Nanodiscs for Immobilization of Lipid Bilayers and Membrane Receptors

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Anal. Chem. 2008, 80, 6245–6252

Nanodiscs for Immobilization of Lipid Bilayers and Membrane Receptors: Kinetic Analysis of Cholera Toxin Binding to a Glycolipid Receptor Jonas Borch,*,† Federico Torta,†,‡ Stephen G. Sligar,§ and Peter Roepstorff† Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, Mass Spectrometry Unit, San Raffaele Scientific Institute, Milan, Italy, and School of Molecular and Cellular Biology and Center of Biophysics and Computational Biology, Department of Biochemistry, University of Illinois, Urbana, Illinois Nanodiscs are self-assembled soluble discoidal phospholipids bilayers encirculated by an amphipathic protein that together provide a functional stabilized membrane disk for the incorporation of membrane-bound and membraneassociated molecules. The scope of the present work is to investigate how nanodiscs and their incorporated membrane receptors can be attached to surface plasmon resonance sensorchips and used to measure the kinetics of the interaction between soluble molecules and membrane receptors inserted in the bilayer of nanodiscs. Cholera toxin and its glycolipid receptor GM1 constitute a system that can be considered a paradigm for interactions of soluble proteins with membrane receptors. In this work, we have investigated different technologies for capturing nanodiscs containing the glycolipid receptor GM1 in lipid bilayers, enabling measurements of binding of its soluble interaction partner cholera toxin B subunit to the receptor with the sensorchip-based surface plasmon resonance (SPR) technology. The measured stoichiometric and kinetic values of the interaction are in agreement with those reported by previous studies, thus providing proof-of-principle that nanodiscs can be employed for kinetic SPR studies. Membrane-associated macromolecules play a key role in the cell. These biomolecules are fundamental in metabolism, cell-cell interactions, signal transduction, and transport of ions and nutrients. However, the study of membrane-associated macromolecules often remains challenging. Membrane proteins have generally been much more difficult to express as recombinant proteins than their soluble counterparts in almost all expression systems tested. Additionally, integral transmembrane proteins require use of detergents during protein purification, which can have negative impact on the yields and stability of the purified protein. Furthermore, crystallization in the presence of detergents as well as the dynamic structure of many membrane proteins with flexible regions reduces favorable crystal contacts and thereby * Corresponding author. E-mail:[email protected]. † University of Southern Denmark. ‡ San Raffaele Scientific Institute. § University of Illinois. 10.1021/ac8000644 CCC: $40.75  2008 American Chemical Society Published on Web 07/11/2008

hampers structure determination.1,2 Several strategies have been developed to overcome the problems inherent in membrane protein analysis. Bioinformatics approaches may reveal why some membrane proteins are better expressed than others, and then a suitable expression system has to be found in order to have the maximum protein expression. In purification, the selection of the right detergent for solubilization while maintaining the activity of the protein requires an extensive screening process.3 Nanodiscs are self-assembled, soluble, and stable structures that consist of a discoidal lipid bilayer encirculated by an amphipathic protein belt4,5 (Figure 1). They are designed by exploiting the properties of a naturally occurring amphipathic protein, apolipoprotein A-I.4,5 A protein expressed by a synthetic gene designed from a truncated form of apolipoprotein A-I, called “membrane scaffold protein” (MSP), provides the framework to encirculate the lipid bilayer, and thus nanodiscs can be designed with varying diameters and phospholipid contents by modifying the length of the MSP. The discs employed in this study measure 10 nm in diameter, are 5.5 nm high, and encirculate approximately 150 phospholipid molecules.6,7 The use of nanodiscs permits the study of membrane proteins in a well-defined, soluble, uniform, and stable environment,8–19 offering an improvement over the use of liposomes as a model membrane system employed in studies of molecular interactions with membranes and membrane proteins.20 Liposomes are large, heterogeneous, and unstable and often induce degree of curvature which can influence phenomena observed at the surface. The nanodisc provides access to both sides of the bilayer structure, obviating the problem of the orientation of the embedded membrane protein as can be the case in vesicles and supported membrane mono- or bilayers. Additionally, the capability of modifying the MSP sequence and attaching different tags at (1) Hunte, C.; von Jagow, G.; Scha¨gger, H. Membrane Protein Purification and Crystallization; Oxford; Academic Press: Oxford, 2003. (2) Selinsky, B. S. Membrane protein protocols: expression, purification, and characterization; Humana Press: Totowa, NJ, 2003. (3) Seddon, A. A.; Curnow, P.; Booth, P. J. Biochim. Biophys. Acta 2004, 1666, 105–117. (4) Bayburt, T. H.; Grinkova, Y. V.; Sligar, S. G. Nano Lett. 2002, 2, 853–856. (5) Nath, A.; Atkins, W. M.; Sligar, S. G. Biochemistry 2007, 46, 2059–2069. (6) Grinkova, Y. V.; Denisov, I. G.; Bayburt, T. H.; Sligar, S. G. Biophys. J. 2004, 86, 252A–252A. (7) Denisov, I. G.; Grinkova, Y. V.; Lazarides, A. A.; Sligar, S. G. J. Am. Chem. Soc. 2004, 126, 3477–3487.

