NANO LETTERS
Self-Assembly of Discoidal Phospholipid Bilayer Nanoparticles with Membrane Scaffold Proteins
2002 Vol. 2, No. 8 853-856
Timothy H. Bayburt,† Yelena V. Grinkova,† and Stephen G. Sligar*,†,‡,§ Department of Biochemistry, Department of Chemistry, and the Beckman Institute, UniVersity of Illinois, Urbana, Illinois 61801 Received May 23, 2002
ABSTRACT Nanoparticulate phospholipid bilayer disks were assembled from phospholipid and a class of amphipathic helical proteins termed membrane scaffold proteins (MSP). Several different MSPs were produced in high yield using a synthetic gene and a heterologous expression system and purified to homogeneity by a one-step purification. The self-assembly process begins with a mixture of the phospholipid and MSP in the presence of a detergent. Upon removal of detergent, 10-nm diameter particles form containing either saturated or unsaturated phospholipid. The ratio of components in the initial mixture was found to be crucial for formation of a monodisperse population of nanoparticles. Exploration of the phase diagram of the lamellar to phospholipid−detergent mixed micelle transition reveals that self-assembly proceeds from the mixed micellar phase. In this case a homogeneous and monodisperse population is formed. In contrast, particle formation from the detergent− phospholipid lamellar phase results in altered size, yield, composition, and heterogeneity of the resultant particles. The nanodisks contain approximately 160 saturated or 125 unsaturated lipids and can be formed from designed amphipathic r-helical scaffold proteins. The 10-nm particles can thus contain two molecules of MSP1 or a single molecule of an MSP1 fusion (MSP2). The phospholipid bilayer main phase transition temperature is preserved in the nanodisks as determined by fluorescence spectroscopy. Scanning probe microscopy shows a monolayer of nanodisks on a mica surface with a diameter of 10 nm and the thickness of a single phospholipid bilayer (5.7 nm), confirming the presence of a bilayer domain. The gentle method of self-assembly and robustness of the resulting nanodisks provides a means for generating soluble lipid bilayer membranes on the nanometer scale and opens the possibility of using these nanostructures to incorporate single membrane proteins into a native-like environment.
Phospholipids have the ability to self-assemble into bilayer structures upon desolvation. A typical form of phospholipid bilayer is the liposome, which is a spherical closed bilayer having an aqueous interior. The resemblance of the liposome to a cell membrane has elevated the liposome to be the model of choice for the study of molecular interactions with membranes and membrane proteins. Liposomes, however, are large aggregates with diameters on the order of microns and contain thousands to millions of phospholipid molecules. The use of detergents to form micelles affords a smaller particle size, but the resultant structure lacks a bilayer and undergoes dynamic fluctuations.1 In addition, detergents are also protein denaturants, rendering the study of macromolecular structure and function problematic. In this communication we present a method for generating a nanometerscale discoidal bilayer structure, or nanodisk, consisting of approximately 150 phospholipid molecules, which is soluble, * Corresponding Author: University of Illinois, 123 Morrill Hall, 505 S. Goodwin, Urbana, IL 61801. Tel: 217-244-7395. Fax: 217-265-4073. E-mail:
[email protected]. † Department of Biochemistry. ‡ Department of Chemistry. § Beckman Institute. 10.1021/nl025623k CCC: $22.00 Published on Web 07/18/2002
© 2002 American Chemical Society
stable, and preserves the general state of the phospholipid bilayer architecture. A class of naturally occurring amphipathic R-helical proteins, the plasma lipoproteins, can be induced to form disklike bilayer structures with synthetic phospholipids.2,3 The amphipathic R-helical protein is associated with the edge of a phospholipid bilayer domain and is believed to stabilize the disk-like bilayer with two or more molecules, each wrapping around the edge of a leaflet of the bilayer.4-6 For lipoprotein-lipid aggregates, however, the predominant natural form is a spherical particle having additional protein domains required for interaction with cellular receptors and enzymes. We have produced a synthetic gene that expresses a class of proteins based on the apolipoprotein A-I sequence but without a globular N-terminal domain present in the native protein. We have termed these proteins “membrane scaffold proteins” or MSP after their ability to self-assemble into discoidal nanoparticles in the presence of synthetic phospholipid. The MSP1 protein consists of a histidine tag and Factor X cleavage site followed by a 200 amino acid sequence consisting of sequential amphipathic alpha helices (see Supporting Information). As will be shown, stoichio-
Figure 1. Gel filtration chromatograms of nanodisks. Preparations with the indicated MSP and phospholipid at the indicated mole ratios were injected onto a calibrated Superdex 200 HR 10/30 gel filtration column equilibrated in disk formation buffer. The column was run at 0.5 mL/minute on a Waters Millenium system with monitoring of protein absorbance at 280 nm.
