Sterol Binding Assay Using Surface Plasmon Fluorescence

The artificial membrane setup is monitored in real time by surface plasmon ... Wiltschi , Vinzenz Kirste , Renate Naumann , Dieter Oesterhelt , Eva-Ka...
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Anal. Chem. 2006, 78, 547-555

Sterol Binding Assay Using Surface Plasmon Fluorescence Spectroscopy Birgit Wiltschi,*,† Michael Schober,‡ Sepp D. Kohlwein,§ Dieter Oesterhelt,† and Eva-Kathrin Sinner†

Department of Membrane Biochemistry, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany, and Institute of Molecular Biosciences, Microbiology Section, and Institute of Molecular Biosciences, SFB Biomembrane Research Center, University Graz, A-8010 Graz, Austria

We describe the design of a novel in vitro assay to study the interaction of soluble proteins with small hydrophobic sterol ligands. The sterol molecules are incorporated in an artificial membrane system in order to mimic their arrangement found in a biomembrane. The artificial membrane setup is monitored in real time by surface plasmon spectroscopy. Binding of fluorescently labeled soluble protein is observed by optical detection with surface plasmon enhanced fluorescence spectroscopy. By application of the novel assay, we demonstrate that four different oxidized sterol molecules are specifically recognized by the yeast protein Osh5p, a presumed oxysterol binding protein. Osh5p from yeast is the first oxysterol binding protein homologue for which oxysterol binding is shown with this new technique. With the design of our novel in vitro oxysterol binding assay, we have solved the technically challenging difficulty of presenting hydrophobic ligands to hydrophilic proteins in aqueous media. Sterols are small hydrophobic molecules that are essential for all eukaryotic organisms. In biomembranes, sterols serve their structural and functional roles by intercalating between the phospholipid fatty acyl chains. Oxidation of sterols is a ubiquitous process that occurs when they are exposed to air, heat, radiation, or enzymatic activities. Oxidized sterol derivatives, so-called oxysterols, are incorporated into biomembranes in a way similar to that of native sterols, but due to their altered chemical structure, they deleteriously affect cells: they are cytotoxic, mutagenic, and carcinogenic; they are membrane distorting and induce apoptosis.1 In 1985, an oxysterol binding protein (OSBP) was identified and was suggested to mediate the intracellular effects of oxysterols.2 Since then, various cytosolic OSBPs have been identified in different types of eukaryotic cells.3-9 While their cellular * To whom correspondence should be addressed. E-mail: wiltschi@ biochem.mpg.de. † Max Planck Institute of Biochemistry. ‡ Institute of Molecular Biosciences, Microbiology Section, University Graz. § Institute of Molecular Biosciences, SFB Biomembrane Research Center, University Graz. (1) Schroepfer, G. J., Jr. Physiol. Rev. 2000, 80, 361-554. (2) Taylor, F. R.; Kandutsch, A. A. Methods Enzymol. 1985, 110, 9-19. (3) Beh, C. T.; Cool, L.; Phillips, J.; Rine, J. Genetics 2001, 157, 1117-1140. (4) Alphey, L.; Jimenez, J.; Glover, D. Biochim. Biophys. Acta 1998, 1395, 159164. 10.1021/ac051388p CCC: $33.50 Published on Web 12/15/2005

© 2006 American Chemical Society

function is still largely unknown, mammalian OSBPs have been shown to interact with oxysterols in in vitro and in vivo binding assays.6,7,10-16 However, the oxysterol binding capacity of the seven OSBP homologues (OSHs) from the yeast Saccharomyces cerevisiae has not been studied yet. Thus, we decided to perform oxysterol binding assays in order to shed light on the oxysterol binding capacities of OSHs. In classical in vitro and in vivo oxysterol binding assays, tritiumlabeled 25-hydroxy-[3H]cholesterol has been used as the ligand of choice. Other oxysterols were tested by competitive displacement of bound 25-hydroxy-[3H]cholesterol.12 The sterol was dissolved in ethanol and added to the aqueous binding protein preparation.6,7,12,13 Unbound radiolabeled sterol was separated from the formed oxysterol/protein complex either by absorption to dextran-treated charcoal,6,7,10,12,13 by sucrose density gradient centrifugation,13,14,16 or, more recently, by size exclusion chromatography.11 All previous oxysterol binding assays introduced the sterol ligand in aqueous solution. Presenting the ligand in this way appears to be inefficient since sterols, e.g., cholesterol or ergosterol, with a lone hydrophilic hydroxyl group that represents less than 5% of the molecular mass are virtually water insoluble because exposure to water of the large nonpolar part would yield a very unfavorable contribution to the free energy.17 Sinensky18 (5) Levanon, D.; Hsieh, C. L.; Francke, U.; Dawson, P. A.; Ridgway, N. D.; Brown, M. S.; Goldstein, J. L. Genomics 1990, 7, 65-74. (6) Dawson, P. A.; Van der Westhuyzen, D. R.; Goldstein, J. L.; Brown, M. S. J. Biol. Chem. 1989, 264, 9046-9052. (7) Dawson, P. A.; Ridgway, N. D.; Slaughter, C. A.; Brown, M. S.; Goldstein, J. L. J. Biol. Chem. 1989, 264, 16798-16803. (8) Taylor, F. R.; Shown, E. P.; Thompson, E. B.; Kandutsch, A. A. J. Biol. Chem. 1989, 264, 18433-18439. (9) Beseme, F.; Astruc, M. E.; Defay, R.; Crastes de Paulet, A. FEBS Lett. 1987, 210, 97-103. (10) Xu, Y.; Liu, Y.; Ridgway, N. D.; McMaster, C. R. J. Biol. Chem. 2001, 276, 18407-18414. (11) Moreira, E. F.; Jaworski, C.; Li, A.; Rodriguez, I. R. J. Biol. Chem. 2001, 276, 18570-18578. (12) Taylor, F. R.; Saucier, S. E.; Shown, E. P.; Parish, E. J.; Kandutsch, A. A. J. Biol. Chem. 1984, 259, 12382-12387. (13) Kandutsch, A. A.; Shown, E. P. J. Biol. Chem. 1981, 256, 13068-13073. (14) Kandutsch, A. A.; Thompson, E. B. J. Biol. Chem. 1980, 255, 10813-10821. (15) Kandutsch, A. A.; Chen, H. W.; Heiniger, H. J. Science 1978, 201, 498501. (16) Kandutsch, A. A.; Chen, H. W.; Shown, E. P. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 2500-2503. (17) Huang, J.; Feigenson, G. W. Biophys. J. 1999, 76, 2142-2157. (18) Sinensky, M. Arch. Biochem. Biophys. 1981, 209, 321-324.

