Letter pubs.acs.org/ac
Isolation of Monodisperse Nanodisc-Reconstituted Membrane Proteins Using Free Flow Electrophoresis Bo Højen Justesen,†,∥ Tomas Laursen,‡,∥ Gerhard Weber,§ Anja Thoe Fuglsang,† Birger Lindberg Møller,*,‡ and Thomas Günther Pomorski*,† †
Section for Transport Biology, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsenvej 40, DK-1871 Frederiksberg C, Denmark ‡ Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsenvej 40, DK-1871 Frederiksberg C, Denmark § FFE Service GmbH, Frankfurter Ring 193a, D-80807 Munich, Germany S Supporting Information *
ABSTRACT: Free flow electrophoresis is used for rapid and highrecovery isolation of homogeneous preparations of functionally active membrane proteins inserted into nanodiscs. The approach enables isolation of integral and membrane anchored proteins and is also applicable following introduction of, e.g., fluorescent tags. Preparative separation of membrane protein loaded nanodiscs from empty nanodiscs and protein aggregates results in monodisperse nanodisc preparations ideal for structural and functional characterization using biophysical methods.
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traditional use of FFE involves the application of a continuous electrical field.5 For the separation of nanodisc samples, we applied a novel interval mode of FFE, interval zone electrophoresis (FFIZE).6 In this mode the electric field is applied for a short period of time during the run, while sample application and the final elution of the separated analytes is performed with the electrical field switched off. Two other modes of FFE commonly used are isoelectric focusing and isotachophoresis. Both are operated with pH gradients or steps in the separation buffer, respectively.7 The separation capacity was initially defined by injection of a mixture of dyes with different electrophoretic mobility. This provides a quality check of the separation capacity and defines the borders toward the anodic and cathodic stabilization buffers. Operative separation can be performed between fractions 16 and 85 (Figure 1b) and allows for a wide field of exploration by adjusting the power and duration of the applied electric field. To evaluate the robustness of FFIZE for separation of nanodiscs, we first studied the charge dependent electrophoretic mobility of empty nanodiscs. When prepared from neutrally charged lipids, the electrophoretic mobility of empty nanodiscs depends on the charge of the two MSPs. Three different MSP constructs were applied for assembling the nanodiscs used in this work: MSP1D1 with a calculated isoelectric point (pI) of 5.83 and a charge of −6.9 at pH 7, MSP1D1(−) with a calculated pI of 5.58 and a charge of −4.5
oluble nanoscale lipid bilayers, termed nanodiscs, are excellent biomimetic systems for studying membraneanchored and integral membrane protein complexes under defined experimental conditions. Assembled from membrane scaffold proteins (MSPs), a nanodisc consists of two MSPs encircling a planar lipid bilayer in a double-belt configuration.1 The diameter of nanodiscs is determined by the length of the MSPs, affording diameters in the 9.8−12.9 nm range.2 Through a self-assembly process, this system can be used as a nanocontainer for membrane proteins, providing a controlled native-like lipid environment. However, the ability to separate empty from membrane protein-containing nanodiscs and to obtain monodispersity are still major challenges during sample preparation. Often a multitude of techniques, e.g., liquid and affinity chromatography are used in combination to obtain welldefined preparations. Free flow electrophoresis (FFE) is a matrix free electrophoresis system operating at nondenaturing conditions and designed to separate biological preparations based on their electrophoretic mobility.3,4 In this study we applied FFE to isolate different classes of membrane proteins inserted into nanodiscs assembled with different types of phospholipids. Separation is performed in aqueous media in the absence of a matrix, thereby reducing loss of material due to unspecific binding or denaturing effects. A continuous flow of buffer is applied in the separation chamber, creating a laminar flow of 0.2 mm in thickness in which the sample is introduced. Through the application of an electric field perpendicular to the flow direction, the analytes are deflected based on their intrinsic charges. At the end of the separation run, the different buffer zones are collected into 96 well plates (Figure 1a). The © 2013 American Chemical Society
Received: January 10, 2013 Accepted: March 4, 2013 Published: March 4, 2013 3497
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integrity of the nanodiscs was preserved upon fractionation as demonstrated by size exclusion chromatography analysis on FFIZE fractions obtained from the separation of nanodiscs containing the fluorescent marker lipid N-rhodamine-dioleoylphosphatidylethanolamine (Figure S-2 in the Supporting Information). On the basis of absorption and fluorescence measurements, the recovery yield of nanodiscs after FFIZE was >80%. The ability of this novel approach to separate nanodiscs was demonstrated by studying two fundamentally different types of membrane proteins reconstituted in nanodiscs. The NADPHdependent cytochrome P450 oxidoreductase (POR) from Sorghum bicolor and the plasma membrane P-type H+-pump (AHA2) from Arabidopsis thaliana were chosen. POR is a ∼78 kDa diflavo protein anchored to the endoplasmic reticulum (ER) membrane system via a single transmembrane spanning α-helix.8 It has a calculated pI of 5.06 and a charge of −22.9 at pH 7. POR transfers electrons from NADPH via the FAD and FMN coenzymes to serve as the obligate electron partner for the large