Directed Assembly of Surface-Supported Bilayers with

Jan 7, 2006 - Finally, bilayers were assembled using lipid vesicle fusion on top of the LB monolayers. The novelty is the incorporation of the peptide...
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Langmuir 2006, 22, 1247-1253

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Directed Assembly of Surface-Supported Bilayers with Transmembrane Helices Mikhail Merzlyakov, Edwin Li, and Kalina Hristova* Department of Materials Science and Engineering, Johns Hopkins UniVersity, Baltimore, Maryland 21218 ReceiVed July 18, 2005. In Final Form: NoVember 30, 2005 The lateral assembly of transmembrane (TM) helices gives rise to membrane proteins with complex folds, which play important roles in biochemical processes. Therefore, the assembly of surface-supported bilayers containing TM helices is the first step toward the development of functional biomembrane mimetics. Here we report novel directed assembly of surface-supported lipid bilayers with laterally mobile TM helices. The TM helices were incorporated into lipid monolayers at the air/water interface, and the monolayers were then transferred onto glass substrates using Langmuir-Blodgett (LB) deposition. Finally, bilayers were assembled using lipid vesicle fusion on top of the LB monolayers. The novelty is the incorporation of the peptides into the monolayer at the first step of bilayer assembly, which allows control over the peptide concentration and orientation. The transmembrane orientation of the peptides was confirmed using oriented circular dichroism (OCD), lateral mobility was assessed using fluorescence recovery after photobleaching (FRAP), and diffusion coefficients were determined using a novel boundary profile evolution (BPE) method. The described directed-assembly approach can be used to develop versatile bilayer platforms for studying membrane proteins interactions in native bilayer environments.

Introduction The transmembrane (TM) helix, a single hydrogen-bonded polypeptide chain designed to stably insert into cellular membranes, is the basic building block of integral membrane proteins.1 It consists of a hydrophobic segment of about 20 amino acids, long enough to cross the ∼30 Å hydrocarbon core of the lipid bilayer. It is flanked by hydrophilic amino acids that are positioned in the bilayer interface and anchor the hydrophobic segment.2 The lateral assembly of TM helices gives rise to membrane proteins with complex folds,3-6 which play crucial roles in cell adhesion, energy production, recognition, cell signaling, motility, and transport of nutrients. Therefore, the assembly of surfacesupported bilayers containing TM helices is the first step toward the development of functional membrane protein arrays that can be used as biosensors, biomimetics of cell surfaces, film catalysts, and tissue engineering substrates.7-12 The incorporation of membrane proteins into surface-supported bilayers has been achieved via self-assembly;13-15 however, the * Corresponding author. Phone: 410-516-8939. Fax: 410-516-5293. E-mail: [email protected]. (1) Jayasinghe, S.; Hristova, K.; White, S. H. J. Mol. Biol. 2001, 312, 927934. (2) White, S. H.; Ladokhin, A. S.; Jayasinghe, S.; Hristova, K. J. Biol. Chem. 2001, 276, 32395-32398. (3) Lemmon, M. A.; Engelman, D. M. Q. ReV. Biophys. 1994, 27, 157-218. (4) Sanders, C. R.; Ismail-Beigi, F.; McEnery, M. W. Biochemistry 2001, 40, 9453-9459. (5) Nagy, J. K.; Lonzer, W. L.; Sanders, C. R. Biochemistry 2001, 40, 89718980. (6) White, S. H.; Wimley, W. C. Annu. ReV. Biophys. Biomol. Struct. 1999, 28, 319-365. (7) Sackmann, E. Science 1996, 271, 43-48. (8) Sackmann, E.; Tanaka, M. Trends Biotechnol. 2000, 18, 58-64. (9) Schmidt, C.; Mayer, M.; Vogel, H. Angew. Chem., Int. Ed. 2000, 39, 3137-3140. (10) Bieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K. P.; Vogel, H. Nat. Biotechnol. 2000, 17, 1105-1108. (11) Bayley, H.; Cremer, P. S. Nature 2001, 413, 226-230. (12) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580-583. (13) Naumann, R.; Jonczyk, A.; Hampel, C.; Ringsdorf, H.; Knoll, W.; Bunjes, N.; Gra¨ber, P. Bioelectrochem. Bioenerg. 1997, 42, 241-247. (14) Schmidt, E. K.; Liebermann, T.; Kreiter, M.; Jonczyk, A.; Naumann, R.; Neumann, E.; Kukol, A.; Maelicke, A.; Knoll, W. Biosens. Bioelectron. 1998, 13, 585-591.