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compared the water solubility of cholesterol and 25-hydroxycholesterol and reported that 25-hydroxycholesterol, which features a second hydroxyl group, exists as a monomer in aqueous solution at physiologically active concentrations. In contrast, the water solubility of cholesterol is much lower so that this molecule would always exist in a membrane-bound or micellar form.18 From these observations, the solubility of different oxysterols in aqueous solution may be expected to vary, which might alter the availability of the various oxysterols to be tested in the present study. Therefore, our principal aim was to develop a novel in vitro oxysterol binding assay that can be performed with any sterol and that provides it in an environment that mimics the arrangement of the sterol molecules found in a biomembrane. For this, we employed artificial peptide tethered membranes, a novel class of model membranes that qualify as an immensely useful tool for biochemical assays as they mimic biomembranes in many respects.19 Such synthetic membrane systems have successfully been used to investigate the biophysical properties of phospholipid/sterol mixtures, e.g., their viscoelastic properties and phase separation behavior. The immobilization of lipids, e.g., sterols, in artificial membranes, as would be necessary for studying interaction of these molecules with native proteins, is a new approach. Therefore, the oxysterols ergosterol epidioxide (EEP), 9(11)dehydroergosterol (DHE), and 5R,6R-epoxy-(22E)-ergosta-8,22dien-3β,7R-diol (8-DED), the nonoxidized main yeast sterol ergosterol, and the mammalian 25-hydroxycholesterol (25OH) were incorporated in an artificial peptide tethered membrane consisting of L-R-phosphatidylcholine (PC). We introduced surface plasmon resonance spectroscopy (SPS) to monitor the setup of this artificial tethered membrane in real time. Surface plasmon fluorescence spectroscopy (SPFS) was used to detect the interaction of a fluorescently labeled soluble protein with the incorporated sterols. Applying this novel sterol binding assay, we demonstrate that Osh5p, a presumed oxysterol binding protein from yeast, binds to the tested oxysterols. EXPERIMENTAL SECTION Yeast Strains, Culture Media, and Materials. Escherichia coli strains were grown in LB medium (5 g/L yeast extract (BD Difco, Franklin Lakes, NJ), 10 g/L tryptone (BD Difco), 5 g/L NaCl (Merck KGaA, Darmstadt, Germany)), plasmids were propagated following standard transformation procedures,20 and ampicillin-resistant transformants were selected on LB medium with 100 mg/L ampicillin (Amresco, Solon, OH). Yeast strains were grown in YPD (10 g/L yeast extract (BD Difco), 20 g/L peptone (BD Difco), 20 g/L glucose (Merck)) medium at 30 °C. Transformation was carried out by the lithium acetate method21 in the Saccharomyces cerevisiae strain cl3-ABYS-86 (MatR pra1-1 prb1-1 prc1-1 cps1-3 ura3∆5 leu2-3,112 His-). The yeast strains BPY15 (cl3-ABYS-86 {pYEX4Tps-OSH5}) and BPY19 (cl3-ABYS86 {pYEX4Tps}) were generated for the expression of GST-Osh5p and GST, respectively. Transformants were selected on appropriate synthetic media (6.7 g/L yeast nitrogen (19) Sinner, E.-K.; Knoll, W. Curr. Opin. Chem. Biol. 2001, 5, 705-711. (20) Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidmann, J. G., Smith, J. A., Struhl, K., Eds. Current Protocols in Molecular Biology; John Wiley & Sons Inc.: Hoboken, NJ, 1987. (21) Gietz, R. D.; Woods, R. A. Methods Enzymol. 2002, 350, 87-96.