metric self-assembly of 10-nm diameter particles requires two MSP1 molecules. A genetic fusion of two MSP molecules was also constructed, termed MSP2, to avoid the necessity of bimolecular assembly. Herein we report the optimized conditions required for self-assembly of MSP with phospholipid to form homogeneous populations of nanodisks, yielding insight into the selfassembly process, and a characterization of the resulting nanoparticle. We foresee the use of nanodisks in producing stable nanoparticulate preparations of membrane proteins in a native-like phospholipid bilayer environment. Membrane proteins contained in the nanodisk environment would be beneficial to research requiring small particle sizes, such as NMR, for single molecule techniques, protein purification, and possibly crystal growth for structure determination. The MSPs also present a means for physical manipulation of nanodisks through the use of protein tags that can be engineered into the nanodisk. The nanodisk also lacks the lumen present in a liposome and obviates the problem of the orientation of the embedded membrane protein, thus providing access to both sides of the bilayer structure. For example, such would be ideal for the study of transmembrane signaling processes in solution as well as on surfaces. The protein sequence for MSP1 and MSP2, expression, and protein purification are provided as Supporting Information. Design of the synthetic gene for expression in the bacterium Escherichia coli follows our previous successes in heterologous expression of eukaryotic proteins7 and provides required bacterial translation sequences, optimizes codon usage, and minimizes secondary structures of the 854
corresponding mRNA. These constructs produce MSPs at a level of about 250 milligrams per liter of culture. Nanodisk self-assembly was initiated by the detergent dialysis technique.3 We titrated MSPs with lipids to identify the optimum lipid-to-MSP ratio for the self-assembly of disks. Purified MSP (∼5 mg/mL protein) was mixed with dipalmitoyl phosphatidylcholine (DPPC) or 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) (Avanti Polar Lipids, Alabaster, AL) cholate solubilized mixed micelles (50 mM stock lipid, 100 mM sodium cholate in 10 mM Tris pH 7.4, 0.1 M NaCl, 0.01% NaN3) followed by incubation at 37 °C for 4-18 h. The self-assembly process was initiated by dialysis against 1000-fold excess of buffer at 37 °C using 10 000 MW cutoff membranes. The nanoparticles were sized on a calibrated8 Superdex 200 HR 10/30 column (Amersham Biosciences, Piscataway, NJ). Figure 1 shows elution profiles for MSP1 and MSP2 formed with DPPC and POPC. As the phospholipid concentration is increased, the particle size coalesces into a main peak corresponding to a 10-nm particle formed at ratios of 75-100 DPPC or 75 POPC per MSP1 and 150-200 DPPC or 150 POPC per MSP2. The peaks are sharp in comparison to homogeneous calibration standards and thus represent a narrow size distribution. This is borne out by single-particle two-photon fluorescence correlation spectroscopy using dye molecules incorporated into the nanodisk (data not shown). The peak appears as a single resolved species for DPPC while nanodisks made with POPC elute with a small shoulder. The number of phospholipids per MSP was measured by doping the lipid with a known amount of tritiated lipid of the same Nano Lett., Vol. 2, No. 8, 2002
Table 1: Properties and Composition of Nanodisks MSP
optimal lipid/ MSP ratio
lipid/ MSP
MSP1 DPPC MSP1 POPC MSP2 DPPC MSP2 POPC
100 75 200 150
78 ( 3 62 ( 5 171 ( 6 129 ( 9
MSP/ diameter bilayer disk (nm) areac(nm2) 2 1
9.3a, 9.9b 9.1a 9.3a, 9.7b 9.0a
42 ( 2 43 ( 3 46 ( 2 45 ( 3
a By size exclusion chromatography. b By SPM. c For one leaflet of the nanobilayer, calculated from the number of lipids per disk.