control over the topology and the orientation of the proteins has been challenging. A proven method of bilayer self-assembly is vesicle fusion (VF) on solid support: the substrate is incubated with liposomes, which break and fuse to form a single bilayer on the substate, sitting on a “cushion” of water molecules.7,16-18 Previously, we showed that fusion of liposomes containing TM helices leads to the formation of peptide/lipid supported bilayers, with the TM helices incorporated into the lipid matrix.19 Imaging Fo¨rster resonance energy transfer (FRET) experiments demonstrated that the interactions between the helices in the supported bilayers are the same as the interactions in suspended liposomes.19 However, the TM helices in these self-assembled supported bilayers exhibited complete lack of lateral mobility, as assessed by fluorescence recovery after photobleaching (FRAP).19 The peptides were always immobile despite full lipid mobility and various attempts to modify the surface and “lift” the bilayer up to minimize interactions with the support. The complete lack of peptide mobility limits the utility of the supported peptide/lipid bilayers. For instance, such self-assembled supported bilayers cannot be used in sensing applications that monitor changes in protein interactions in response to environmental changes. Furthermore, the directionality of the TM helices cannot be controlled in the liposomes prepared by hydrating protein/lipid mixtures,20 and therefore it is random in the selfassembled surface-supported bilayers. This is a severe limitation for the design of functional biomimetic membranes, because proteins in cellular membranes are unidirectionally oriented, and lateral interactions occur only in this unidirectional context (Cterminus-to-C-terminus and N-terminus-to-N-terminus interactions). Random distribution of directionalities in the bilayer may give rise to nonbiological C-terminus-to-N-terminus interactions. (15) Naumann, R.; Johczyk, A.; Kopp, R.; van Esch, J.; Ringsdorf, H.; Knoll, W.; Graber, P. Angew. Chem., Int. Ed. Engl. 1995, 34, 2056-2058. (16) Cremer, P. S.; Boxer, S. G. J. Phys. Chem. B 1999, 103, 2554-2559. (17) Vontscharner, V.; Mcconnell, H. M. Biophys. J. 1981, 36, 409-419. (18) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105-113. (19) Li, E.; Hristova, K. Langmuir 2004, 20, 9053-9060. (20) You, M.; Li, E.; Wimley, W. C.; Hristova, K. Anal. Biochem. 2005, 340, 154-164.

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Surface-supported bilayers have been previously prepared by depositing a lipid Langmuir-Blodgett (LB) monolayer, followed by fusion of vesicles containing the proteins.21,22 In this approach, protein concentration, directionality, and mobility could be difficult to control. Here we show that bilayers with TM helices can be prepared from peptide/lipid monolayers using LB deposition, followed by fusion of lipid vesicles. The novelty in this approach is the incorporation of the peptides into the LB monolayer, at the first step of bilayer assembly. As we discuss here, this approach allows precise control over the peptide concentration in the bilayer, the overall architecture of the structure, and the topology of the helices in the bilayer. The TM orientation of the helices is confirmed using oriented circular dichroism (OCD). OCD is a well-established method that is routinely used to assess the orientation of helices in multilayer samples,23-27 but it has never been used previously to measure helix orientation in single surface-supported bilayers. Lipids and peptides in the supported bilayers are mobile, as assessed by FRAP and a novel “boundary profile evolution” (BPE) data analysis method, which offers an alternative for quantitative diffusion measurements when state-of-the-art FRAP or single molecule setups are not available. In addition, the LB deposition/ vesicle fusion method has previously been shown to produce bilayers with asymmetric lipid composition28 that mimic the asymmetry of the cellular membrane. Therefore, the described directed-assembly method can lead to the development of versatile bilayer platforms with various potential applications, such as novel tools to study the lateral interactions between TM helices in biological context. Materials and Methods Materials. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was obtained from Avanti Polar Lipids (Alabaster, AL). Tetramethylrhodamine maleimide and N-(7-nitrobenz-2-oxa-1,3diazol- 4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (NBD-PE) were purchased from Molecular Probes (Eugene, OR). Lipids were used without further purification. Lipid stock solutions were prepared in chloroform at a concentration of ∼1 mg/mL and were stored in the freezer for future use. All experiments were performed with the 33 amino acid long TM domain of FGFR3, a protein implicated in growth disorders and cancers.29 This TM domain is very hydrophobic: the free energy of transfer from water to octanol for the membrane-embedded segment is estimated as -9.4 kcal/mol.30 FGFR3 TM domain (TMFGFR3, sequence DEAGSVYAGILSYGVGFFLFILVVAAVTLCRLR) was synthesized using solid phase peptide synthesis. The protocol for the synthesis of the peptide and its initial characterization have been described previously.30 The peptides were purified using reversed-phase HPLC (Varian Analytical Instruments, Walnut Creek, CA) and a water/acetonitrile gradient. MALDI-TOF mass spectrometry confirmed the correct molecular weight. The single Cys residue in the peptide was labeled with rhodamine maleimide and purified as previously described.31 The peptides collected from the (21) Wagner, M. L.; Tamm, L. K. Biophys. J. 2000, 79, 1400-1414. (22) Wagner, M. L.; Tamm, L. K. Biophys. J. 2001, 81, 266-275. (23) Wu, Y.; Huang, H. W.; Olah, G. A. Biophys. J. 1990, 57, 797-806. (24) de Jongh, H. H. J.; Goormaghtigh, E.; Killian, J. A. Biochemistry 1994, 33, 14521-14528. (25) Hristova, K.; Wimley, W. C.; Mishra, V. K.; Anantharamaiah, G. M.; Segrest, J. P.; White, S. H. J. Mol. Biol. 1999, 290, 99-117. (26) Hristova, K.; Dempsey, C. E.; White, S. H. Biophys. J. 2001, 80, 801811. (27) Vogel, H. Biochemistry 1987, 26, 4562-4572. (28) Crane, J. M.; Kiessling, V.; Tamm, L. K. Langmuir 2005, 21, 13771388. (29) L’Horte, C. G. M.; Knowles, M. A. Exp. Cell Res. 2005, 304, 417-431. (30) Iwamoto, T.; You, M.; Li, E.; Spangler, J.; Tomich, J. M.; Hristova, K. Biochim. Biophys. Acta 2005, 1668, 240-247. (31) Li, E.; You, M.; Hristova, K. Biochemistry 2005, 44, 352-360.

