Archives of Biochemistry and Biophysics 450 (2006) 215–222

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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 20–30% of all cal micelle concentration to avoid aggregation during puri-
open reading frames from archaebacterial, bacterial, and Wcation, manipulation, assay, and structural studies.
eukaryotic organisms and are perhaps among the most Maintenance of activity also often requires the presence of
challenging and potentially rewarding subjects of study [1]. phospholipid or lipid components [3]. Finally, membrane
Indeed, a majority of currently marketed therapeutics are protein oligomers exist whose association is concentration-
active against membrane protein targets, and the large dependent and potentially critical for function.
number of membrane associated orphan receptors and The G-protein coupled receptors, seven-transmembrane
enzymes of unknown function, as revealed by the human helix proteins with roles in cellular signal transduction, are
genome sequencing eVorts, are thought to represent excit- of great interest in human disease and as therapeutic tar-
ing pharmacological opportunities. A central problem gets. These receptors are believed to form hetero- and
encountered by biochemists and biophysicists is the solubi- homo-oligomers of functional consequence, and methods
lization of membrane proteins in a functional state using for controlling and isolating monomeric and oligomeric
detergents (for a recent review, see [2]). states would be a signiWcant contribution to understanding
Detergent–membrane and detergent–protein interaction the functional role of oligomerization [4,5]. Clearly, tech-
depends on molecular properties and the physicochemical niques are needed to maintain stability and oligomerization
make-up of the solution and detergent–protein interactions state in the absence of deleterious eVects of detergent.
are often destabilizing. Once solubilized, the protein We have recently introduced a new type of stable
requires the continued presence of detergent above its criti- phospholipid bilayers termed Nanodiscs, which have a
monodisperse size, into which membrane proteins can be
*
Corresponding author. Fax: +1 217 265 4073. self-assembled [6–10]. Nanodiscs are composed of
E-mail address: s-sligar@uiuc.edu (S.G. Sligar). 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

and structural studies of refractory membrane proteins. The potential uses of Nanodiscs for stabilization. storage at 4 °C. respectively. CD. circular dichroism.4. MSP1E1. bacteriorhodopsin. Addition of a complete protease inhibitor . Bacteriorhodopsin was initially solubilized with 4% w/v Materials and methods Triton X-100 as described [15]. Membrane ScaVold Pro. and suggest a role for a reagents. removed by centrifugal Wltration. CA. The native Link-Sulfo-NHS-LC-LC-biotin (Pierce Biotechnology. Billerica. In some cases. ity of Nanodiscs for solubilization of membrane proteins in dissolved in chloroform and quantitated by phosphate amounts necessary for physical characterizations of the analysis [14]. MSP.1 M NaCl. [10]. The use of bR as cients [13]. Bayburt et al. detergent was removed by treatment for 3–4 h gation at 35. 1 tities and therefore bacteriorhodopsin (bR). lized. a seven-trans. pathic helices and was termed MSP1. 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 Fig. The N-terminal polyhistidine tag was solubilized protein. and MSP1E3 were grown and puriWed as In this work we used a model seven-transmembrane helix described previously [10]. either by dialysis or treat- ment with hydrophobic detergent-removal media [7.01% [11] thereby presenting the possibility of addressing the NaN3 unless stated otherwise. linker and residues 1–11 found not to participate in the helical with 200 amino acids which formed the encircling amphi. cumference of the »280 Å helical belt by 30. critical number of phospholipids necessary to preserve a native oligomerization state in the Nanodisc bilayer struc. Sucrose was removed by centrifu. with the exception the size of Nanodiscs on the oligomerization state of the of MSP1D1(¡). They form reproducibly and in high yield through a directed self- assembly process in which detergent is removed from initial detergent–phospholipid micelles. IL. All buVers con- ple membrane into a two-dimensional