type and scintillation counting the column fractions. Below the optimum ratios, nonspecific aggregates of MSP and lipid are produced. Above these ratios, larger aggregates containing MSP and phospholipid are formed to a much greater extent. The numbers of lipids per MSP are presented in Table 1 for the main nanodisk peak along with corresponding particle sizes. The number of lipids in sequential fractions across the peak was found to be constant, again attesting to the homogeneity of the sample. As the lipid to MSP ratio is increased, the peak shifts slightly to larger values along with a slightly increased number of lipids per MSP (data not shown). The molecular area of a nanobilayer leaflet can be estimated from the known molecular area of phospholipid in a bilayer, and values are given in Table 1. For egg PC (which consists mostly of POPC) the molecular area is 0.7 nm2 and for DPPC, 0.54 nm2.9,10 Thus the bilayer areas of all of the particles are the same, despite the presence of differing numbers of phospholipids. The number of MSP molecules per disk was determined by chemical crosslinking.11 Two molecules of MSP1 or one molecule of the genetically fused MSP2 are found to be present in a single 10-nm nanodisk. MSP is mostly R-helical, and a slight increase in helicity is induced by lipid-protein interaction. Circular dichroism (CD) was used in conjunction with spectral deconvolution using the program SELCON312 to estimate the fraction of R-helix for the scaffold proteins and self-assembled nanodisks. Lipid-free MSPs are heterogeneous aggregate structures, and it is difficult to fit the CD spectra at all wavelengths. The lipid-free forms of MSP1 and MSP2 are roughly 52% and 58% R-helical, respectively. The selfassembled nanodisks display CD spectra that can be cleanly deconvoluted. MSP1 in discoidal bilayers is 66% and 69% R-helical when formed from DPPC and POPC, respectively. The corresponding values for MSP2 10 nm disks are 71% and 70% respectively. The DPPC gel phase in the 10-nm nanodisk has a main melting transition at 43 °C as determined by fluorescence polarization of diphenylhexatriene incorporated into the disks (data not shown). The main transition corresponds to the melting temperature of hydrated DPPC bilayers (41.5 °C) but appears to be broadened due to acyl chain disorder and reduced motion of the boundary lipids interacting with the R-helices of MSP at the edge of the nanodisks. The initial state of the lipid-detergent mixture is important for self-assembly of nanodisks. We have explored the phases of dilute aqueous cholate-DPPC vesicle mixtures by optical density measurements13 in the standard disk formation buffer Nano Lett., Vol. 2, No. 8, 2002
Figure 2. Phase diagram of liposome solubilization by sodium cholate. Lines are fit to onset of solubilization (circles) and completion of solubilization (squares). Formation of nanodisks (solid triangles) or altered particles (open triangles) were determined by gel filtration. Altered particles have different particle sizes and do not contain the normal amount of phospholipid.
at 37 °C (Figure 2). The boundaries demarcating the onset and completion of solubilization of vesicles are linear, as expected. The boundaries can be interpreted as having the form DT ) DW + Reff‚L where DT is the total detergent molar concentration, DW is the aqueous detergent monomer molar concentration, Reff is the mole ratio of detergent to lipid in the micelle or bilayer, and L is molar phospholipid concentration. DW values of 5.2 mM and 6.1 mM were found at the onset and completion of solubilization, respectively, and Reff values were 0.38 and 0.47. Formation of disks appears to proceed from the mixed micelle phase because formation from the lamellar phase results in drastically altered particles, as evidenced by size exclusion chromatography and lipid analysis. Notably, a population of particles of slightly larger size than disks are formed that contain approximately half the number of phospholipid molecules per MSP compared with the well-characterized disk formed from the micelle phase. Formation from the lamellar phase also results in more heterogeneous particle distributions. A possible mode of disk formation is the displacement of cholate by MSP from the edges of discoidal phospholipidcholate micelles. While the model of disk-like micelles in aqueous DPPC-bile salt mixtures has existed for several decades,14 from more recent X-ray scattering measurements it has been found that the predominant species are elongated micelle structures that form due the propensity for lower spontaneous curvature of the structures with increasing ratios of phospholipid to detergent.15,16 However, disk-like micelles have been observed as a transient species upon dilution of globular mixed micelles.16 Work is in progress to further understand the mechanism and kinetic pathway of selfassembly of nanodisks from mixed micelles. Scanning probe microscopy was performed with a Digital Instruments Nanoscope IIIa SPM using a fluid cell in contact mode using a sharpened SiN probe with a rectangular cantilever having a nominal spring constant of 0.03 N/m 855
particles, which self-assemble from mixed cholate-phospholipid micelles and MSP upon removal of detergent. The nanodisks are homogeneous, approximately 10 nm in diameter, and contain a phospholipid bilayer domain consisting of a quantized amount of lipid. The formation and characterization of the nanodisks through self-assembly paves the way for incorporation of membrane proteins into a soluble nanoparticulate and native-like environment for use in physical, structural, biochemical, and biotechnological research.
Figure 3. Scanning probe microscopy of nanodisks. Nanodisks formed with DPPC and MSP1 (panels A and C) or MSP2 (panels B and D) were imaged under buffer in contact mode on a mica surface. The nanodisks are approximately 10 nm in diameter and sit flat on the mica surface.