MerzlyakoV et al. HPLC column (Varian) were lyophilized and redissolved in a solution of hexafluoro-2-propanol and methanol (HFIP/MeOH, 1:2). The solvents were removed under a stream of nitrogen gas, and the peptides were redissolved in 2,2,2-trifluoroethanol (TFE) to a concentration of ∼30 µM. The helicity of the peptides in TFE was confirmed by circular dichroism (CD) using a J-710 spectropolarimeter (Jasco, Easton, MD). Stock solutions of lipids in chloroform and peptides in TFE were mixed together in the required proportions to obtain the desired peptide-to-lipid mol ratio. The mixtures were stored in the freezer for future use. Substrate Preparation. Cover glass slides (Fisher Scientific) were cleaned by sonication for 10 min in 2-propanol (IPA), followed by acetone and IPA. After the second sonication in IPA, slides were rinsed with deionized (DI) water and soaked in 30% hydrogen peroxide, 70% sulfuric acid for >30 min. Slides were extensively rinsed with and stored in DI water until future use. Quartz slides (25 × 25 × 0.5 mm3) from SPi Supplies (West Chester, PA) were used for oriented circular dichroism (OCD) experiments and were cleaned following the same procedure. Liposome Preparation. To prepare unilamellar vesicles (liposomes), POPC in chloroform was added to a test tube, the solvent was evaporated under a stream of nitrogen gas, and the lipids were redissolved in 10 mM phosphate buffer, 500 mM NaCl, pH 7 to a final concentration of 1 mg/mL of POPC. Samples were vortexed, freeze-thawed, and extruded using a 100 nm pore diameter membrane (Avanti) to produce unilamellar vesicles.32 Bilayer Assembly. A lipid monolayer containing the peptides was deposited on a glass substrate by the Langmuir-Blodgett (LB) method, as described in detail below. To form the bilayer, a second monolayer was assembled via vesicle (lipids only) fusion on top of the LB monolayer. For the LB deposition, two clean wet coverslips were stacked together and were immersed vertically into the clean subphase of a Langmuir-Blodgett trough (Nima Technologies, Coventry, England). The subphase was Milli-Q purified (Millipore, Molsheim, France) water with resistivity >18 MΩ cm, kept at 20 °C. A lipid/ peptide solution in chloroform/TFE was spread dropwise at the airwater interface of the open trough (600 cm2). A total of ∼20 nmol of lipids with the desired peptide concentration were spread (Figure 1A). Solvents were allowed to evaporate for 30 min, and the monolayers were compressed to 32 mN/m. Monolayers were deposited on the outer surfaces of the coverslips using the Langmuir-Blodgett method, at a rate of 15 mm/min (Figure 1B). During deposition the surface pressure was maintained at 32 mN/m. A thin water layer between the coverslips was stabilized by capillary forces, such that no lipid or peptide transfer occurred on the “inner” surfaces of the coverslips. The transfer ratio for each deposition was 1, as determined by the decrease of the monolayer area on the trough and the coated area of the coverslips. The coated coverslip was placed on top of two adhesive tape spacers attached to a microscope slide. In this geometry, a narrow gap formed between the coverslip and the slide, with the coated side of the coverslip facing the slide (Figure 1C). POPC vesicles were added to the gap and were incubated for 5 min at room temperature (Figure 1D). Excess vesicles were removed by flushing water through the gap opening. After the rinse, silicon grease was used to seal the chamber. The bilayers were stable for at least 1 day. OCD Experiments. For OCD measurements, monolayers were deposited on both sides of quartz slides using the Langmuir-Blodgett method. For OCD of dry monolayers, a total of 7 slides (14 monolayers) were stacked together and placed in the spectropolarimeter normal to the beam. CD spectra were measured at eight different angles and averaged, and corrected for the lipid background as described.23 For OCD measurements of fluid bilayers, the monolayer-coated slides were stacked together with tape spacers at the corners (the spacers did not block the CD beam). Twenty-six coated slides were stacked between two clean quartz slides and placed in the (32) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161-168.