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Figure 1. Schematic illustration of nanodisc capture and cholera toxin subunit B (CTB) binding to sensorchips. A sensorchip with immobilized capture reagent, e.g., Ni-NTA, antibody, binds discs with an appropriate tag and ganglioside GM1 (middle) that interacts with CTB (right). Corresponding surface plasmon resonance sensorgrams are displayed below. The response measured in resonance units (RU) is linearly dependent on the mass bound to the sensorchip when there is no significant difference in the refractive index of the sample and running buffers and to sensor chips.

specifically engineered sites opens opportunities for the immobilization of membrane bilayers and membrane-associated molecules for affinity chromatography media and measurements on protein chips. By doing so the membrane and its receptors are not part of anchoring to the solid support, as is the case for vesicles bound by hydrophobic anchors or specific anchoring through binding of molecules embedded in the phospholipid bilayer. Another difference from other membrane capture methods is that lateral diffusion is limited to the area of the nanodisc as defined by the MSP. Thus, the oligomerization state of embedded receptors can be controlled by selecting the right phospholipid/ receptor ratio and the right length of MSP. In vesicles, on the other hand, the receptors are allowed to diffuse over much longer distances and are thus able to oligomerize to larger aggregates. The enterotoxin from Vibrio cholerae consists of one A subunit (Mr 27 kDa), responsible for ADP ribosylating catalysis, and five B subunits (Mr 11.6 kDa per monomer), which contain the binding region for the glycolipid receptor ganglioside GM1 (ganglioside GM1 (II3NeuAcGgOse4Cer)). Entoxication begins when the B subunits recognize and bind to the pentasaccharide moiety of GM1. The A subunit can enter the cell, ADP-ribosylating the signal transduction protein Gs-R. This modification abolishes GTP hydrolysis, and Gs-R remains constitutively active, increasing the (8) Bayburt, T. H.; Leitz, A. J.; Xie, G. F.; Oprian, D. D.; Sligar, S. G. J. Biol. Chem. 2007, 282, 14875–14881. (9) Alami, M.; Dalal, K.; Lelj-Garolla, B.; Sligar, S. G.; Duong, F. EMBO J. 2007, 26, 1995–2004. (10) Denisov, I. G.; Baas, B. J.; Grinkova, Y. V.; Sligar, S. G. J. Biol. Chem. 2007, 282, 7066–7076. (11) Shaw, A. W.; Pureza, V. S.; Sligar, S. G.; Morrissey, J. H. J. Biol. Chem. 2007, 282, 6556–6563. (12) Boldog, T.; Grimme, S.; Li, M. S.; Sligar, S. G.; Hazelbauer, G. L. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 11509–11514. (13) Bayburt, T. H.; Grinkova, Y. V.; Sligar, S. G. Arch. Biochem. Biophys. 2006, 450, 215–222. (14) Leitz, A. J.; Bayburt, T. H.; Barnakov, A. N.; Springer, B. A.; Sligar, S. G. BioTechniques 2006, 40, 601. (15) Duan, H.; Civjan, N. R.; Sligar, S. G.; Schuler, M. A. Arch. Biochem. Biophys. 2004, 424, 141–153.