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base (BD Difco), 10 g/L glucose (Merck)) supplemented with all amino acids and nucleic acid bases (Merck) except uracil or leucine. For solid media plates, 20 g/L agar (BD Difco) was added. Plasmid pYEX4T-1 was purchased from Clontech Laboratories Inc. (Palo Alto, CA). Plasmid pAZ7 was kindly provided by Andriy Zakalskiy (University of Graz). Ergosterol and 25-hydroxycholesterol were from Fluka (Buchs, Switzerland). Ergosterol epidioxide, DHE, and 8-DED were kindly provided by Kevin Barrow (University of New South Wales). Glass beads (diameter 0.4-0.6 mm) were purchased from Sigma. Restriction enzymes and T4 DNA ligase were from New England Biolabs (Beverly, MA). Unless otherwise indicated, all other chemicals were from Sigma or Merck. Gene Expression and Protein Preparation. The GST fusion protein expression vector pYEX4Tps was constructed by excising the insert and 483 bp downstream on the pYEX4T-1 descendant plasmid pAZ722 with BamHI and KpnI and religation with the original pYEX4T-1 multiple cloning site cut with the same enzymes. pYEX4Tps contains the PreScission Protease cleavage site from plasmid pAZ7 instead of the original thrombin cleavage site of pYEX4T-1. A full-length GST fusion construct of Osh5p was obtained by subcloning the 1330-bp BamHI-SalI fragment encompassing OSH5 from plasmid pEG(KT)-OSH5 (kindly provided by Christopher Beh, University of California, Berkeley) into pYEX4Tps cleaved with the same restriction enzymes, yielding plasmid pYEX4Tps-OSH5. Main cultures of 50 mL of leucine-free synthetic medium in a 500-mL baffled flask were inoculated with 0.5 mL of a 48-h preculture and incubated at 30 °C with vigorous shaking for 1820 h. Cells were harvested (3000g, room temperature, 5 min) under sterile conditions and resuspended in 50 mL of fresh, prewarmed leucine-free medium. The cells were again cultured in 500-mL baffled flasks at 30 °C with vigorous shaking. After 2 h, gene expression was induced by addition of CuSO4 to a final concentration of 0.5 mM. The optimum induction period was determined empirically. After 4-h induction of GST-Osh5p or GST expression, cells were harvested by low-speed centrifugation (3000g, room temperature, 5 min), washed once with deionized water, and transferred to thick-walled glass tubes. Cell pellets were frozen at -20 °C until used for protein isolation. For the isolation of soluble proteins, the frozen cell pellets were mixed with 1 mL of glass beads/g of cell wet weight and 2 mL of phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4‚7H2O, 1.4 mM KH2PO4, pH ∼7.3) per mL of glass beads. Cells were disrupted by vigorous mixing (1 min) and chilling on ice (1 min). Mixing and chilling were repeated five times. Glass beads and cell debris sedimented during chilling on ice, and the supernatant was transferred to a centrifuge vessel. The remaining glass beads were washed once with 500 µL of PBS/ mL of glass beads and the supernatants pooled. Insoluble cell debris was sedimented by high-speed centrifugation (∼20000g, 4 °C, 20 min) and the clear supernatant transferred to a fresh tube. The protein lysate was used immediately for subsequent applications. (22) Zakalskiy, A.; Hogenauer, G.; Ishikawa, T.; Wehrschutz-Sigl, E.; Wendler, F.; Teis, D.; Zisser, G.; Steven, A. C.; Bergler, H. J. Biol. Chem. 2002, 277, 26788-26795.

Binding Assays Using Surface Plasmon Resonance Spectroscopy and Surface Plasmon Fluorescence Spectroscopy. For SPS measurements, surface plasmon resonance was excited in the so-called Kretschmann configuration. In this setup, a prism was used to couple the p-polarized light into a thin, typically, ∼4550-nm-thick gold film evaporated onto a glass slide (“gold chip”; Berliner Glas KGaA, Berlin, Germany), which was then indexmatched (Cargille Laboratories, Cedar Grove, NJ) to the base of the prism. The energy coupling in the surface plasmon was observed as a deep minimum in the reflectivity of the p-polarized light as the surface plasmon resonance angle of incidence was incremented. Reflectivity versus angle of incidence scans, i.e., plasmon scans, yielded the optical thickness of the adsorbed coating. Binding kinetics were recorded by monitoring the reflected light intensity at a fixed angle of incidence as a function of time. For SPFS measurements, the Cy5 fluorophore (absorption maximum at 650 nm) was excited with a helium-neon laser (JDS Uniphase Corp., San Jose, CA) at a wavelength of 632.8 nm. The experimental setup for SPS and SPFS was described in detail by Liebermann and Knoll.23 All experiments were performed at room temperature. Ergosterol, 8-DED, DHE, EEP, and 25OH were employed as sterol substrates for binding by incorporation into protein-tethered artificial membranes. For the preparation of the protein-tethered membrane, 5 µg of anchor peptide (CSRARKQAASIKVAVSADR; Cys-Laminin A chain 2091-2108, Sigma; aqueous solution), acting as an anchor for the artificial membrane on the gold surface, was applied onto the gold chip (15-20 min). After rinsing with 1 mL of water, the carboxy groups of the peptide were activated for amino coupling in 500 µL of a solution containing 0.2 M N-ethylN′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC; Fluka) and 0.05 M N-hydroxysuccinimide (NHS; Fluka) for 10 min. A hydrophobic surface was obtained by addition of 500 µg of dimyristoyl-L-R-phosphatidylethanolamine (DMPE; Sigma) dissolved in PBS with 0.1% Triton-X100 (Sigma; 30-60 min). Excess EDC/NHS and DMPE were removed by rinsing the surface once with 1 mL of water. Mixed vesicles containing PC (from soybean, type III-S; Sigma) and the sterol of interest were prepared by mixing the chloroform-dissolved lipids in equal amounts (w/w). The solvent was evaporated under a stream of nitrogen and the lipids resuspended in PBS to a final concentration of 1 mg/mL. Vesicles were formed in an extruder (Avestin Inc., Ottawa, Canada) equipped with a polycarbonate membrane (50-nm pore size; Avestin). Typically, 500 µL of the vesicle suspension was applied onto the hydrophobic surface on the gold chip and spread spontaneously (30-60 min). Excess vesicles were discarded by rinsing the membrane with 1 mL of PBS. Protein lysates, i.e., 100 µg of soluble protein prepared each from the GST-Osh5p and GST expressing strains, were reacted with the artificial PC/sterol membrane until a steady state was monitored (30-60 min). Nonspecifically bound proteins were removed by rinsing with 1 mL of Western Blocking Reagent (Roche) in PBS. Surface-bound GST fusion proteins were specifically identified with anti-GST antibody from goat (Amersham Biosciences AB, Uppsala, Sweden; 30 min, diluted 1:1000 in Western Blocking Reagent in PBS). Using an anti-goat Cy5-labeled (23) Liebermann, T.; Knoll, W. Colloids Surf., A 2000, 171, 115-130.