hexagonal crystal sisted of 10 mM Tris–HCl. 1. USA) per ml of solution. MSP stock solutions (200– 400 M) and a DMPC/cholate mixture (200/400 mM in Materials buVer) were added to bR (typically »190 M) at mole ratios of two MSP to three bR with varying amounts of Purple membrane was isolated from H. dimyristoyl phosphatidylcholine. Self-assembly of nanodiscs with bacteriorhodopsin ture. samples DMPC. The MSPs used in the experi- membrane protein to investigate the eVects of increasing ments had N-terminal polyhistidine tags. with gen- tle agitation to keep the beads suspended [16]. obtained from Avanti Polar Lipids (Alabaster. puriWca- tion. lyophi- pared to MSP1 [10] corresponding to increases in the cir. MSP1D1. All other establish the utility of Nanodiscs for solubilizing large inte. USA) as described [8]. pH 7. AL.H. per particle. and stored at ¡20 °C. USA). and 100 Å. MSP1E2. and MSP1E3 have 22. The extended MSP constructs MSP1E1. has often been used as a model GPCR absorbance at 280 nm using calculated extinction coeY- with multiple membrane spanning helices. belt [10]. Rockford. Here. were found to be proteolytically degraded upon prolonged tein. MSP1E2. removed from MSP1D1 to make MSP1D1(¡) as described GPCRs are notoriously diYcult to obtain in large quan. salinarum JW-3 phospholipid to optimize self-assembly. Hercules. USA). 44. The MSP constructs are shown schematically in Fig. Inc. 0. After 1 h at room cultures as described [12]. materials were commercially available high-quality gral membrane protein complexes. enzymes and receptors are exciting prospects. we Milli-Q system (Millipore.000 rpm in a Beckman Ti-45 rotor for 15 min at room temperature with »500 mg wet Biobeads SM-2 followed by resuspension in water.216 T. temperature. 0. MSP1. manipulation. / Archives of Biochemistry and Biophysics 450 (2006) 215–222 of an amphipathic -helical “Membrane ScaVold Protein” (MSP)1 that wrap around the periphery of the bilayer. small angle X-ray scattering.. Water was puriWed with a introduction of protein oligomers into Nanodiscs.10]. This process was (Bio-Rad. Beads were 1 Abbreviations used: bR. hence potentially allowing an increase in the MSP1E2. The Nanodisc structure consists of a discoidal phospholipid bilayer domain encircled by two MSPs in a belt fashion. Concentrations were determined from the purple membrane. com. 70. form of bR is a trimer that is further organized in the pur. SAXS. and MSP1E3 have the indicated 22. 44. Schematic of MSP constructs used in this work. Our original sequence insertions. repeated three times and the sample was aliquoted. Dimyristoyl phosphatidylcholine (DMPC) was a model membrane protein allowed assessment of the util. and 66 additional amino acid residues. and full amino acid sequences are available as Supplemen- membrane proton pump from Halobacterium salinarum tary material. MA. compared to MSP1. and 66 amino acid buried volume of a membrane protein target. MSP1D1(¡) lacks work with membrane scaVold proteins used a construct the His tag. Bacteriorhodopsin was biotinylated with EZ- Nanodiscs containing monomeric protein [8]. MSP1E1. respectively.

56–99. The extended MSP con- Spectroscopy structs were obtained by inserting a repeat of amino acids 56–77.19] and scattering intensities of sample modiWed bR were eluted with 2. T. Peak elution was monitored at 280 Co.10]). Scattering curves were Wt with 70% mostly due to incomplete elution of the sample.1 M phos. encod- ing the amphipathic -helical MSP. The ratios of bR to MSP in Nano. The momentum trans- phate buVer. Fred- erick. mer MSP1E3 samples were puriWed by gel Wltration. The recovery of biotin–bR nanodiscs fer is deWned as s D 4sin()/. The number of phospholipids .826 Å. NJ.0. a sample to detector distance of 1. were measured with a Varian Cary 300 or Hewlett Packard olis. Sam- USA) run at 0. Bayburt et al. The concentration determined number of DMPC molecules (see Results) and of MSP was calculated by subtracting contributions of bR the estimated area of lipid in MSP1E3 Nanodiscs [10]. The column was calibrated as described [10]. same size and lipid stoichiometry [10] and can be consid- ered interchangeable. as these N-terminal amino acids do not contribute to the Phosphate analysis of aYnity-puriWed bR Nanodisc perimeter of Nanodiscs [10].826 Å and  is from the monoavidin resin