. / Archives of Biochemistry and Biophysics 450 (2006) 215222

Fig. 5. Small angle X-ray scattering of bR Nanodiscs. (A) Scattering curves were Wt using GNOM (see Materials and methods). Upper curve, bR trimer
Nanodisc formed with MSP1E3, lower curve plain MSP1E3 Nanodisc. (B) Distance distribution functions for bR trimer Nanodisc (thick solid line), and
bare MSP1E3 Nanodisc (thin solid line) corresponding to the data shown in (A) Also shown are the distance distribution functions obtained using atom-
istic models of a bR trimer in a Nanodisc with a weighted sum of distributions in the plane of the bilayer (dashed line) and a model of a bare Nanodisc
(dotted line).

Solution small angle X-ray scattering
Small angle X-ray scattering measurements were per-
formed on MSP1E3 Nanodisc samples with and without
bR (Fig. 5A). Samples with bR were formed at a stoichiom-
etry to yield an average of three bR per disc. The radius of
gyration (Rg) determined by Wtting of the entire scattering
curve [20] for trimer Nanodiscs is 50.6 while the Rg value
for plain Nanodiscs is 57.2 . A smaller Rg is expected for
the bR Nanodisc due to increased electron density within
the bilayer of the Nanodisc structure containing a bR tri-
mer. Distance distributions calculated from a Wt to the
experimental curves are shown in Fig. 5B. Nanodiscs with-
out bR have a distance distribution very similar to
phospholipid bilayers, with negative contrast correspond-
ing to the acyl group termini in the middle of the bilayer, a
maximum corresponding to the thickness of the bilayer,
and a maximum distance value that approximates the
Nanodisc diameter [10,20,27]. The distance distribution for
the bacteriorhodopsin Nanodisc shows increased contrast
in the 24 nm region, as would be expected for added elec-
tron density near the center of the bilayer, and a maximum
near that of plain Nanodiscs. Also shown is the distance
distribution for a weighted sum calculated from molecular
models with diVering positions of the trimer in the plane of
the bilayer.
Discussion
Assembly of oligomeric bacteriorhodopsin was explored
using Nanodiscs of diVerent sizes to establish the factors
governing successful oligomer incorporation into nanopar-
ticles of the expected composition and overall organization.
A bacteriorhodopsin trimer would theoretically Wt into
each of the Nanodisc constructs used and multiple bR mol-
ecules were found to self-assemble into particles formed
with each of the MSPs. The sizes of the resultant particles
are consistent with assembly of the bR into Nanodiscs with
a slightly increased apparent hydrodynamic radius com-
pared to plain Nanodiscs. When assembly was performed
at a ratio of three bR per Nanodisc and variable amounts
of lipid, the optimal lipid ratio based on qualitative con-
straints was consistent with displacement of approximately
135 molecules of DMPC per bR trimer in the assembly as
compared to plain Nanodiscs. The area per lipid for Nano-
discs composed of dimyristoyl phosphatidylcholine
(DMPC) has been estimated at 5258 2 [28]. The bR unit
cell determined by electron diVraction has P3 symmetry
with unit cell dimension of 62.45 in the plane of the mem-
brane [29,30] and an area of 3380 2 so that a bR trimer
with its associated archaeal lipid is predicted to displace
approximately 116130 DMPC molecules. The results of
the optimized ratio of lipid to MSP are thus in reasonable
agreement with the expected values. The amount of lipid
phosphate associated with each of the Nanodisc particles
was also determined. Purple membrane contains approxi-
mately 78 phospholipid phosphates per retinal [31], and
assuming preservation of the full complement of native
phospholipid in the Nanodiscs as suggested [9], trimer
incorporation displaces approximately 110 molecules of
DMPC from the MSP1E3 Nanodiscs containing three bR
molecules, also reasonably close to the predicted value of
116130 DMPC molecules. Thus to maximize assembly of
large integral membrane proteins or complexes into Nano-
disc structures account must be made for the numbers of
phospholipids displaced.
The presence of trimeric bR as determined by CD spec-
troscopy was consistently found only when MSP1E2 and
MSP1E3 were used to form Nanodiscs. There are several
potential explanations for these observations. The mini-
mum number of phospholipids that would be required to
eYciently solvate a bR trimer with one layer of lipids is
on the order of 60, a condition that may be limiting for the
MSP1 and MSP1E1 scaVolds. Monomers may be sterically
 T.H. Bayburt et al. / Archives of Biochemistry and Biophysics 450 (2006) 215222
trapped in the wrong orientation in smaller bilayers during
Nanodisc formation and are unable to reorient to assemble
a trimer. The temperature-induced increase in size of the
MSP1 particles and trimer formation suggests that fusion
and rearrangement of Nanodiscs could relieve the problem
of bR orientation or solvation by lipid. The instability of
the bRMSP1 Nanodiscs also suggests that they are imper-
fectly packed, possibly due to the lack of suYcient lipid.
The timescales for the assembly of discrete Nanodiscs, and
of bR assembly into trimers are not known, but the gradual
removal of detergent from the system occurs over a few
hours and is probably rate-limiting. The results of Fig. 4
indicate that the majority of the bR is in a trimeric form at