Figure 1. Representative SPS kinetics illustrating the setup of an artificial membrane tethered to a gold surface. Single steps of the assembly kinetics (1-7) are described in the cartoon.

antibody (Chemicon International Inc., Temecula, CA; 30 min, diluted 1:10 in Western Blocking Reagent in PBS), the SPFS technique was employed to measure the emitted fluorescence of the bound, labeled antibody. During incubation with the secondary Cy5-labeled antibody, the laser was shut off in order to avoid photobleaching of the fluorophore. Unspecifically bound anti-goat Cy5-labeled antibody was removed by rinsing three times with 1 mL of Western Blocking Reagent in PBS. The kinetics of anchor peptide binding, EDC/NHS activation, DMPE coupling, vesicle spreading, and binding of protein lysate were recorded at a fixed angle of incidence starting at 30% reflectivity. Plasmon and fluorescence scans were recorded from the gold surface exposed to air and water in order to characterize the gold layer. In addition, fluorescence scans were recorded after incubation with the protein lysate, after addition of the anti-GST and the anti-goat Cy5-labeled antibody, and after the three final rinses with Western Blocking Reagent in PBS. Fluorescence data were evaluated by subtracting the fluorescence scan after addition of the anti-GST antibody (background fluorescence) from the fluorescence scan after the three final rinses (specific fluorescence) to yield a fluorescence difference scan. The reflectivity increase for each layer was deduced from corresponding SPS kinetics as follows (refer to Figure 1): anchor peptide from steps 2 to 3; DMPE from steps 3 to 5; PC/oxysterol membrane from steps 6 to 7. Layer thickness was calculated by Analytical Chemistry, Vol. 78, No. 2, January 15, 2006

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quantitative modeling (WINSPALL software, Max Planck Institute for Polymer Research, Mainz, Germany) of the corresponding plasmon scans with Fresnel’s equations.24 Refractive indices for peptide and lipid layers were assumed n ) 1.45 and n ) 1.5, respectively. Miscellaneous Methods. Protein was quantified with the BioRad Laboratories (Hercules, CA) protein assay based on the Bradford dye binding procedure25 using bovine serum albumin as a standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by the method of Laemmli.26 Since nonreduced total protein lysates were used for oxysterol binding studies, proteins were separated by SDS-PAGE under nonreducing conditions. Western blot analysis was performed as described previously.27 Immunoreactive GST was detected by ELISA using commercially available anti-GST IgG from goat (Amersham Biosciences AB) as the primary antibody and a secondary, horseradish peroxidase linked anti-(goat IgG) IgG (Bio-Rad Laboratories). After chemiluminescent detection (Pierce, Rockford, IL) of GST-Osh5p and GST on the Western nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany), the blots were exposed to Kodak X-OMAT film (Sigma). The films were scanned and the band intensities determined using E.A.S.Y. Win32 analysis software (Herolab, Wiesloch, Germany). RESULTS Setup of a Tethered Artificial Membrane: Immobilizing the Sterol Ligands. Our intention to mimic a native environment for hydrophobic sterol molecules localized in biomembranes was realized by the use of an artificial phospholipid bilayer that was attached to a gold surface by a peptide spacer and that contained the sterols to be tested. SPS allowed real-time monitoring of each single setup step and of the multiple layer architecture of the tethered membrane. A typical kinetics curve illustrating the different steps for tethering an artificial membrane to a gold surface is shown in Figure 1. A short hydrophilic anchor peptide of the sequence CSRARKQAASIKVAVSADR was attached to the gold surface (Figure 1, step 1) through chemisorption of the thiol group on its amino-terminal cysteine moiety (Figure 1, step 2). Since sulfur has a high affinity for gold,28-30 the peptide molecules formed a layer that was firmly attached to the surface, with their carboxy termini remaining accessible to subsequent chemical coupling. Peptide layer formation yielded a mean SPS reflectivity increase of 5 ( 2%. The corresponding layer thickness was calculated to be 16 ( 7 Å by mathematical simulation, where the peptide layer on the gold surface was assigned a refractive index of n ) 1.4531 (see Experimental Section for layer thickness evaluation). The (24) Raether, H. In Springer Tracts in Modern Physics; Ho ¨hler, G., Niekisch, E. A., Eds.; Springer-Verlag: Berlin, 1988; Vol. 111, pp 4-39. (25) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254. (26) Laemmli, U. K. Nature 1970, 227, 680-685. (27) Haid, A.; Suissa, M. Methods Enzymol. 1983, 96, 192-205. (28) Wagner, P.; Hegner, M.; Kernen, P.; Zaugg, F.; Semenza, G. Biophys. J. 1996, 70, 2052-2066. (29) Sellers, H.; Ulman, A.; Shnidman, Y.; Eilers, J. E. J. Am. Chem. Soc. 1993, 115, 9389-9401. (30) Bain, C. D.; Troughton, E. B.; Tao, Y. T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335. (31) Bunjes, N.; Schmidt, E. K.; Jonczyk, A.; Rippmann, F.; Beyer, D.; Ringsdorf, H.; Graber, P.; Knoll, W.; Naumann, R. Langmuir 1997, 13, 3, 6188-6194.