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intracellular levels of cAMP. The consequence is fluid loss in the small intestine and, if untreated, death (see refs 21–23 for reviews). The affinity of the interaction between CT and GM1 has been explored using different techniques.24–27 The kinetics of the system has also been studied by surface plasmon resonance (SPR), immobilizing gangliosides by capture of vesicles to the dextran matrix of sensorchips, for example, via hydrophobic anchors or by creating supported membrane monolayers on a hydrophobic coating on the sensorchip.28–34 Recently, nanodiscs (16) Civjan, N. R.; Bayburt, T. H.; Schuler, M. A.; Sligar, S. G. BioTechniques 2003, 35, 556. (17) Bayburt, T. H.; Sligar, S. G. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6725– 6730. (18) Bayburt, T. H.; Carlson, J. W.; Sligar, S. G. Langmuir 2000, 16, 5993– 5997. (19) Bayburt, T. H.; Carlson, J. W.; Sligar, S. G. J. Struct. Biol. 1998, 123, 37–44. (20) Torchilin, V. P.; Weissig, V. Tiposomes: A Practical Approach; Oxford Press: Oxford, 2003. (21) Moss, J.; Vaughan, M. Curr. Top. Cell. Regul. 1992, 32, 49–72. (22) Fishman, P. H.; Pacuszka, T.; Orlandi, P. A. Adv. Lipid Res. 1993, 25, 165187. (23) Lencer, W. I.; Hirst, T. R.; Holmes, R. K. Biochim. Biophys. Acta 1999, 1450, 177–190. (24) Shi, J. J.; Yang, T. L.; Kataoka, S.; Zhang, Y. J.; Diaz, A. J.; Cremer, P. S. J. Am. Chem. Soc. 2007, 129, 5954–5961. (25) Lopez, P. H. H.; Schnaar, R. L. Methods Enzymol. 2006, 417, 205-220. (26) Janshoff, A.; Steinem, C.; Sieber, M.; el Baya, A.; Schmidt, M. A.; Galla, H. J. Eur. Biophys. J. 1997, 26, 261–270. (27) Turnbull, W. B.; Precious, B. L.; Homans, S. W. J. Am. Chem. Soc. 2004, 126, 1047–1054. (28) Cooper, M. A.; Hansson, A.; Lofas, S.; Williams, D. H. Anal. Biochem. 2000, 277, 196–205. (29) Catimel, B.; Scott, A. M.; Lee, F. T.; Hanai, N.; Ritter, G.; Welt, S.; Old, L. J.; Burgess, A. W.; Nice, E. C. Glycobiology 1998, 8, 927–938. (30) Ohlsson, P. A.; Tjarnhage, T.; Herbai, E.; Lofas, S.; Puu, G. Bioelectrochem. Bioenerg. 1995, 38, 137–148. (31) Puu, G. Anal. Chem. 2001, 73, 72–79. (32) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993, 9, 1361– 1369. (33) MacKenzie, C. R.; Hirama, T.; Lee, K. K.; Altman, E.; Young, N. M. J. Biol. Chem. 1997, 272, 5533–5538. (34) Kuziemko, G. M.; Stroh, M.; Stevens, R. C. Biochemistry 1996, 35, 6375– 6384.

have also been used for an SPR experiment of protein binding to phospholipids, however, only as equilibrium measurements.11 The scope of the present study is to investigate capture methods of nanodiscs and demonstrate the utility of nanodiscs in kinetic binding studies using SPR. For this purpose we immobilized ganglioside GM1-containing nanodiscs on sensorchips by different reversible capture methods and obtained kinetic and affinity constants for the interaction between these receptors and cholera toxin subunit B (CTB) by measuring SPR responses in the microfluidically controlled Biacore system. The measured values for the GM1-CTB interaction, after insertion of GM1 into nanodiscs, are in agreement with those reported previously. EXPERIMENTAL SECTION Materials. Ganglioside GM1 was obtained from Axxora, U.S.A. Cholera toxin B subunit (CTB) was obtained from Sigma. Dimyristoylphosphatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC) were purchased from Avanti Polar Lipids, U.S.A. Other solvents and chemicals were analytical grade from various manufacturers. Membrane Scaffold Protein Production. 6xHis-tagged MSP MSP1T2 was expressed in Escherichia coli and purified by Ni-NTA metal chelate chromatography as described in ref 7. Likewise, FLAG-tagged MSP was expressed in E. coli and purified by FLAG antibody affinity chromatography employing FLAG antibody coupled to agarose (Sigma, St. Louis, MO) according to the manufacturers recommendations. Nanodiscs Preparation. The method was modified from refs 7 and 35. Nanodiscs with varying degrees of ganglioside GM1 were prepared by mixing the desired ratios of DMPC (solubilized in chloroform) and GM1 (solubilized in a 1:1 mixture of chloroform/ methanol), keeping the total GM1 + DMPC molar amount constant. The mixture was then dried under a gentle stream of argon and placed under vacuum for at least 4 h to remove traces of organic solvents. The lipid film was then resolubilized in standard buffer (100 mM NaCl, 20 mM Tris-HCl, 1 mM EDTA, pH 7.4) supplied with 25 mM sodium cholate by sonication for 10 min at 50 °C. After cooling to room temperature MSP was added to obtain a final 1:80 MSP/(lipid + GM1) ratio. To remove detergent and thereby start assembly of the discs an equal volume of biobeads SM-2 (Biorad) was incubated with the mixture for at least 3 h at room temperature. To collect correctly assembled discs, 20 µL was separated on a Superdex 200 PC 3.2/30 gel filtration column (Pharmacia Biotech, Uppsala, Sweden) with standard buffer as eluent at a flow rate of 40 µL/min, and the major peak, eluting at 38 min, was collected. Surface Plasmon Resonance Analysis. Binding kinetics was determined by SPR using a Biacore 3000 instrument (Biacore AB, Uppsala, Sweden). HEPES-buffered saline (HBS), which contained 10 mM HEPES, pH 7.4, 150 mM NaCl, 3.33 mM EDTA, was used as running buffer. All buffers were filtered (0.22 µm) and thoroughly degassed. Nanodiscs were immobilized on research grade CM5, nitrilotriacetic acid (NTA), or streptavidin (SA) coated sensorchips (Biacore AB, Uppsala, Sweden). All analyses were performed at 25 °C. (35) Denisov, I. G.; McLean, M. A.; Shaw, A. W.; Grinkova, Y. V.; Sligar, S. G. J. Phys. Chem. B 2005, 109, 15580–15588.