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Figure 1. Directed-assembly of a surface-supported bilayer with TM helices (not drawn to scale). (A) A mixture of lipids and peptides in chloroform and TFE is spread on the water/air interface. (B) After solvent evaporation, the monolayer is compressed and deposited on a glass coverslip using the Langmuir-Blodgett method. (C) The coverslip is placed on top of a clean glass slide, with the monolayer facing the slide. (D) The monolayer is incubated with a solution of vesicles, the excess vesicles are rinsed out, and the chamber is sealed.

Figure 2. Sequence of FRAP images showing peptide mobility in bilayers assembled as shown in Figure 1. These are images of rhodamine-labeled TMFGFR3 (0.05 mol %) in a POPC bilayer. The scale bar is 100 µm. An octagonal spot (30 µm in diameter) was bleached, and images were taken right after bleaching (A), 1 h (B), and 16 h after bleaching (C). The immobile fraction, if existent, is less than 2% and cannot be resolved. Similarly, the immobile lipid fraction, if existent, is less than 2% (not shown). specropolarimeter cuvette holder such that they were normal to the beam, with the beam passing through the center of each slide. POPC vesicles were added between the slides and incubated for 5 min. The excess vesicles were removed by rinsing with DI water. The bilayers formed on the surface of the slides were never exposed to air; the gaps between the slides were kept hydrated due to capillary forces. Thus, a total of 52 fluid supported bilayers were formed, each oriented normal to the beam. Ten different CD spectra were recorded and averaged. Background CD spectra were collected from bilayers composed of lipids only and subtracted from the peptide/lipid spectra. Fluorescence Recovery After Photobleaching (FRAP). FRAP experiments were performed using an Eclipse E600 microscope (Nikon) equipped with a mercury lamp and a SPOT RT camera (Diagnostic Instruments). NBD and rhodamine were excited and observed using the appropriate filter cubes: NBD, excitation 465495 nm, emission 515-555 nm; rhodamine, excitation 530-560 nm, emission 573-648 nm (Nikon). To follow recovery (as in Figure 2) and determine the mobile fraction, an octagonal region (spot) of ∼30 µm diameter was bleached for 1 min. The spot size was set by adjusting the field stop of the microscope using a 40× objective.19 Fluorescence recovery was monitored by taking successive images after photobleaching. To minimize photobleaching during focusing and exposure, the illumination intensity was lowered with a neutral density filter (ND4). Boundary Profile Evolution (BPE) Method. A novel method was used to determine the diffusion coefficients. This method allows quantitative diffusion coefficient measurements even with a standard fluorescence microscope equipped with a mercury lamp. The bleached spot was set to ∼100 µm, and a sequence of images (every 5 min

for peptides, every 15 s for lipids) was taken after bleaching (Figure 3A). The intensity profile of the boundary region between bleached and unbleached areas was plotted for each image (Figure 3B). The γ factor (the exponent in the power-law transformation between input and output intensity of the camera33) was set to one, such that the recorded intensity was proportional to the concentration of unbleached dyes. At the initial stages of fluorescence recovery, when the diffusion depth w (see eqs 1 and 2 below for definition) is smaller that the size of the bleached spot, diffusion can be treated as onedimensional. If the intensity profile right after bleaching is described by a step function, then the profile evolution with time, F(x, t), is given by a Gaussian error function,34 2