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Figure 2. Chemical structures of the sterols used in this study. Abbreviations given in parentheses; MW, molecular weight.

“inner” leaflet of the artificial membrane was obtained by chemical coupling of the anchor peptide with DMPE via the primary amine of the ethanolamine moiety. For this amino coupling, the carboxy termini of the anchor peptide were activated with EDC/NHS. The addition of the EDC/NHS solution evoked a substantial increase in reflectivity (Figure 1, step 3). This effect, however, is caused by a shift in the refractive index and does not represent mass transfer since reflectivity decreased again after coupling of DMPE and rinsing with buffer, due to a similar refractive index shift (Figure 1, steps 4 and 5). The physical layer thickness increase caused by DMPE coupling was 4 ( 3%, which corresponds to 8 ( 4 Å, assuming a refractive index of n ) 1.5 for the lipid layer.31 DMPE coupling provides the highly hydrophobic surface necessary for subsequent vesicle spreading. To prepare a “sterolfunctionalized” lipid membrane, one of the five sterols, the structures of which are given in Figure 2, was incorporated into vesicles containing PC. These PC/sterol vesicles spread on the DMPE by spontaneous fusion and formed the “outer” leaflet of the artificial membrane that is in contact with the bulk aqueous solution (Figure 1, step 6). Ergosterol, EEP, DHE, 8-DED, and 25OH were incorporated into the artificial membranes at a ratio of 1:1 (w/w) with PC. This ratio was chosen because sterols are highly abundant in eukaryotic plasma membranes comprising up to half of all the lipids.32,33 Spreading of the different PC/sterol vesicles yielded lipid layers of varying thickness (Table 1), ranging from 12 ( 6 (PC/25OH) to 33 ( 3% (PC/DHE) reflectivity increase, which corresponds to 30 ( 8 and 86 ( 19 Å, respectively. PC/sterol layer thicknesses were calculated using a refractive index of n ) 1.5 for the lipid layers31 and are summarized in Table 1. (32) Zinser, E.; Paltauf, F.; Daum, G. J. Bacteriol. 1993, 175, 2853-2858. (33) Bloch, K. In Biochemistry of Lipids, Lipoproteins and Membranes; Vance, D. E., Vance, J. E., Eds.; Elsevier: Amsterdam, 1991; pp 363-381.

Table 1. Thicknesses of the Individual Layers of Tethered Artificial Membranesa layer

reflectivity increase (%)

layer thickness (Å)

anchor peptide DMPE PC/25OH PC PC/ergosterol PC/EEP PC/8-DED PC/DHE

5(2 4(3 12 ( 6 15 ( 10 17 ( 6 22 ( 12 29 ( 6 33 ( 3

16 ( 7 8(4 30 ( 8 48 ( 27 41 ( 26 53 ( 21 68 ( 11 86 ( 19

a Reflectivity increases were deduced from SPS kinetics; layer thicknesses were calculated from plasmon scans as described in the Experimental Section. Data represent mean ( SD of at least three independent experiments for each layer.

Figure 3. Western blot of GST-Osh5p and GST. 1, GST (26 kDa); 2, GST-Osh5p (77 kDa). A total of 30 µg of soluble yeast protein was loaded per lane and separated by SDS-PAGE under nonreducing conditions. GST-Osh5p and GST were detected using a polyclonal anti-GST antibody. Molecular masses of standard proteins are indicated on the left margin.