is typically in the range of 50– half the scattering angle. MA. IL).600 M¡1 cm¡1 teriorhodopsin trimer (PDB ID 1C3W) [22] was positioned based on the amino acid composition of bR and a mea. USA) and placed in a sample holder with and 560 nm. Gels were scanned and quantitated Results using Scion Image for Windows (Scion Corporation. and MSP1E3.5 ml/min at room temperature with collec. USA) to the self-assembly mixture and stored sam. Solution small angle X-ray scattering Size exclusion and monoavidin aYnity chromatography Small angle X-ray scattering measurements were per- Self-assembled mixtures were concentrated. gram FIT2D [18. Calibration of staining intensities were Optimization of bR reconstitution mixtures performed with standards consisting of mixtures bR and MSP at known ratios. 8453 spectrophotometer at room temperature. The MSP constructs used are depicted schematically in disc samples were determined from least squares Wts to the Fig. Intensities were mea- AYnity puriWcation of Nanodiscs containing biotinylated sured at ambient temperature using a wavelength of bR was performed using a 5 ml soft-release monoavidin 0. A absence of bR was determined from the amount of lipid monolayer consisting of 148 molecules of DMPC in a hex- phosphate and the concentration of MSP in the sample agonal array and molecular area of 59 Å2 was placed in the based on the absorbance at 280 nm. A silver behenate standard was used for calibration column (Promega.5 mM biotin in 0. Nanodiscs containing biotin. where  D 0.22 m Wlters and injected onto a Superdex 200 Laboratory. generated with the program CRYSOL [23] and the curves Use of the MSP1D1(¡) construct was necessary because were summed using weighting factors based on the frac- MSP1 does not separate eYciently from bR on SDS– tional area encompassed by concentric circular bounding PAGE. USA) according to the of the detector array. J-720 spectrapolarimeter at 20 °C at a sample OD560 of respectively [10]. The value for the 560 D 56. pH 7. ples prevents degradation. WI. to make MSP1E1. IN. Indianap. consisting of buVer were subtracted. Madison. molecular area was chosen based on the experimentally tion of bleached Nanodisc samples [17]. MD. at the center of the Nanodisc structure and models were sured value of 42. 1 and consist of an N-terminal polyhistidine tag. Samples contained non-biotinylated two membrane scaVold proteins and a Wxed number of phos- bR and were puriWed by gel Wltration. UV–visible spectra pholipids (Table 1 and [7. The amount of lipid center of a circular MSP1E3 molecule and juxtaposed with per bR was determined using an extinction coeYcient itself to form a Nanodisc structure. MSP1E2. GNOM [20]. Calculated scattering curves were 20% SDS–PAGE and staining with Coomassie blue R-250. / Archives of Biochemistry and Biophysics 450 (2006) 215–222 217 cocktail without EDTA (Roche Applied Science.5 m. Model structures were made using an MSP1E3 sequence Protein quantitation and determination of lipid stoichiometry without polyhistidine tag and residues 1–11 (see Fig. Nanodiscs result from the self-assembly of approximately one. 1). Natick.5 mm glass capillaries (Charles Supper tion of 1 min fractions. USA). The phi and psi angles were samples was performed after extensive dialysis to remove adjusted to form a 3 to 11 helical pitch so that hydrophobic phosphate buVer [14].H. Argonne. Piscataway. ples were sealed in 1. Data were processed with the pro- manufacturer’s protocol. MSP to center of the Nanodisc. Bac- to absorbance at 280 nm using values of 59.000 § 1200 M¡1 cm¡1 for contribution by constructed in which the trimer was translated from the bound retinal obtained from the retinal titration. linker/protease recognition sequence and a sequence. The bare MSP1E3 and bR tri- HR 10/30 column (Amersham Biosciences. MSP1D1(¡) and MSP1 form Nanodiscs of the intervals for each model.600 § 1200 M¡1 cm¡1 measured using retinal titra. The trimer displaced 90 molecules bR ratios were also estimated after separation on 4–15 or of DMPC on average. Wltered formed at the Advanced Photon Source (Argonne National through 0. a calibration curves. The amount of lipid per MSP in the residues line one side of the amphipathic helix [21]. and 56–121 of the parent MSP1 sequence after Circular dichroism spectra were measured with a Jasco residue 55.