( )

x - xb F(x, t) - Fbleached ) erf +1 Funbleached - Fbleached 2w

(1)

In this equation, Fbleached and Funbleached are the fluorescence intensities inside and outside of the bleached spot, xb is the position of the boundary between the bleached and unbleached areas, and (x - xb) is the distance to this boundary. The diffusion depth w is defined as w ) xDt

(2)

where D is the diffusion coefficient and t is the lapse time after bleaching. Note that eq 1 is valid for homogeneous diffusion. It can be modified to account for an immobile fraction or populations with varying diffusion coefficients. The intensity profiles (Figure 3B) were fitted to eq 1, and w2 was plotted against t (Figure 3C). Since w2 ) Dt, the slopes of the lines in parts C and D of Figure 3 are equal to the diffusion coefficients of peptides and lipids. We note that the intensity distribution right after photobleaching is not a perfect step function (Figure 3B), due to diffusion occurring during bleaching (and partly due to the limited resolution of the optics). However, as shown by the straight lines in parts C and D of Figure 3, the diffusion depth does obey eq 2. The lateral diffusion of bleached and unbleached dyes during bleaching leads to an intensity distribution that is similar to the distribution created by a flash bleach after a certain time lapse. The w2 versus time points fall on a straight (33) Gonzalez, R. C.; Woods, R. E. Digital Image Processing; Prentice Hall: Upper Saddle River, NJ, 2002. (34) Schakelford, J. F. Diffusion. In Introduction to Materials Science for Engineers; Prentice Hall: Upper Saddle River, NJ, 2005; pp 157-182.

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Figure 3. Measurements of peptide diffusion coefficients using the boundary profile evolution (BPE) method described in the Materials and Methods. (A) A sequence of images is taken during recovery at t ) 0, 5, and 10 min. Intensity profiles are calculated for the highlighted rectangular area. The scale bar is 100 µm. (B) The intensity profile across the boundary between the bleached and unbleached area is plotted (t ) 0, solid line; t ) 5 min, dashed line; t ) 10 min, dotted line) and is fitted to a Gaussian error function (eq 1), yielding the diffusion depth, w, and its corresponding standard error. (C) The diffusion coefficient D for the peptides is determined from the plot of w2 vs t. Since w2 ) Dt, the slope of the line is equal to D. (D) Calculation of the lipid diffusion coefficient using the same method. line for long lapse times, until the diffusion depth becomes comparable to the size of the bleached spot. We also note that in the BPE method, the diffusion coefficient is determined from the slope of w2 versus time, not from the value of w2 itself. Therefore, it is not required to define t ) 0. Furthermore, while fitting the intensity profile to eq 1, the intensities of the unbleached and bleached areas (Funbleached and Fbleached) are allowed to vary from image to image in order to account for possible photobleaching during image acquisition. To the best of our knowledge, the described BPE method has not been used previously to determine diffusion coefficients in membranes. However, similar profile evolution methods, in which the size of the bleached spot is smaller than the diffusion depth, have provided valuable information about cellular processes, such as the dynamics of GFP-modified proteins35,36 and photosynthetic complex diffusion in thylakoid membranes.37

Results A directed-assembly method was used to produce surfacesupported bilayers with laterally mobile FGFR3 TM peptides. The method is described in detail in the Materials and Methods and illustrated in Figure 1. Briefly, the assembly consists of two steps: (1) a monolayer of peptides and lipids is deposited on a clean glass surface by the LB technique; (2) a second lipid monolayer is produced via vesicle fusion. Lateral mobility of peptides and lipids in the bilayer was measured using fluorescence recovery after photobleaching (FRAP) (Figure 2). Diffusion (35) Yokoe, E.; Meyer, T. Nat. Biotechnol. 1996, 14, 1252-1256. (36) Sowa, G.; Liu, J.; Papapetropoulos, A.; Rex-Haffner, M.; Hughes, T. E.; Sessa, W. C. J. Biol. Chem. 1999, 274, 22524-22531. (37) Mullineaux, C. W.; Tobin, M. J.; Jones, G. R. Nature 1997, 390, 421424.