Measurements with the NTA Sensor Chip. Immobilization. In each cycle for measurements employing the NTA chip, 0.5 mM NiCl2 was injected over both the sample and reference flow cells at 5 µL/min for 1 min followed by injection of nanodiscs containing DMPC or 98% DMPC with 2% GM1 for 4 min as described in the figure legends. To block Ni2+-dependent binding, 8000 resonance units (RU) of nanodisc without GM1 was immobilized in both flow cells, whereas 150 RU of DMPC/GM1 nanodiscs were immobilized in the sample flow cell only. For binding analysis of CTB it was injected in HBS at a flow rate of 20 µL/min across the two flow cells. After 4 min of binding the flow was changed to buffer without CTB to monitor the dissociation of the interaction, and finally the chip surface was regenerated by a 2 min pulse of 0.33 M EDTA. This treatment stripped off Ni2+ and associated nanodisc/CTB complexes, leaving the NTA surface ready for another cycle of coating with Ni2+, capture of nanodiscs, binding of CTB, and regeneration. Measurements with Antibody-Coated Chips. Antibodies anti-tetra-His (Qiagen, Hilden, Germany) or FLAG M2 (SigmaAldrich, St. Louis, MO) were immobilized covalently on CM5 sensorchips by amine coupling with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (Pierce, Rockford, IL) according to the guidelines of Biacore AB. To capture the discs they were injected at 5 µL/ min for 4 min over the sample flow cell, and subsequently CTB was injected for 4 min at 20 µL/min. To monitor the dissociation, the flow was changed to buffer without CTB, and finally the chip was regenerated by injection of two 1 min pulses of 50 mM glycine-HCl, pH 2.2, stripping off discs and CTB, leaving the antibody ready for another round of capture of discs, binding of CTB, and regeneration. Data Analysis. Data from the reference flow cell were subtracted from those of the sample flow cell to remove bulk responses caused by different refractive indexes of sample and running buffer. The baselines were adjusted to zero on the y (SPR response) axis and aligned to the injection time on the x (time) axis. In kinetic experiments each toxin binding response was then corrected for a drifts and other systematic variations by subtracting the response of a buffer injection substituting for a CTB injection. Data were analyzed using BIAevaluation software 4.1 by fitting the data globally to a Langmuir 1:1 interaction model. The equilibrium dissociation constants were calculated from the rate constants kd and ka as KD ) ka/kd. Toxin concentrations were calculated using the molecular weight of the pentameric B subunit. RESULTS AND DISCUSSION Toxin Binding Kinetics to Nanodiscs Immobilized on a Nickel Ion Coated Nitrilotriacetic Acid Sensorchip. To test the use of chip-based SPR biosensor technology we produced nanodiscs encircled by an MSP containing a 6xHis tag. The binding of His tags to Ni2+ chelated by NTA is widely used for purification of recombinant proteins,36 and we wanted to exploit this for nanodiscs immobilization over an NTA SPR-active sensorchip surface. SPR is a label-free optical technology that measures local refractive index changes in the vicinity of a dextran-coated (36) Sharma, S. K. In Affinity Separations, A Practical Approach; Matejtschuk, P., Ed.; IRL Press: Oxford, 1997.