These results show that we were able to immobilize sterols by incorporation in an artificial PC membrane thereby generating a surrounding that mimics their native arrangement. Expression of Osh5p as a GST-Fusion Protein in Yeast. The Cu2+-inducible expression vectors, pYEX4Tps and pYEX4TpsOSH5, were constructed for high-yield expression of GST and GST-Osh5 fusion protein, respectively, in the yeast S. cerevisiae. The molecular weight of the expressed full-length GST-Osh5p was estimated to be 77 000. GST has a calculated molecular mass of 26 kDa and was introduced in the assay as a negative control since it was anticipated not to interact with sterols. GST-Osh5p and GST were soluble as they were highly enriched in membrane-depleted cell lysates, and on a Western blot they showed apparent molecular weights in agreement with the predicted values (Figure 3). Relative expression levels of GST-Osh5p and GST were quantified by densitometry of the bands on the Western blot shown in Figure 3. GST (set to 100%) was expressed at an ∼2fold higher level than GST-Osh5p (41%). To rule out unspecific binding, the negative control GST was introduced in the binding assays at twice the amount of GST-Osh5p. Oxysterol Binding Studies. We used soluble protein extracts containing GST-Osh5p or GST for the interaction studies with the tethered artificial membranes with incorporated oxysterol

Figure 4. Model of the specific detection of GST-Osh5p bound to a tethered artificial membrane using SPFS. GST tags are recognized by anti-GST/Cy5-labeled anti-IgG antibody complexes. The fluorescence of the Cy5-labeled secondary antibody is enhanced only within the evanescent field of the plasmon in proximity to the gold surface, i.e., within ∼1000 Å perpendicular to the gold layer. Fluorescence of labeled GST-Osh5p/anti-GST antibody complexes bound to the membrane is enhanced, whereas antibody complexes in the bulk solution are negligibly excited. The size of the evanescent field relative to the membrane with the bound protein complexes is not to scale.

molecules. Since oxysterol binding conditions for yeast OSHs have not been assessed so far, we did not apply purified GST-Osh5p to avoid loss of putative binding cofactors. Moreover, specific detection of binding events of a single protein species contained in the polypeptide mixture of soluble protein extracts is an important feature of our novel oxysterol binding assay. While SPS is insufficient to give evidence for the specific binding of GSTOsh5p from soluble protein extracts to a particular sterol in the membrane, SPFS offers the option to monitor this interaction using a fluorescently labeled protein. For this specific detection with SPFS (see model in Figure 4), an anti-GST antibody was employed that recognizes the GST tag and a secondary Cy5-labeled anti-IgG antibody that binds to the primary anti-GST antibody. The fluorescence of this secondary antibody is enhanced only within the evanescent field of the plasmon in proximity to the gold surface (Figure 4). Thus, enhanced fluorescence is detected only from labeled GSTOsh5p/anti-GST antibody complexes bound to the membrane, whereas antibody complexes in the bulk solution are negligibly excited (Figure 4). The interaction between GST-Osh5p or GST and the artificial bilayers with the particular incorporated sterol molecules was analyzed simultaneously with SPFS and SPS. Plasmon or fluoresAnalytical Chemistry, Vol. 78, No. 2, January 15, 2006

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Figure 5. Binding studies of GST-Osh5p and GST with different oxysterols incorporated in artificial tethered PC membranes. (a-f) Plasmon scans, GST-Osh5p (0), GST (O); fluorescence scans, GST-Osh5p (9), GST (b). Interactions of GST-Osh5p and GST with (a) PC/erg; (b) PC/DHE; (c) PC/EEP; (d) PC/8-DED; and (e) PC/25OH. (f) Interaction of GST-Osh5p with PC. Fluorescence scans of GST-Osh5p and GST interacting with PC membranes that contain different sterols are summarized in (g) and (h), respectively. For clarity, every fourth data point is shown. The relative positions of the plasmon minimums on the y-axis are dependent on the optical properties of the gold layer. Relative positions on the x-axis reflect layer thickness. Representative experiments are shown. 552 Analytical Chemistry, Vol. 78, No. 2, January 15, 2006

cence scans were obtained by recording reflectivity or fluorescence intensity, respectively, over increment angles. The fluorescence scans in Figure 5 represent difference scans of the angledependent plasmon-enhanced fluorescence emitted before and after the addition of Cy5-labeled antibody. Plasmon (open symbols) and fluorescence scans (full symbols) of GST-Osh5p (squares) versus GST (circles) interacting with different sterols are shown in Figure 5a-e. Distinct relative positions of plasmon minimums and respective fluorescence maximums on the x-axis reflect the varying layer properties in different experiments due to the fact that independent tethered membranes were prepared on separate gold-layered glass slides. The slight shift of the fluorescence maximum relative to the corresponding plasmon minimum angle observed in Figure 5b-e can be explained by inhomogeneous distribution of dye molecules bound to the surface. In this case, the fluorescence maximum is observed at a smaller angle than the reflectivity minimum of the surface plasmon. Negative fluorescence values were obtained when high background fluorescence was subtracted from lower observed fluorescence signals. GST (full circles) did not show a fluorescence signal with any of the artificial bilayers (Figure 5ae, summarized in Figure 5h). We observed that interaction of GST-Osh5p (full squares) with 25OH yielded a strong fluorescence signal (Figure 5e), which was usually more intensive than that excited by binding of GST-Osh5p to the yeast oxysterols DHE, EEP, and 8-DED (Figure 5b-d) or ergosterol (Figure 5a). The fluorescence scans from interactions of GST-Osh5p with tethered artificial membranes containing different sterols are summarized in Figure 5g. In general, absolute fluorescence values differed noticeably. To rule out unspecific binding of GST-Osh5p to the artificial membrane, a control experiment was performed using an artificial membrane consisting exclusively of PC. No binding of GSTOsh5p to the PC membrane was observed in the absence of sterols (see Figure 5f). In summary, we established a novel sterol binding assay based on artificial membranes and SPFS detection of binding events that represents a reliable method to investigate interactions of soluble macromolecules with their small hydrophobic ligands. As proof of principle, we were able to demonstrate the oxysterol binding capacity of Osh5p.