coefficients were determined as described in Materials and Methods using the BPE method (see Figure 3 for details). The lapse time between captured images was chosen in accordance with the diffusion rates. It was set to 5 min for peptides and ∼15 s for lipids, but further data processing was identical. The diffusion coefficients for lipids and peptides in bilayers composed of POPC, 0.5 mol % NBD-PE and 0.05 mol % peptides (1:2000 peptideto-lipid ratio) were 2.3-2.7 µm2/s and 0.006-0.007 µm2/s, respectively. The BPE method offers advantages for both slow- and fastmoving molecules over the “traditional” half-time of recovery trajectory. The advantage is obvious for the slow-moving peptides, since the half-time of recovery is hours (see Figure 2), and therefore experiments that measure recovery half-times take many hours to complete (as compared to 10 min for the BPE method). For the fast-moving lipids, the BPE method presents an alternative for quantitative diffusion measurements when state-of-the-art laser-based FRAP or single molecule setups are not available. The BPE method works even if the bleaching time is comparable to the recovery time in the FRAP experiments, allowing us to conduct experiments on a standard fluorescence microscope without a laser. Note that the traditional half-time recovery method gives the correct diffusion coefficient only if the bleaching time is much shorter than the recovery time.39-41 The lipid diffusion (38) Deverall, M. A.; Gindl, E.; Sinner, E. K.; Besir, H.; Ruehe, J.; Saxton, M. J.; Naumann, C. A. Biophys. J. 2005, 88, 1875-1886. (39) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W. Biophys. J. 1976, 16, 1055-1069. (40) Merkel, R.; Sackmann, E.; Evans, E. J. Phys. (Orsay, Fr.) 1989, 50, 1535-1555. (41) Zhang, L.; Longo, M. L.; Stroeve, P. Langmuir 2000, 16, 5093-5099.

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Table 1. Lipid Diffusion Coefficients in Supported Fluid Bilayersa lipid

D(µm2/s)

ref

POPC POPC eggPC eggPC SOPC eggPC eggPC

2.3-2.7 1.3 4 2.46 2.1 6 1.4

this study 21 47 48 38 49 50

a The lipid diffusion coefficients, calculated using the BPE method, are similar to previously reported values.

coefficients calculated using the BPE method are very similar to previously reported values (Table 1) obtained using other methods, such as flash bleaching and single molecule measurements.38 The measured diffusion coefficients show that while lipids in the assembled bilayer move very fast, peptides move relatively slowly. Even slow mobility, however, is a substantial improvement over the complete lack of peptide mobility in self-assembled mixed peptide/lipid bilayers.19 We note that in our hands the alternative approach of fusing vesicles containing the peptides to a lipid-only LB monolayer42 produced bilayers with immobile peptides. We therefore conclude that the directed-assembly approach illustrated in Figure 1 is the only method that ensures the lateral mobility of the particular peptide that we work with, FGFR3 TM domain. Thus, this approach offers an important advantage over the various self-assembly methods that we have previously explored.19 This advantage would be real, however, only if the directed assembly leads to transmembrane orientation of the peptides. As discussed above, a TM helix consists of a hydrophobic segment of about 20 amino acids, long enough to span the ∼30 Å hydrocarbon core of the lipid bilayer, and charged flanks. This is why the helix incorporates in liposomes in a transmembrane orientation, driven by the hydrophobic effect. If the liposomes containing the peptides are fused onto a solid substrate to form bilayers,19 the TM topology is expected to be preserved. However, in the directed-assembly approach we cannot have such expectations. The TM helix is designed to span two monolayers, and it is not clear how it will orient within a single monolayer at the air/water interface. Therefore, it is not clear if the orientation of the helices would be transmembrane once bilayer formation is completed via vesicle fusion. The method of OCD measures helix tilt with respect to the bilayer normal.43,44 The OCD signal is dramatically different for helices that are normal and parallel to the beam, such that TM orientation is easy to confirm once a high-quality OCD spectrum is available.23 OCD is used routinely to verify the TM orientation of self-assembled multilayers, deposited from organic solvent.30,31 Here we examined if this technique can be used to confirm TM orientation in supported lipid bilayers. The OCD signal for a TM helix is low, and we have previously found that, in multilayers, one needs at least a few mol % peptide to observe a high-quality signal over the lipid background.30,31 Therefore, fully hydrated surface-supported bilayers were prepared by LB/vesicle fusion deposition with 2.5 mol % total peptide (5 mol % peptide in the LB monolayer), as described in the Materials and Methods. Using FRAP, we confirmed that the peptides are mobile at this concentration (Figure 4). Thus, the 50-fold increase in peptide concentration (up to 2.5 mol %) did not have a significant effect on the diffusion of lipids and peptides. (42) Kalb, E.; Tamm, L. K. Thin Solid Films 1992, 210, 763-765. (43) Olah, G. A.; Huang, H. W. J. Chem. Phys. 1988, 89, 2531-2538. (44) Olah, G. A.; Huang, H. W. J. Chem. Phys. 1988, 89, 6956-6962.