(Veeco Metrology Group, Sunnyvale, CA) on a mica surface. Imaging buffer consisted of 10 mM Tris pH 8.0, 0.15 M NaCl, 10 mM MgCl2. Disks were allowed to adsorb to the mica surface followed by washing with 10 mM Tris pH 8, 0.5 M NaCl, 0.5 M imidazole for 15 min in the fluid cell and rinsing with imaging buffer. This washing procedure prevented difficulties due to the presence of a six-histidine chelating tag (used for purification) on the MSP, which caused formation of aggregates on the surface in the presence of divalent cation. Figure 3 shows MSP1 and MSP2 nanodisks assembled from DPPC. The disks adsorb tightly to mica to form a monolayer of disks. The disk diameters were estimated from Fourier transforms of the images using the NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http:// rsb.info.nih.gov/nih-image/), which provided measurements of 9.7 nm for MSP2 and 9.9 nm for MSP1. These are the same size within the error of the measurement. The thickness of the bilayer was determined by scraping away the nanodisks from a small area of the sample with the SPM probe at high force (nN range as opposed to an imaging force of ∼50 pN) and low feedback gain of the SPM until a smooth area of mica could be imaged alongside an area of nanodisks (Supporting Information). The thickness for both MSP1 and MSP2 DPPC nanodisks measured within the range of 5.65.9 nanometers, corresponding to the thickness of a DPPC bilayer. The disks also tended to fuse into large smooth domains if high force loads were used in the SPM imaging. The fused domains maintained a height constant with the top of the disks, providing further evidence of a bilayer domain present in the nanodisks. Thus the plane of the nanobilayer domain sits flat on the mica surface. In summary, nanoscale particles were formed with membrane scaffold proteins and phospholipid. Lipid ratios and concentrations were optimized for the formation of the 856
Acknowledgment. This research was supported by a grant from the National Institutes of Health GM33775 and the National Science Foundation Nanoscale Science and Engineering Center SBC NW 0830 520N602 and MCB0115068. The CD and fluorescence correlation spectroscopy measurements reported in this paper were performed at the Laboratory for Fluorescence Dynamics (LFD) at the University of Illinois at Urbana-Champaign (UIUC) with the assistance of Qiao Qiao Ruan. The LFD is supported jointly by the Division of Research Resources of the National Institutes of Health (PHS 5 P41-RRO3155) and UIUC. We thank Aretta Weber for excellent editorial assistance, Tom Sarna for assistance in optimizing MSP expression, and Andrew Leitz for measurements of phase transition temperatures. Supporting Information Available: Detailed descriptions of MSP sequence, expression and purification, size exclusion chromatography, phospholipid analysis, CD, SPM thickness measurement, and detergent micelle phase measurements are available. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Nichols, J. W. Biochemistry 1988, 27, 3925-3931. (2) Brouillette, C. G.; Jones, J. L.; Ng, T. C.; Kercret, H.; Chung, B. H.; Segrest, J. P. Biochemistry 1984, 23, 359-367. (3) Jonas, A. Methods Enzymol. 1986, 128, 553-582. (4) Atkinson, D.; Smith, H. M.; Dickson, J.; Austin, J. P. Eur. J. Biochem. 1976, 64, 541-547. (5) Koppaka, V.; Silvestro, L.; Engler, J. A.; Brouillette, C. G.; Axelsen, P. H. J. Biol. Chem. 1999, 274, 14541-14544. (6) Wlodawer, A.; Segrest, J. P.; Chung, B. H.; Chiovetti, R., Jr.; Weinstein, J. N. FEBS Lett. 1979, 104, 231-235. (7) Beck von Bodman, S.; Schuler, M. A.; Jollie, D. R.; Sligar, S. G. Proc. Natl Acad. Sci. U.S.A. 1986, 83, 9443-9447. (8) Short protocols in molecular biology, 4th ed.; Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K., Eds.;Wiley: New York, 1992. (9) Parsegian, V. A.; Fuller, N. L.; Rand, R. P. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 2750-2754. (10) Marsh, D. CRC Handbook of Lipid Bilayers; CRC Press: Boca Raton, Florida., 1990. (11) Leroy, A.; Toohill, K. L.; Fruchart, J. C.; Jonas, A. J. Biol. Chem. 1993, 268, 4798-4805. (12) Sreerama, N.; Venyaminov, S. Y.; Woody, R. W. Anal. Biochem. 2000, 287, 243-251. (13) Paternostre, M.-T.; Roux, M.; Rigaud, J.-L. Biochemistry 1988, 27, 2668-2667. (14) Small, D. M. The physical chemistry of cholanic acids. In The Bile Acids; Nair, P. P., Kritchasky, D., Eds.; Plenum Press: New York, 1971; Vol. 1, p 332. (15) Almgren, M. Biochim. Biophys. Acta 2000, 1508, 146-163. (16) Egelhaaf, S. U.; Schurtenberger, P. Phys. ReV. Lett. 1999, 82, 2804-2807.
NL025623K Nano Lett., Vol. 2, No. 8, 2002