I. and absorbance after puriWcation by size exclusion and tein [7]. high yields of Nanodiscs require self-assembly at Re-injections of the pooled main peaks are shown in a Wxed lipid to MSP ratio. ues of molar extinction for bR. The errors in bR to MSP ratios based on roughly 10:1. / Archives of Biochemistry and Biophysics 450 (2006) 215–222 Table 1 Composition and size of bare and bR incorporated Nanodiscs Bare Nanodiscs bR Nanodiscs ScaVold protein Dia. 3A Nanodiscs. with yields of absorbance are very sensitive to errors in the measured val- bR–nanodiscs of about 60%. larger. samples were injected onto a Superdex 200 HR 10/30 column (Amersham Biotech). Devia- bR–MSP mixtures at a ratio of 3:2 were used to provide an tions in the empirically determined calibration plot can average of three bR molecules per Nanodisc. Optimization of Nanodisc self-assembly with bR. 55:1. MSP. The sizes based on calibration of the column with a mixture should result in lower optimal ratios of lipid to MSP set of standard proteins are given in Table 1 and are slightly due to the displacement of lipid. Size exclusion chromatograms were obtained with MSP1 (A).3 § 0.1 295 § 4 12. 10:1. and 80:1.4) 24 § 3 MSP1E2 10.9 § 0. MSP1E1.H. 2. (nm) Lipids Dia.7 244 § 6 12.0 2. values using SDS–PAGE quantitation in parentheses.4 (3. larger aggregates. monoavidin aYnity chromatography to isolate the main MSP1E2. Fig.4) 69 § 7 a calculated from absorbance spectrum. Representative chromatograms are shown in Fig.5–1. MSP1E3 deWne the correct lipid ratio for bR trimer assembly into assemblies elute shortly after ferritin (12.6 25 § 2 MSP1D1(¡) 9.8 § 1.6 § 0. larger than Nanodiscs formed in the absence of bR. Nanodisc peak. per Nanodisc is related to the length of the MSP and the lipid Size and stoichiometries of bR-containing nanodiscs cross-sectional area [10].7 § 0. Inclusion of bR in the assembly Fig.0 § 0. II. quantitation by SDS–PAGE phospholipid to form Nanodiscs with no incorporated pro. for example.6 2. respectively.218 T. To empirically result in systematic errors and so.4 (3. Void volume.5) 55 § 4 MSP1E3 12. and MSP1E3 determined in this manner were bR population. MSP1E2 (C) and MSP1E3 (D) assembled in the presence of the indicated mole ratio of DMPC to MSP. Bayburt et al. At less than optimal ratios of DMPC to and without a bR trimer.1 2. bR and MSP were held at mole ratios of 1. which are in turn sensitive .7 4. The optimal ratios of DMPC to MSP1. to alter the diVusion coeYcient of the Nanodisc by a small The optimal ratio was chosen as the ratio at which the main amount. As a starting point.0 205 § 20 11.6 § 0. MSP1E1 (B). Modeling of the hydrodynamic radius with molec- peak I of solubilized bR is the major component with a mini.2 nm) in Fig.3) — MSP1E1 10. species of smaller size appear. self-assembly was performed with varying but the size based on calibration data appears somewhat amounts of DMPC and assessed by gel Wltration chromatog. This The stoichiometries of bR. (nm) bR per Nanodisca Lipids per bR MSP1 9. 2.4 (2. Because of the Wxed geometry of Nanodiscs. and lipid were deter- behavior is also seen in the reconstitution of MSP and mined by phosphate analysis. ular models using the program HYDROPRO [24] yields mum amount of large species II and species eluting in the approximately the same Stokes radii for Nanodiscs with void volume (v).6 § 0. 3A. v. smaller species are marked with asterisks (¤).2 — (2. The small extramembrane loops of bR are expected raphy. Following self- assembly.1 154 § 4 10. MSP1E2 and MSP1E3.2 — 11.