Figure 4. Sequence of FRAP images of rhodamine-labeled TMFGFR3 (2.5 mol %) in a POPC bilayer, taken at t ) 0, 5, and 10 min after photobleaching. The scale bar is 100 µm. Diffusion coefficients are 0.004 and 2.4 µm2/s for peptides and lipids, respectively. A 50-fold increase in peptide concentration (from 0.05 to 2.5 mol % peptide) does not have a significant effect on lateral mobility.

Figure 5. Oriented circular dichroism (OCD) spectrum of TMFGFR3 in surface-supported fluid bilayers. The peptides were incorporated into 52 fluid POPC bilayers stacked between 28 quartz slides, as described in Materials and Methods. The peptide-to-lipid ratio was 1:40. Comparison of the measured spectrum (solid line) with the theoretical spectra of helices that are normal (dashed line) and parallel (dotted line) to the bilayer indicates that the orientation of TMFGFR3 is transmembrane.

Figure 5 shows the predicted OCD spectra for helices that are normal and parallel to the bilayer plane.23 These spectra are calculated using parameters given in ref 23 as previously reported,30,45 and are very similar to earlier experimental OCD spectra.27 We see that the OCD spectrum of a helix that is parallel to the membrane plane exhibits two minima at 205 and 225 nm and a maximum around 192 nm. Transmembrane helices, however, exhibit a single minimum around 230 nm and a maximum around 200 nm.23 The experimental OCD spectrum of TMFGFR3 in supported bilayers, collected from 52 bilayers stacked between 28 slides, is shown in Figure 5. Each bilayer was composed of POPC and 2.5 mol % peptides (1:40 peptideto-lipid ratio). The measured CD spectrum (solid line in Figure 5) exhibits a single minimum, indicating that the peptides are normal to the bilayer plane. To the best of our knowledge, this is the first direct quantitative assessment of helix tilt in surfacesupported bilayers using OCD. A major challenge in OCD measurements of self-assembled multilayers is the determination of the signal amplitude.30 It depends on the concentration of peptide per unit area and, ultimately, on the thickness of the sample. For self-assembled multilayers, the thickness is hard to control and estimate. Thus, one has to rely primarily on the shape of the OCD spectrum, rather than the amplitude, to estimate helix tilt with respect to the bilayer normal. During LB deposition, however, the transfer ratio is measured, and therefore the peptide concentration in the supported bilayer is accurately known. Thus, the ellipticity amplitude can be measured reliably for peptides that are transferred using LB deposition. As seen in Figure 5, the amplitude of the measured TMFGFR3 spectrum is very similar to the (45) Wimley, W. C.; White, S. H. Biochemistry 2000, 39, 4432-4442.

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Figure 6. Pressure-area isotherms of monolayers composed of POPC only (dashed line) and of POPC and TMFGFR3 (2:1 mol ratio, solid line) at 20 °C. Isotherms were recorded during compression and expansion at a rate of 100 cm2/min.

theoretical one, further confirming the transmembrane orientation of the peptides. We therefore conclude that the described directed-assembly method produces bilayers containing peptides with TM orientation. This assertion is based on OCD measurements in fully assembled bilayers. We believe that the TM orientation is a direct consequence of the peptide sequence encoding a TM domain. Next, we investigated how the FGFR3 TM topology is achieved in the process of directed assembly. When spread from the organic solvent at the air/water interface, the helices probably adopt various orientations (parallel, normal, tilted) in the gaseous phase. We hypothesize that, upon compression, all helices in the LB monolayer will orient normal to the air/water interface. This topology is then preserved during LB deposition and vesicle fusion. To assess the validity of the above hypothesis, we followed changes in the orientation of the TM helices upon compression by measuring the area-pressure isotherms of lipid monolayers containing the peptides. For these experiments, we increased the concentration of the peptides in order to observe changes in molecular area associated with changes in peptide conformation. The pressure-area isotherms are shown in Figure 6 for POPC (dashed line) and POPC with TMFGFR3 (1:2 peptide-to-lipid ratio). As seen in Figure 6, the area per molecule at 32 mN/m is ∼100 Å2 in the mixed peptide/lipid monolayer and ∼81 Å2 in the pure lipid monolayer. Assuming that the molecular areas do not change due to peptide-lipid interactions, the area occupied by the peptide for a known lipid-to-peptide ratio can be calculated as