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Figure 2. Binding of 20 nM CTB on Ni-NTA sensor chips without discs (panel A). Capture of 6xHis-tagged discs to Ni-NTA sensor chips followed by binding of 20 nM CTB to discs that contain 0% GM1 (panels B-D) and/or 2% GM1 (panels C and D). The injections of discs and CTB are indicated by double arrows. Ni2+ is injected between 60 and 120 s, and EDTA is injected between 1695 and 1815 s. The curve in panel D represents a close up of the sensorgram obtained from the sample flow cell shown in panel C after subtraction of the reference sensorgram from the reference flow cell shown in panel B.

sensorchip to which a receptor, in this case nanodisc-embedded, is covalently coupled or captured reversibly. By injections of analyte the interaction can be measured because the refractive index, and thus the measured SPR response, increases linearly with the mass bound to the sensorchip. The flow system of Biacore instruments can switch between injections of analyte and flow of buffer; consequently, both association and dissociation kinetics can be measured by plotting the responses against time.37,38 See Figure 1 for a schematic representation of an SPR binding experiment. During the initial trials of binding of CTB to nanodiscs, it was observed that a large amount of DMPC nanodiscs could be immobilized on the flow cells with an efficient and stable binding after activation of the sensorchip surface with Ni2+; however, a dominant proportion of the binding was independent of the presence of gangliosides (data not shown). To estimate if the major proportion of binding was caused by binding to Ni2+ or to (37) Fagerstam, L. G.; Frostellkarlsson, A.; Karlsson, R.; Persson, B.; Ronnberg, I. J. Chromatogr. 1992, 597, 397–410. (38) Karlsson, R.; Lo ¨fås, S. In Immobilized Biomolecules in Analysis, A Practical Approach; Cass, T., Ligler, F. S., Eds.; Oxford University Press, 1998.

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nanodiscs, 20 nM CTB was injected over the NTA chip after activation with Ni2+, but without nanodiscs. The toxin was found to bind directly to the chip surface as shown in Figure 2A. In fact, also in previous works CTB has been demonstrated to bind Ni2+.39 This effect must be avoided to be able to correctly analyze the kinetics of the CTB-ganglioside interaction without an error caused by the interaction between the toxin and Ni2+. To deal with the problem, high levels of receptor-free nanodiscs were captured on two flow cells with the intention to mask the Ni2+ from the toxin. On one of the two flow cells, the sample flow cell, nanodiscs containing 2% GM1 was captured as well. Thus, the flow cell without GM1 discs served as a reference to determine the degree of remaining Ni2+ binding. However, CTB still bound the control surface (Figure 2B), although to a lower extent than the surface containing GM1 discs (Figure 2C) and to a lower extent than to the surface without discs (Figure 2A). Thus, the (39) Dertzbaugh, M. T.; Cox, L. M. Protein Eng. 1998, 11, 577–581. (40) Karlsson, R.; Mo, J. A.; Holmdahl, R. J. Immunol. Methods 1995, 188, 63– 71. (41) Marushchak, D.; Gretskaya, N.; Mikhalyov, I.; Johansson, L. B. A. Mol. Membr. Biol. 2007, 24, 102–112.

Figure 3. Capture of discs with antibodies directed against tetra-His tags (panels A and B) or against FLAG tags (panels C and D) and binding of 20 nM CTB to discs that contain 0% GM1 (panels A and C) or 2% GM1 (panels B and D). The injections of discs and CTB are indicated by double arrows. The presented data are overlaid duplicates.

reference-subtracted curve shown in Figure 2D displays a positive slope during CTB injection. However the curve appears more complex than desired for kinetic analysis due to the remaining binding to the reference surface and the leakage of discs observed when high levels of discs are captured. Capture of Nanodiscs Immobilized via 6xHistidine Tags on Sensorchips Coated with Tetra-His Antibody. To alleviate the problem of analyte binding to Ni2+ we also investigated the possibility to capture 6xHis-tagged nanodiscs with immobilized antibodies directed against the 6xHis tag. An amount of 10.600 RU of tetra-His antibodies immobilized on the sensorchip by covalent amine coupling captured 576 RU of nanodiscs after a 4 min injection of 10 µg/mL nanodiscs that contained 2% GM1. Injections of 20 nM CTB over two flow cells, one with antibodies capturing GM1-nanodiscs and one with antibodies capturing nanodiscs without GM1, revealed that the captured GM1-nanodiscs bound 238 RU of CTB while not binding to the antibody capturing discs without GM1 (Figure 3, parts A and B). Thus, antibody-mediated capture of His-tagged nanodiscs seems to be a viable method for SPR studies. Capture of Nanodiscs Immobilized via FLAG Tags on Sensorchips Coated with FLAG Epitope Antibody. As an alternative to 6xHis tags we also tested a commercially available antibody against FLAG tags. The FLAG tag is an epitope consisting of the amino acid sequence DYDDDDK that was inserted in the C-terminus of the MSP surrounding the lipid bilayer of the nanodisc by genetic engineering. When 11.350 RU of FLAG antibody was immobilized on a sensorchip by covalent amine coupling it captured 468 RU of nanodiscs after a 4 min injection of 10 µg/mL nanodiscs that contained 2% GM1. Injections of 20 nM CTB over two flow cells, one with antibody capturing