DISCUSSION In the present study, we describe the design of a new strategy for the detection of interactions between soluble proteins, e.g., Osh5p, and highly hydrophobic molecules, i.e., oxysterols. Therefore, we set up a novel biomimetic sterol binding assay that involved application of combined SPS/SPFS analyses to tethered artificial membranes containing oxysterols. SPS kinetic measurements allowed the visualization of membrane formation and the potentials of SPFS enabled us to specifically detect binding events of a single protein species, here Osh5p contained in the polypeptide mixture of cell lysate, to oxysterols of interest. Biacore (Uppsala, Sweden) has commercialized efficient surface chemistries together with SPS for biomolecular interaction analysis. However, Biacore technology does not exploit SPFS and is, therefore, inapplicable to the described oxysterol binding assay.

Performing a “classical” binding assay that introduced radiolabeled 25OH in aqueous solution, we were unable to obtain clear results on the interaction of GST-Osh5p with oxysterols (B.W., unpublished observation). Therefore, we applied the novel binding assay to elucidate whether the yeast Osh5p binds oxysterols as do the mammalian OSBPs. A GST-Osh5p fusion protein was successfully expressed in yeast cells. Soluble protein lysate containing the GST-Osh5 fusion protein was isolated from yeast and used in the binding assays without further purification. The GST tag allowed specific detection with an anti-GST antibody. The results from the SPS/SPFS sterol binding assays clearly demonstrate that Osh5p functions as an oxysterol binding protein in vitro. Osh5p is the first yeast OSH for which oxysterol binding is shown. The artificial tethered membrane setup occurs in a selfassembly process starting with the formation of a peptide layer on a gold surface. In the present study, we applied the 19-amino acid anchor peptide CSRARKQAASIKVAVSADR with good results. The peptide was chosen because of its good water solubility and the thiol moiety of the amino-terminal cysteine residue that allowed self-assembly of the peptide molecules on the gold surface. The peptide layer was calculated to be 16 ( 7 Å thick, a thickness that disfavors multilayer formation.31 Freely accessible carboxy groups within the peptide matrix were activated with a mixture of EDC and NHS to form Nhydroxysuccinimide esters. These esters undergo a spontaneous chemical reaction with nucleophilic amine groups as present, e.g., in the subsequently coupled DMPE, to form covalent links.34 DMPE was introduced into the reaction in a detergentsolubilized form. Most probably, DMPE forms mixed micelles with the detergent since the detergent was present in concentrations 1 order of magnitude higher than its critical micelle concentration.35 These mixed detergent/phospholipid micelles might be linked to the anchor peptide layer by amine coupling of their DMPE molecules rather than single phospholipid molecules. In aqueous buffer, the activated carboxy termini are eventually deactivated by hydrolysis of the N-hydroxysuccinimide esters; however, hydrolysis occurs slower than amine coupling.36 The hydrophobic myristoyl residues of DMPE avoid contact with the bulk aqueous solution; thus, they most probably do not protrude perpendicularly away from the surface but more likely form a nonordered layer that might contain residual detergent. The 8 ( 4 Å thick DMPE layer appears much thinner than a lipid monolayer of the expected theoretical thickness of 20-25 Å. The small thickness increment found after DMPE coupling supports the notion that the fatty acyl chains of the phospholipid are not perpendicularly arranged. Moreover, it might reflect inhomogeneous patchlike attachment of the DMPE molecules since SPS yields only laterally averaged thicknesses. Nonetheless, contact angle measurements indicated that lipid coupling generates a hydrophobic surface that allows vesicle spreading (data not shown). Techniques with high lateral resolution, such as AFM, will unveil the arrangement of DMPE upon coupling to the peptide tether. (34) Johnsson, B.; Lo¨fas, S.; Lindquist, G. Anal. Biochem. 1991, 198, 268-277. (35) Neugebauer, J. M. Methods Enzymol. 1990, 182, 239-253. (36) Grabarek, Z.; Gergely, J. Anal. Biochem. 1990, 185, 131-135.