0. MSP1E1 (diamonds). suggesting that most if not all bR is present as a bR molecules are present in MSP1 and MSP1E1 Nanodiscs trimer. Therefore measurements (Table 1). Heating of a MSP1 or MSP1D1(¡) sample SDS–PAGE gels. Circular dichroism spectra are shown used to measure assembly and disassembly of the bR lattice in Fig. and MSP1E3 (circles) were assembled at the optimal ratio of DMPC and bR and subjected to a size exclusion puriWcation step. while monomeric forms of bR to account for displacement of lipids by the bR. the positive and negative MSP1E2 and MSP1E3 Nanodiscs have a negative peak amplitudes for the exciton splitting appear approximately near 600 nm. The numbers of bR per Nanodisc are to 35 °C for ten minutes results in appearance of a trimer- given in Table 1 and are approximately three for all MSP like CD spectrum with concomitant particle fusion as mea- constructs. titra- ence of trimeric bacteriorhodopsin since bR in purple mem. CD spectra are often the results of Table 1. 2. (A) CD spectra of samples formed at ratios of 5. brane exhibits a positive and a negative peak in the visible The amount of DMPC in each reconstitution was adjusted circular dichroism spectrum. 280.2 bR per Nanodisc (DMPC to bR mole ratios of 8. tion of bR into the assembly with MSP1E3 was performed. and 1640). 3. 4A with titration proWles at wavelengths of 520 and in purple membrane and in liposomes composed of syn. indicating the existence of a trimer. 593 nm given in Fig. Fig. 4.26]. The chromatographic traces are reinjections of the pooled elution peak. Spectra of bR solubilized by tion appears to saturate at approximately four bR mole- MSP’s at the optimal lipid ratios are shown in Fig.5. cules per Nanodisc. MSP1E2 (squares). To assess the amount of trimer versus monomer. The CD spectra and gel Wltration elution proWle of the MSP1E2 and Circular dichroism of bR containing nanodiscs MSP1E3 Nanodiscs. 110. / Archives of Biochemistry and Biophysics 450 (2006) 215–222 219 Fig. and 0. T. 3B. Nanodiscs formed at the optimized ratio of lipid. 4. sured by gel Wltration (data not shown).H. and MSP1E3 bR Nanodiscs showing the presence of a bilobed trimer spectrum for MSP1E2 and MSP1E3 Nanodiscs. 25. 53. The characteristic trimer CD titra- thetic phospholipid [25. 620. Circular dichroism is often used to probe for the pres. Thus. but don’t always appear to form an ordered tri- were also undertaken using an analysis of stained meric structure. 1. do not change when incu- bated at the elevated temperature. (B) Titration proWle of spectral amplitudes at 520 nm (circles) and 593 nm (squares). 4B. (A) MSP1 (triangles). . Bayburt et al. MSP1E2. however. 3. Multiple symmetric. MSP1E1. to the bR microenvironment. based on exhibit a single positive peak. Qualitatively. (B) Visible circular dichroism of MSP1.