GM1-nanodiscs and one with antibody capturing nanodiscs without GM1, revealed that the captured GM1-nanodiscs apparently bound 195 RU of CTB while not binding to the antibody capturing discs without GM1 (Figure 3, parts C and D). However, the FLAG-tagged nanodiscs leaked from the FLAG antibody as seen by the negative slop of the sensorgram (best seen in Figure 3C) when the injection of discs was stopped and the flow changed to buffer. Thus, a significant proportion of discs leaks from the surface during binding of CTB to the captured nanodiscs, increasing the complexity of kinetic or other quantitative analysis. Kinetics of CTB Binding to Nanodiscs with Different GM1/ DMPC Ratios. One of the advantages of nanodiscs is that the stoichiometry of receptors in nanodiscs can be controlled by careful preparation of the nanodiscs. The incorporated receptors are restricted by the area of the discs and thus cannot diffuse over large distances as is the case with receptors in vesicles. Thus, the aggregation state is expected to be controllable in nanodiscs.8,12,13 To test how different amounts of GM1 per nanodisc affected CTB binding, discs with GM1/DMPC ratios between 1:400 (0.25% GM1) and 1:12.5 (8%) were prepared in 6xHis-tagged MSP. Amounts of 270-500 RU of discs were captured on the sample flow cell of the sensorchip by the tetra-His antibody that we found was most likely to be reliable for quantitative analysis, whereas no discs were captured on the other flow cell that also contained antibody. Subsequently, binding experiments with 20 nM CTB and nanodiscs with different GM1/DMPC ratios were conducted. In order to correct for the different nanodisc immobilization levels, the obtained binding data were overlaid and normalized according to the level of immobilized discs. As seen in Figure 4, parts A and B, the amount of captured CTB correlated linearly with the GM1/ DMPC ratio so that twice the amount of CTB was bound when Analytical Chemistry, Vol. 80, No. 16, August 15, 2008

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the amount of GM1 per disk was doubled. To investigate if the CTB binding kinetics is independent of amount of GM1 per nanodisc, the data were further normalized to the maximum binding of CTB. If the kinetics is independent of the number of binding sites then the normalized curves should be overlapping. However, this is not the case; the association as well as the dissociation rates are both higher for the lower GM1/DMPC ratios compared to higher GM1/DMPC ratios (Figure 4C). This effect is most pronounced for the dissociation rates. To validate this observation CTB in concentrations ranging from 1.48 to 40 nM were injected over discs that contain 1% or 8% GM1. The data were then fitted to interaction models that describe the binding kinetics of a first order 1:1 interaction (Figure 5, parts A and C). The model with the association rate constant ka ) 8.4 × 105 M-1 s-1 and dissociation rate constant kd ) 4.7 × 10-4 s-1 and thus the equilibrium dissociation constant KD ) kd/ka ) 5.7 × 10-10 M describes the data 1% GM1 as shown in Figure 5A. The model with the association rate constant ka ) 2.3 × 105 and a dissociation rate constant that could not be measured due to the very slow dissociation describes the data for the 8% GM1 as shown in panel C of Figure 5. Because CTB is multivalent we also investigated an existing model for the binding of a bivalent analyte (AA) to monovalent ligands (B) immobilized on a surface.40 The bivalent analyte may bind one or two ligands where the first forward reaction has the association rate constant ka1 and the dissociation rate kd1 and the second reaction has the association rate constant ka2 and the dissociation rate constant kd2:

AA + B a AAB AAB + B a AABB The first equation is the major contribution to the SPR response, but the second can contribute to stabilization of the complex. We are aware that CTB is pentavalent, but the existing bivalent model is a good approximation of the first steps involved in the binding reaction. The bivalent analyte model with ka1 ) 2.3 × 105 M-1 s-1, kd1 ) 0.026, ka2 ) 1.9 × 103, kd2 ) 7.1 × 105 (Figure 5B) was a slightly better fit to the data with 1% GM1 discs than the 1:1 monovalent binding model, but both fitted acceptably (χ2 ) 0.47 for the 1:1 model and χ2 ) 0.31 for the bivalent model; χ2 describes the average squared residuals per point, χ2 < 10 is generally acceptable for Biacore data). Thus, the interaction kinetics of the discs with 1% incorporated GM1 can be described as monovalent and the CTB pentamers as mono- or bivalent. The bivalent analyte model with ka1 ) 6.1 × 104 M-1 s-1, kd1 ) 0.036 s-1, ka2 ) 9.7 × 105, kd2 ) 0 (Figure 5D) fitted slightly better than the 1:1 monovalent binding model to the data of the kinetic experiment with 8% GM1 discs. However, neither of the models was in good agreement with the kinetic experiment with 8% GM1 discs (χ2 ) 23 for the 1:1 model and χ2 ) 14) for the bivalent model). One explanation can be that GM1 ligands in discs with more than one GM1 molecule can not be considered monovalent, so it will appear as the reaction AA + BB a AABB

Figure 4. Amounts and kinetics of CTB binding to GM1 incorporated in nanodiscs vary with the GM1/lipid ratio. CTB is injected over 6xHistagged captured by approximately 10.000 RU of anti-tetra-His antibody immobilized on the sensorchip. (A) Overlaid sensorgrams of 20 nM CTB injected over approximately 300 RU of nanodiscs that contain GM1 ranging from 0.25% to 8% of DMPC. The sensorgrams are normalized according to the level of discs captured in each cycle. (B) Maximum response read from panel A plotted against the GM1/DMPC ratio. (C) Data from panel A further normalized to the maximum response of each binding curve. The higher GM1 percentage the darker shading. The sensorgrams in panels A and B represent two overlaid duplicates. 6250

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Figure 5. (A) Full lines: overlaid sensorgrams of 40, 13.3, 4.44, and 1.48 nM (top to bottom) CTB injected over approximately 300 RU of discs that contain 1% GM1. The presented sensorgrams are normalized according to the level of discs captured in each cycle and fitted to a 1:1 interaction model represented by dotted lines. (B) Same data as panel A but fitted to the bivalent model described in the text. (C) Full lines: overlaid sensorgrams of 40, 13.3, 4.44, and 1.48 nM (top to bottom) CTB injected over approximately 170 RU of discs that contain 8% GM1. The presented sensorgrams are normalized according to the level of discs captured in each cycle and fitted to a 1:1 interaction model described in the text and represented by dotted lines. (D) Same data as panel C but fitted to the bivalent model described in the text.

Table 1 immobilization strategy

gangliosides % of lipids

ka M-1 s-1

kd s-1

KD M

ref

nanodiscs nanodiscs hydrophobic anchors in dextran matrix hydrophobic monolayer LPS antibodies in dextran matrix hydrophobic adsorption to dextran matrix

1 8 0.1 5 2 100

8.4 × 105 2.0 × 105 6.2 × 105 1.3 × 106 6.2 × 105 N.D.b

4.7 × 10-4 N.A.a 1.60 × 10-4 5.00 × 10-4 4.50 × 10-4 N.D.

5.7 × 10-10 N.A. 2.6 × 10-10 4.6 × 10-12 7.3 × 10-10 1.9 × 10-7

present study present study 28 34 33 29

a

N.A.: not applicable because no dissociation could be observed within the time frame of the experiment. b N.D.: not determined.

There are on average 1.6 GM1 molecules per disk at 1% GM1 and 12.8 GM1 molecules per disk at 8% GM1. Thus, the probability that disk-incorporated GM1 will perform as multivalent ligands is increased for 8% GM1 discs. An overview of the kinetic constants obtained for the 1:1 interaction model in this study compared to other published studies is given in Table 1 because the other studies only reported kinetic constants for 1:1 interactions. The values obtained with 1% discs are in good agreement with the kinetic parameters previously determined for CTB-GM1 interaction. MacKenzie et al.33 found KD, ka, and kd values of 7.3 × 10-10 M, 6.2 × 105, and 4.5 × 10-4, respectively, using artificial liposomes that contained 2% glycolipid receptors and immobilized on the chip using an antibody capture technique. Cooper et al.28 studied the same system and measured values of 2.6 × 10-10 M, 6.2 × 105, and 1.6 × 10-4 for KD, ka, and kd, using liposomes containing 0.1% GM1 captured on a sensor chip via hydrophobic anchors. The kinetic constants that we obtained with the 8% discs match those of Kuziemko at al.34 that reported an affinity of 4.6 × 10-12 M, and