number of ER localized cytochrome P450 (P450) enzymes catalyzing highly diverse and specialized functions. Electron transfer to the P450 is accompanied by major conformational changes of POR.9 Electrostatic interaction with P450s via a highly negatively charged patch affects the overall charge profile of POR.10 Therefore, POR serves as an ideal candidate for charge dependent separation by FFIZE. The proton pump AHA2 is a member of the P-type ATPase superfamily and responsible for establishing and maintaining the electrochemical proton gradient across the plant plasma membrane that energizes secondary active transport of nutrients into plant cells.11 AHA2 has a molecular mass of ∼100 kDa and consists of three large cytoplasmic domains and 10 transmembrane segments. It has a calculated pI of 6.48 and a charge of −3.5 at pH 7. The individual characteristics of POR and AHA2 represent a large variety of membrane-associated proteins and therefore display the flexibility of the FFIZE separation technology of native membrane proteins reconstituted in nanodiscs. Self-assembly of both POR and AHA2 into nanodiscs results in a mixture of empty and protein-loaded nanodiscs. Classic matrix based gel-filtration does not have the resolution to separate the two nanodisc species (Figures S-3 and S-4 in the Supporting Information). POR was reconstituted in nanodiscs containing only neutral PC lipids, thus separation was based on greater electrophoretic mobility of the highly negatively charged POR enzyme. A comparison with an empty nanodisc preparation clearly demonstrated that the POR assembled nanodiscs was separated into two distinct populations, i.e., empty and loaded, monitored by absorption at 280 nm (Figure 2a). The loaded nanodiscs displayed a significant increase in mobility, which was verified by following FMN and FAD absorbance and by SDS-PAGE. Only the MSP1E3 protein was detected in the empty nanodiscs with the main peak in fraction ∼60, whereas POR nanodiscs peaked in fractions ∼45−47 as demonstrated by the presence of both MSP1E3 and POR proteins. The catalytic activity of separated POR nanodiscs was verified by the cytochrome-c reduction assay.12 A similar approach using PC for AHA2 reconstitution into nanodiscs did not result in any significant separation of empty and loaded nanodiscs, even when increasing the duration of the applied electrical field (Figure S-5 in the Supporting Information). This can be explained by the nearly neutral charge of the proton
Figure 1. Free flow interval zone electrophoresis (FFIZE) for separation of nanodiscs with different charge densities. (a) Schematics of the FFIZE separation process. (Step 1) Sample is injected into the continuous laminar flow and allowed to continue into the chamber. (Step 2) The electrical field is applied in an optional time interval depending on the electric mobility of the compound of interest. (Step 3) After separation of the sample, fractions are collected into a 96 well plate and are available for analysis. (b) Empty nanodiscs were assembled from PC alone or with increasing amounts of negatively charged PG (10 or 25 mol %). The mobility of the individual nanodiscs toward the anode was monitored by absorption at 280 nm. On top of the three nanodisc samples is an illustration of a nanodisc containing neutral lipids (blue) and negatively charged lipids (red). A mixture of six pI markers (violet) with differing electric mobility was included demonstrating the boundaries toward the cathodic and anodic stabilization buffers. The blank sample displays a characteristic peak at the boundary of the anodic stabilization buffer as well as the expected background noise throughout the fractions.
at pH 7, and MSP1D1E3 with a calculated pI of −5.44 and a charge of −5.4 at pH 7. On the basis of the negative charge of the three MSPs at pH 7, the empty nanodiscs are expected to migrate in the direction of the anode at pH 7. Empty nanodiscs were assembled from MSP1D1, pure phosphatidylcholine (PC) lipids or mixtures of PC, and negatively charged phosphatidylglycerol (PG; 10 or 25 mol %; Figure S-1 in the Supporting Information) lipids and then subjected to FFIZE. Separate injections of the preparations were analyzed by recording the absorption at 280 nm of the collected fractions. The mobility of nanodiscs within the electric field was charge dependent, resulting in distinct elution profiles (Figure 1b). The structural 3498
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Figure 2. Purification of empty and membrane-protein containing nanodisc complexes using FFIZE. (a) Nanodiscs assembled in the presence of POR using PC lipids and membrane scaffold protein MSP1E3. Separation is based on greater electrophoretic mobility resulting from the negative charge of POR. The empty nanodiscs (green) elute in fractions with the main peak at ∼60, whereas POR containing nanodiscs (yellow) peak at fractions ∼45−47, demonstrated by the presence of both MSP1E3 and POR proteins in SDS-PAGE and by measurement of cytochrome-c reduction (red) and flavin determination (blue). (b) Nanodiscs assembled in the presence of AHA2 using a 3:2 stoichiometric ratio of PC/PG lipids and membrane scaffold protein MSP1D1(−). Separation is based on reduced electrophoretic mobility of the AHA2-containing nanodiscs (red) compared to empty nanodiscs (blue), due to the displacement of negatively charged PG lipids by insertion of AHA2. SDS-PAGE and BN-PAGE on fractions covering the peak of the separation of AHA2-containing nanodiscs displays a clear separation of empty and loaded nanodiscs. ATPase activity measured for fractions covering the AHA2-containing nanodiscs peak (indicated by an asterisk) confirmed the presence of active nanodiscreconstituted AHA2 in the eluted fractions.