20. 116–130 DMPC molecules. a condition that may be limiting for the with each of the MSPs. as would be expected for added elec. assuming preservation of the full complement of native tron density near the center of the bilayer. cell determined by electron diVraction has P3 symmetry mer.45 Å in the plane of the mem- experimental curves are shown in Fig. The amount of lipid and a maximum distance value that approximates the phosphate associated with each of the Nanodisc particles Nanodisc diameter [10. 5A). Monomers may be sterically .H. Upper curve. The results of ing to the acyl group termini in the middle of the bilayer. Assembly of oligomeric bacteriorhodopsin was explored The presence of trimeric bR as determined by CD spec- using Nanodiscs of diVerent sizes to establish the factors troscopy was consistently found only when MSP1E2 and governing successful oligomer incorporation into nanopar. MSP1E3 were used to form Nanodiscs. the optimal lipid ratio based on qualitative con- etry to yield an average of three bR per disc. lower curve plain MSP1E3 Nanodisc. brane [29. / Archives of Biochemistry and Biophysics 450 (2006) 215–222 Fig. The bR unit the bilayer of the Nanodisc structure containing a bR tri. a the optimized ratio of lipid to MSP are thus in reasonable maximum corresponding to the thickness of the bilayer. The area per lipid for Nano- for plain Nanodiscs is 57. and a maximum phospholipid in the Nanodiscs as suggested [9]. bR trimer Nanodisc formed with MSP1E3. approximately 116–130 DMPC molecules.220 T. A smaller Rg is expected for discs composed of dimyristoyl phosphatidylcholine the bR Nanodisc due to increased electron density within (DMPC) has been estimated at 52–58 Å2 [28]. The distance distribution for was also determined. When assembly was performed formed on MSP1E3 Nanodisc samples with and without at a ratio of three bR per Nanodisc and variable amounts bR (Fig. agreement with the expected values. pared to plain Nanodiscs. and in the 2–4 nm region. Samples with bR were formed at a stoichiom. 5. Distance distributions calculated from a Wt to the with unit cell dimension of 62. Bayburt et al.27]. Thus to maximize assembly of large integral membrane proteins or complexes into Nano- Discussion disc structures account must be made for the numbers of phospholipids displaced. also reasonably close to the predicted value of the bilayer. Also shown is the distance incorporation displaces approximately 110 molecules of distribution for a weighted sum calculated from molecular DMPC from the MSP1E3 Nanodiscs containing three bR models with diVering positions of the trimer in the plane of molecules. 5B. trimer near that of plain Nanodiscs. Nanodiscs with.6 Å while the Rg value compared to plain Nanodiscs. potential explanations for these observations. eYciently “solvate” a bR trimer with one layer of lipids is ecules were found to self-assemble into particles formed on the order of 60. The radius of straints was consistent with displacement of approximately gyration (Rg) determined by Wtting of the entire scattering 135 molecules of DMPC per bR trimer in the assembly as curve [20] for trimer Nanodiscs is 50. (A) Scattering curves were Wt using GNOM (see Materials and methods). Solution small angle X-ray scattering are consistent with assembly of the bR into Nanodiscs with a slightly increased apparent hydrodynamic radius com- Small angle X-ray scattering measurements were per. Small angle X-ray scattering of bR Nanodiscs. The mini- A bacteriorhodopsin trimer would theoretically Wt into mum number of phospholipids that would be required to each of the Nanodisc constructs used and multiple bR mol. (B) Distance distribution functions for bR trimer Nanodisc (thick solid line). Purple membrane contains approxi- the bacteriorhodopsin Nanodisc shows increased contrast mately 7–8 phospholipid phosphates per retinal [31].30] and an area of 3380 Å2 so that a bR trimer out bR have a distance distribution very similar to with its associated archaeal lipid is predicted to displace phospholipid bilayers. of lipid.2 Å. There are several ticles of the expected composition and overall organization. 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). with negative contrast correspond. The sizes of the resultant particles MSP1 and MSP1E1 scaVolds.

Bacteriorhodop. but the gradual chemical analysis. Acknowledgments ids. at doi:10. and rearrangement of Nanodiscs could relieve the problem In conclusion. exhibits the bilobed CD at amount of lipid present in the Nanodisc and overall Nano- an average of three bR per Nanodisc suggests that bR is disc diameter. ratios of 3–4 per Nanodisc. .H. reproduce the features seen in the experimental data. The 2006. Supplementary data periphery of the bilayer disk. / Archives of Biochemistry and Biophysics 450 (2006) 215–222 221 trapped in the wrong orientation in smaller bilayers during model in which the trimer is positioned randomly within Nanodisc formation and are unable to reorient to assemble the plane of the discoidal bilayer better reproduces the a trimer. The results of Fig. and Dr. the U. 4 presence of a native-like trimer is found to be aVected by indicate that the majority of the bR is in a trimeric form at the stoichiometries