ratios of 34 per Nanodisc. The fact that more than
one-fourth of the bR, the expected value based on a ran-
dom orientational distribution, exhibits the bilobed CD at
an average of three bR per Nanodisc suggests that bR is
not randomly oriented heads or tails but that oligomeri-
zation occurs before Nanodiscs are completely formed.
Monte Carlo simulation of the assembly of trimers
shows a transition from monomer to trimer versus area
fraction corresponding to a pairwise short-range attractive
well depth of about 5 kT [32] that is similar to that shown in
Fig. 4. In this study the authors concluded that attractive
interactions between monomers as well as associated lipid
in the interstitial space are responsible for trimer formation.
In addition, there is experimental evidence for a speciWc
role of archaeal lipids in assembly of bacteriorhodopsin
and archaeal lipids are observed tightly associated with bR
in the crystal structure (PDB ID 1C3W) [22,33]. Again, it
should be noted that these reconstitutions performed with
Nanodiscs used whole purple membrane with its native lip-
ids. The assembly of bacteriorhodopsin into higher order
structures as probed by CD spectroscopy has been studied
in DMPC liposomes [25,34,35]. Below a ratio of roughly 80
lipids per bR molecule the bR is trimeric, both above and
below the phase transition temperature, and above that
ratio the trimer formation is temperature-dependent. Con-
sistent with the literature, the spectra in Fig. 4 indicate that
the amount of trimeric species decreases substantially
above 50110 DMPC per bR when Nanodiscs were assem-
bled near the phase transition of DMPC. Bacteriorhodop-
sin trimers (7.5 nm diameter particles) have also been
detected in egg phosphatidylcholine/phosphatidic acid (9:1
mol ratio) liposomes by freeze-fracture electron microscopy
at lipid protein ratios similar to those in Fig. 4, but for some
reason do not exhibit a bilobed CD spectrum [36].
The experimental small angle X-ray scattering data and
scattering calculated from models that are based on the
stoichiometry of MSP, bR, and phospholipid in Nanodiscs
are consistent with an arrangement of a bilayer structure
containing a bR trimer that displaces on average 3040
molecules of DMPC per bR molecule, with MSP at the
periphery of the bilayer disk. Though the models are imper-
fect, particularly with respect to bilayer thickness and acyl
chain conformations, the derived distance distributions
reproduce the features seen in the experimental data. The
221
model in which the trimer is positioned randomly within
the plane of the discoidal bilayer better reproduces the
experimental scattering than models in which it is all cen-
tered or exists at the edge of the Nanodisc.
In conclusion, ordered trimers of bR having 21 total
transmembrane helices were assembled into Nanodisc
phospholipid bilayer structures, which provide a native-like
phospholipid environment for solubilizing membrane pro-
teins. Characterizations by size exclusion chromatography,
chemical analysis, and small angle X-ray scattering are con-
sistent with the predicted organization of the particles. The
presence of a native-like trimer is found to be aVected by
the stoichiometries of components and, in a general sense,
reXects the lipid to bR ratio during formation as shown by
the results of Fig. 4. A critical factor appears to be the
amount of lipid present in the Nanodisc and overall Nano-
disc diameter. Circular dichroism measurements indicate
that an ordered trimer structure assembles in Nanodiscs
with a bilayer diameter of about 95 or greater. This value
is approximately the diameter of a bR trimer with a con-
centric ring of two lipid molecules at its outer edge. The
results presented herein enable the use of Nanodiscs for
controlling the oligomerization state of membrane proteins
having multiple transmembrane helices. We are currently
exploring the incorporation and isolation of monomers and
dimers of G-protein coupled receptors for evaluation of the
functional role of receptor dimerization in cell signaling, a
fairly recent topic of intense debate and wide speculation
for which Nanodiscs appear to be optimally suited as an
experimental system.
Acknowledgments
Portions of this work were performed at the DuPont-
Northwestern-Dow Collaborative Access Team (DND-
CAT) Synchrotron Research Center located at Sector 5 of
the Advanced Photon Source. DND-CAT is supported by
the E.I. DuPont de Nemours & Co., The Dow Chemical
Company, the U.S. National Science Foundation through
Grant DMR-9304725 and the State of Illinois through the
Department of Commerce and the Board of Higher Educa-
tion Grant IBHE HECA NWU 96. Use of the Advanced
Photon Source was supported by the U.S. Department of
Energy, Basic Energy Sciences, OYce of Energy Research
under Contract No. W-31-102-Eng-38. We gratefully
acknowledge the help and support provided by Dr. J. Quin-
tana, Dr. D. Keane, and Dr. S. Weigand while working at
Argonne. We thank Dr. I. Denisov for helpful discussions.
This material is based upon work supported by the
National Institutes of Health under Award No. GM33775
and R41 GM075362.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.abb.
2006.03.013.
222 T.H. Bayburt et al. / Archives of Biochemistry and Biophysics 450 (2006) 215222

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