ka, and kd of 1.27 × 106 and 5 × 10-6, respectively, working with a sensor chip where the lipid was captured on a hydrophobic surface and 5% GM1 content in the phospholipids mixture. Presumably, the observed lower dissociation rates in discs with a higher number of GM1 molecules per disk are a consequence of stabilization by multivalent binding due to the pentavalency of CTB. To investigate if a higher number of binding sites were available when CTB was captured by discs with 1% GM1 than on discs with 8% GM1 we injected a solution of 2 µM GM1 over the immobilized discs after capturing CTB from a 40 nM solution. After a 4 min injection of free GM1 over 1% GM1 discs, an average of 14 RU (n ) 3, ±0.5 RU standard deviation) of GM1 bound per 100 RU of CTB. For the 8% discs 8.9 RU (n ) 3, ±0.1 RU standard deviation) of GM1 bound per 100 RU of CTB (data not shown). The higher amount of bound GM1 per CTB bound to discs with 1% compared to discs with 8% GM1 suggests that unoccupied GM1 binding sites are available when CTB is bound to discs with low GM1 incorporation. It also shows that binding sites of CTB bound to discs with 8% GM1 are already involved in multivalent binding. Following the Analytical Chemistry, Vol. 80, No. 16, August 15, 2008

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injection of free GM1 the dissociation rate of the CTB-free GM1 complex from the discs with 8% GM1 was increased. Unfortunately, we cannot distinguish if the apparent accelerated dissociation is caused by dissociation of free GM1 from CTB or by dissociation of CTB-free GM1 complex from the nanodiscs. The slightly decreased association rates in discs with a higher number of GM1 molecules per disk may be caused by GM1 clustering. A recent study investigated the effect of GM1 clustering on the equilibrium binding to CTB.24 In contrast to our observations, they observed that the apparent equilibrium dissociation constant increased with increasing GM1/lipid rations, i.e., the binding weakened. They suggested that the decreased binding is a consequence of clustering of GM1 molecules on the supported membranes. We did not investigate the discrepancy further, but it is likely to be an effect of the restriction of GM1 molecules in nanodiscs imposed by the limited diameter of the nanodiscs. Consequently, it is less likely that GM1 molecules cluster at lower GM1/lipid ratios in nanodiscs than in supported bilayers where the gangliosides can diffuse freely and cluster if they can selfagggregate. Additional support for GM1-GM1 aggregation is supported in ref 41. CONCLUSIONS In this work we demonstrate that nanodiscs and their incorporated membrane glycolipid receptors can be attached to Biacore sensorchips by different capture methods and used to measure the kinetic constants of the cholera toxin-GM1 interaction. We studied this well-known model system to explore and verify the potential of the association between nanodiscs and the SPR technique. Due to the stable binding and low nonspecific binding the best methods for capture are antibodies that recognize 6xHistagged MSP of the nanodiscs. The other tested capture systems had problems of leakage from the sensorchip (FLAG tag/FLAG tag antibody) or binding of the analyte to the capturing molecule

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(6xHis tags/Ni-NTA). For other interaction partners Ni-NTA capture might be suitable if the unspecific or irrelevant binding is caused by unspecific binding to, for example, the antibodies used for immobilization of the discs. In general, capture of the discs needs to be optimized for each specific interaction study. The measured kinetic values obtained in this study are in agreement with those found by previous studies on the interaction of the cholera toxin with the GM1 receptor embedded in different membrane systems. Thus, we have provided proof-of-principle that discoidal membrane bilayer nanodiscs can be anchored to solid supports and used for chip-based kinetic SPR studies of receptors embedded in the discs. One difference from other model membrane systems is the limited diffusion of lipids and receptors imposed by the restraints of the nanodisc as compared to liposomes and supported membrane systems. This restraint limits lateral diffusion and thus receptor-receptor interactions so that oligomerization is more controllable. In broader perspectives, the ability to anchor membrane bilayer discs with inserted receptors opens up the possibility to use them for applications such as screening for interaction partners of membrane receptors with chip-based technologies and optimization of procedures for subsequent isolation of interaction partners using affinity chromatography. ACKNOWLEDGMENT We are thankful to the members of the Sligar Laboratory, especially Drs. Grimme and Shaw for initial experiments and purification of membrane scaffold proteins. Financial support from the Human Frontiers in Science Programme (Grant No. RGP0065/ 2005-C) is acknowledged. Received for review January 10, 2008. Accepted April 15, 2008. AC8000644