of components and. a role of archaeal lipids in assembly of bacteriorhodopsin fairly recent topic of intense debate and wide speculation and archaeal lipids are observed tightly associated with bR for which Nanodiscs appear to be optimally suited as an in the crystal structure (PDB ID 1C3W) [22. DND-CAT is supported by ratio the trimer formation is temperature-dependent. W-31-102-Eng-38. of bR assembly into trimers are not known. In this study the authors concluded that attractive having multiple transmembrane helices. in the online version. Basic Energy Sciences.S. the spectra in Fig. Dr. but for some acknowledge the help and support provided by Dr. should be noted that these reconstitutions performed with Nanodiscs used whole purple membrane with its native lip. Denisov for helpful discussions. Department of Commerce and the Board of Higher Educa- bled near the phase transition of DMPC. and teins. Con. I. The temperature-induced increase in size of the experimental scattering than models in which it is all cen- MSP1 particles and trimer formation suggests that fusion tered or exists at the edge of the Nanodisc. The fact that more than reXects the lipid to bR ratio during formation as shown by one-fourth of the bR. ordered trimers of bR having 21 total of bR orientation or solvation by lipid. Below a ratio of roughly 80 Northwestern-Dow Collaborative Access Team (DND- lipids per bR molecule the bR is trimeric. dimers of G-protein coupled receptors for evaluation of the In addition.33]. The hours and is probably rate-limiting. the derived distance distributions found. scattering calculated from models that are based on the This material is based upon work supported by the stoichiometry of MSP.S.013. tana. the E. Bayburt et al. and small angle X-ray scattering are con- removal of detergent from the system occurs over a few sistent with the predicted organization of the particles. 4. and phospholipid in Nanodiscs National Institutes of Health under Award No. and above that the Advanced Photon Source. it experimental system.. with MSP at the Appendix A.abb. with a bilayer diameter of about 95 Å or greater. We are currently interactions between monomers as well as associated lipid exploring the incorporation and isolation of monomers and in the interstitial space are responsible for trimer formation. that an ordered trimer structure assembles in Nanodiscs zation occurs before Nanodiscs are completely formed. J. National Science Foundation through the amount of trimeric species decreases substantially Grant DMR-9304725 and the State of Illinois through the above 50–110 DMPC per bR when Nanodiscs were assem. Again. phospholipid environment for solubilizing membrane pro- The timescales for the assembly of discrete Nanodiscs. possibly due to the lack of suYcient lipid. Weigand while working at The experimental small angle X-ray scattering data and Argonne.35]. 4 indicate that Company.1016/j. This value Monte Carlo simulation of the assembly of trimers is approximately the diameter of a bR trimer with a con- shows a transition from monomer to trimer versus area centric ring of two lipid molecules at its outer edge. OYce of Energy Research mol ratio) liposomes by freeze-fracture electron microscopy under Contract No. The assembly of bacteriorhodopsin into higher order structures as probed by CD spectroscopy has been studied Portions of this work were performed at the DuPont- in DMPC liposomes [25. Though the models are imper- fect.03. We gratefully at lipid protein ratios similar to those in Fig. Circular dichroism measurements indicate not randomly oriented “heads or tails” but that oligomeri. 4. Quin- reason do not exhibit a bilobed CD spectrum [36]. Characterizations by size exclusion chromatography. The instability of transmembrane helices were assembled into Nanodisc the bR–MSP1 Nanodiscs also suggests that they are imper. bR.34. We thank Dr. A critical factor appears to be the dom orientational distribution. there is experimental evidence for a speciWc functional role of receptor dimerization in cell signaling. particularly with respect to bilayer thickness and acyl Supplementary data associated with this article can be chain conformations. Department of detected in egg phosphatidylcholine/phosphatidic acid (9:1 Energy. The Dow Chemical sistent with the literature. both above and CAT) Synchrotron Research Center located at Sector 5 of below the phase transition temperature. GM33775 are consistent with an arrangement of a bilayer structure and R41 GM075362. T. Use of the Advanced sin trimers (7.5 nm diameter particles) have also been Photon Source was supported by the U. 4. the results of Fig. the expected value based on a ran. S. DuPont de Nemours & Co. The fraction corresponding to a pairwise short-range attractive results presented herein enable the use of Nanodiscs for well depth of about 5 kT [32] that is similar to that shown in controlling the oligomerization state of membrane proteins Fig. Keane. tion Grant IBHE HECA NWU 96. which provide a native-like fectly packed.I. containing a bR trimer that displaces on average 30–40 molecules of DMPC per bR molecule. phospholipid bilayer structures. in a general sense. D.

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