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The artificial membrane was completed by fusion of PC/ oxysterol vesicles with the DMPE layer that had been generated on top of the anchor peptide. Performing an appropriate number of repetitive experiments, we achieved reasonable reproducibility for layer formation by vesicle spreading (Table 1). Nevertheless, the thickness of the PC/oxysterol layer varied noticeably with the different incorporated oxysterols (see Table 1 for details). As SPS is an optical method, a simple explanation for the varying layer thicknesses would arise from discriminate refractive indices of these oxysterols. However, the change of the refractive index in a dilute sterol solution with changing concentration, i.e., the specific refractive index increment dn/dc, is very similar for cholesterol (dn/dc ) 0.0803 mL/g), ergosterol (dn/dc ) 0.0928 mL/g), and 25OH (dn/dc ) 0.0895 mL/g). From these findings, we assumed similar dn/dc values for the oxysterols used in this study. Thus, the refractive index of an oxysterol incorporated in our tethered membrane apparently does not account for the differing PC/oxysterol layer thicknesses observed by SPS. The different sterol molecules might influence vesicle spreading, e.g., by exerting a more or less strong condensing effect on PC, thereby influencing the membrane fluidity of the vesicles.37 For certain PC/oxysterol mixtures, this could lead to pronounced adhesion of undamaged vesicles instead of or in parallel to vesicle fusion yielding layers whose thicknesses vary with the incorporated oxysterol. Performing SPFS analysis on the artificial membranes, we observed the plasmon minimums and the corresponding fluorescence maximums at shifted angles in different experiments (Figure 5a-e and Figure 5g). This observation reflects the varying overall film thicknesses of independent membrane preparations on separate gold surfaces. Therefore, we are currently working on the removal of the used lipid bilayer in order to recycle the lipid-modified peptide tether for the setup of a fresh membrane on the same gold surface. With this approach, we might be able to obtain more reproducible overall film thicknesses between individual experiments. Using this artificial membrane system, we demonstrated that GST-Osh5p binds to all five provided oxysterols. GST-Osh5p did not bind to a plain PC membrane (Figure 5f), indicating that the interaction with the membrane is dependent on the presence of oxysterols. Binding of GST-Osh5p to membranes was independent of the GST tag since we observed no interaction of GST with any of the artificial membranes incorporating different oxysterols (summarized in Figure 5h). However, from our results, we cannot deduce a mechanism for the interaction of Osh5p with the sterols, e.g., whether cofactors are required for binding. Preliminary data from binding assays performed with purified GST-Osh5p and 25OH suggest direct interaction of Osh5p with the sterol. Comparable to GST-Osh5p contained in soluble protein extracts used for the experiments presented here, isolated GST-Osh5p bound to a PC/25OH surface whereas it did not interact with a plain PC membrane (data not shown). The interaction of GST-Osh5p with 25OH evoked a markedly higher fluorescence signal (Figure 5e) than that with the other sterols, i.e., 8-DED, EEP, DHE (Figure 5d, c, b), and ergosterol

(Figure 5a). This finding suggests a distinct preference of GSTOsh5p from yeast for mammalian 25OH. It is possible that 25OH, due to its particular chemical structure (Figure 2), is inserted in the artificial membrane in a way that results in a favorable presentation of the binding epitope. SPS and SPFS display high sensitivity perpendicular to the gold surface, but little lateral resolution. Thus, with both techniques, it is impossible to gain information on the lateral distribution of the oxysterols within the PC bilayer. The lateral distribution of the oxysterols in the PC membrane could bias GST-Osh5p binding and, subsequently, fluorescence signal intensity and might differ with the individual sterol. We presume that at a ratio of 1:1 (w/w) PC forms homogeneous mixtures with the oxysterols used in this study. This assumption relies on the observation that lipid-phase separation is absent in monolayers formed by similar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesterol.38 However, we cannot rule out that phase separation occurs in our artificial membrane and that domain formation, i.e., lots of small patches versus few large patches, depends on the particular oxysterol. The lateral distribution might influence the availability of the sterol molecules for binding, thus yielding the observed higher fluorescence signal with 25OH but not with the other oxysterols. GST-Osh5p was expressed in the cl3-ABYS-86 strain background carrying wild-type alleles of all seven OSH genes. Thus, endogenously expressed Osh5p is a component of the cell lysate applied in the binding assay and Osh5p (and other OSHs) might compete with GST-Osh5p for the sterol molecules in the artificial membrane. Sterical hindrance due to crowding at the binding sites, phase separation, or both might affect GST-Osh5p binding to oxysterols, and thus, the intensity of the fluorescence signal varies with the particular oxysterol incorporated in the PC bilayer. The fluorescence signal intensity in SPFS is greatly dependent on the distance of the fluorophore to the gold surface due to the exponential decay of the exciting surface plasmon field.23 Therefore, it will be of outstanding importance to systematically analyze the different layers of the membrane setup with techniques alternative to SPS and SPFS, since characterization of membrane topology can shed light on the lateral distribution of the sterol molecules in the artificial membrane and on possible phase separations induced by the oxysterols.

(37) Theunissen, J. J.; Jackson, R. L.; Kempen, H. J.; Demel, R. A. Biochim. Biophys. Acta 1986, 860, 66-74.

(38) Dietrich, C.; Bagatolli, L. A.; Volovyk, Z. N.; Thompson, N. L.; Levi, M.; Jacobson, K.; Gratton, E. Biophys. J. 2001, 80, 1417-1428.

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CONCLUSION By immobilizing the oxysterol ligands in an artificial membrane instead of immobilizing the receptors, as usually performed in binding assays, we not only avoided the introduction of small hydrophobic molecules in an aqueous environment but eluded difficulties related to the detection of interactions with mobile, low molecular mass ligands. With the design of our novel in vitro oxysterol binding assay we have solved the technically challenging difficulty of presenting hydrophobic ligands to hydrophilic proteins in aqueous media.

ACKNOWLEDGMENT We thank Eva Mu¨ller, Nediljko Budisa, and Douglas Griffith for carefully reading the manuscript, Beate Mu¨ller for performing dn/dc measurements, and Fatma Nese-Ko¨k for the contact angle measurements. We are also indebted to Kevin Barrow for kindly providing yeast oxysterols. Special thanks are extended to Andreas Scheller for excellent software support and maintenance. We are

grateful to Wolfgang Knoll for fruitful discussions. This research was supported by grant F706 of the Austrian Science Fund to S.D.K. and grant SFB533. Received for review August 4, 2005. Accepted October 28, 2005. AC051388P

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