applicable following modification of the membrane proteins, e.g., by addition of affinity tags to align the discs on surfaces or by addition of fluorescent tags to monitor conformational changes. The possibility to independently vary several parameters during the nanodisc self-assembly process, such as lipid charges and disc sizes, opens up a range of different strategies to be employed in the FFIZE based separation. This is demonstrated here by the application of charged lipids for nanodisc assembly with AHA2, basing the separation on reduced electrophoretic mobility compared to the more traditional approach used for purification of the POR containing nanodiscs. In principle, this strategy can serve as a general approach for neutrally charged membrane proteins. Its feasibility is further indicated by reports of increased stability of nanodiscs supplemented with PG lipids13 and the fact that a lipid bilayer consisting of charged lipids more accurately reflects the in vivo environment of membrane proteins. A major challenge in characterization of membrane-anchored and integral membrane protein complexes is to obtain these in a functionally active, water-soluble, and monodisperse form enabling structural and functional characterization using biophysical methods such as small-angle X-ray scattering, neutron diffraction, surface plasmon resonance, nuclear magnetic resonance spectroscopy, as well as electron paramagnetic resonance spectroscopy. The FFIZE approach reported here overcomes this major challenge by enabling isolation of monodisperse preparations of integral and membrane anchored proteins inserted into nanodiscs. The FFIZE technology is based on a matrix free electrophoresis system operating at nondenaturing mild conditions and is rapid
pump at pH 7, resulting in only a small difference in electrophoretic mobility between empty and loaded nanodiscs. Although separation at a higher pH value would result in an increased negative charge of the proton pump and thus increased electrophoretic mobility of loaded compared to empty nanodiscs, this approach was not used to avoid potential detrimental effects on the reconstituted proton pump. To optimize the separation, a different approach was used instead based on reconstitution using a lipid mixture of PC/PG with a molar ratio of 3:2. In this approach, FFIZE separation was based on the partial displacement of lipids, including negatively charged PG, by the proton pump resulting in a less negatively charged particle with a reduced electrophoretic mobility. Comparison of FFIZE separation of empty and a mixture of empty and AHA2-containing nanodiscs prepared in this way displayed a shift of migration as monitored by absorption at 280 nm (Figure 2b). As expected, the AHA2containing nanodiscs exhibited reduced electrophoretic mobility compared to empty nanodiscs, verified by SDS-PAGE and BN-PAGE, displaying a clear separation between empty and loaded nanodiscs. Analysis of the ATP hydrolytic activity of the proton pump confirmed the presence of active nanodiscreconstituted AHA2 in the eluted fractions. Our results from nanodisc separation using FFIZE demonstrate the potential of this method as an alternative to traditional approaches. As exemplified from nanodisc reconstitutions containing the membrane proteins POR and AHA2, this method is applicable for both highly and close to neutral charged membrane proteins with distinctly different structures and properties. Accordingly, the FFIZE approach is also 3499
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Letter
and highly efficient and the membrane proteins are obtained in a native-like lipid bilayer and thereby ideally suited for biophysical characterization.
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ASSOCIATED CONTENT
S Supporting Information *
Protein purification and nanodisc preparation protocols, FFE instrumental parameters, and supporting figures. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*B.L.M.: e-mail,
[email protected]; phone, +45-35333352. T.G.P.: e-mail,
[email protected]; phone, +45-35333373. Author Contributions ∥
B.H.J. and T.L. contributed equally to this work.
Notes
The authors declare the following competing financial interest(s): Gerhard Weber is the Chief Executive Officer of the FFE service GmbH and has a financial interest in the FFE device used in this work.
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ACKNOWLEDGMENTS We are grateful to Anne-Mette Bjerg Petersen for excellent technical assistance. This work was supported by the UNIK research initiative of the Danish Ministry of Science, Technology and Innovation through the “Center for Synthetic Biology” at the University of Copenhagen and by the Danish National Research Foundation through the PUMPKIN Center of Excellence (DNRF85) and by the Villum Foundation to the Center of Excellence “Pro-Active Plants”. We thank Carlsbergfondet and Augustinus Fonden for equipment grants.
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