Ap )

A h - xlAl xp

(3)

where A h is the mean area per molecule in the lipid/peptide monolayer, xl and xp are the molar fractions of lipids and peptides (two-thirds and one-third, respectively), and Al is the area per molecule in the pure lipid monolayer. With the use of eq 3, the area per peptide is calculated as 138 Å2. Thus, the “thickness” of the helix can be estimated as ∼12 Å. On the other hand, a typical R-helix diameter in a peptide crystal, void of any thermal fluctuations, is 10 Å. We expect that thermal fluctuations in the monolayer can account for the ∼2 Å difference. Therefore, the measured area per helix in the monolayer at 32 mN/m (high compression) is consistent with the idea that all helices are normal to the air/water interface. At 5 mN/m (low compression), the surface area per peptide is calculated as 223 Å2 (Figure 6). This value allows us to estimate

Figure 7. OCD spectra of TMFGFR3 in dry monolayers, deposited on quartz slides at surface pressures of 5 (solid line) and 32 mN/m (dashed line). The monolayers consisted of POPC and TMFGFR3 (2:1 lipid-to-peptide mol ratio). These spectra of dry peptides do not follow exactly the theoretical predictions in Figure 5. Still, the single minimum and low amplitude of the spectrum at 32 mN/m (dashed line) suggest that the peptides are likely perpendicular to the monolayer plane at the time of deposition. The higher amplitude and the appearance of two minima suggest that the helices are tilted (not 100% transmembrane) when the monolayer is deposited at 5 mN/m.

the average tilt with respect to the air/water interface as follows: The area of a helix that is parallel to the interface is predicted to be ∼500 Å2 (10 Å (helix diameter) × 33 (number of residues) × 1.5 Å (rise per residue)). On the basis of these values, the average tilt of the helix at 5 mN/m is estimated to be ∼60° with respect to the air/water interface. Thus, the pressure-area isotherm suggests that the helices, upon increasing compression, gradually orient normal to the air/water interface. Note that the pressurearea isotherm cycles in the presence of the peptide exhibit hysteresis, most probably due to attractive peptide-peptide and/ or peptide-lipid interactions that persist during area expansion. The helix tilt in monolayers compressed at different pressures (5 and 32 mN/m) was also assessed by OCD (shown in Figure 7). These were dry monolayers, and it was not clear if the OCD spectra of such monolayers can be used for quantitative assessment of topology. As seen in Figure 7, a monolayer deposited at 32 mN/m (dashed line) still shows a single minimum and a low amplitude, reminiscent of a helix orientation that is normal to the monolayer (see Figure 5 for reference). However, the maximum occurs at ∼190 nm, rather than the expected ∼200 nm. Therefore, the OCD spectra of dry monolayers, by themselves, are not sufficient to infer normal-to-the-monolayer orientation at high lateral compression. However, the area-pressure isotherms strongly suggest that such orientation occurs, and the OCD spectrum does not point to the contrary by suggesting a helix orientation that is parallel to the bilayer. For comparison, when the monolayers are deposited at 5 mN/m (solid line), the OCD spectrum exhibits two minima and a higher amplitude, suggestive of a helix tilt with respect to the substrate plane. Next, pressure-area isotherms were recorded for pure peptide monolayers. TMFGFR3, dissolved in organic solvent, was deposited on the open trough. After solvent evaporation, the peptide monolayer was gradually compressed up to 50 mN/m, the maximum pressure that the peptide monolayer could withstand before collapse. The compression/expansion cycle shown in Figure 8 revealed a large hysteresis in the pressure-area isotherm, indicative of strong peptide-peptide interactions in the monolayer. The area per peptide at 32 mN/m was ∼165 and ∼128 Å2 at compression and expansion, respectively, as compared to a peptide surface area of 138 Å2 in the lipid/peptide monolayer (see Figure 6). It therefore appears that the lipids facilitate the transmembrane orientation of the peptide upon monolayer

Surface-Supported Bilayers with TM Helices

Figure 8. Pressure-area isotherm cycle for a TMFGFR3 monolayer. The area per helix in the solid monolayer at 50 mN/m is 100 Å2, in excellent agreement with helix cross-sectional areas in protein crystals. The large hysteresis in the pressure-area isotherm indicates strong peptide-peptide interactions.

compression. At 50 mN/m, the monolayer appeared to be in a solid phase, as indicated by the complete lack of molecular flow around the Wilhelmy plate, resulting in a slight plate tilt during compression. The area per helix in the solid monolayer at 50 mN/m was 100 Å2, in excellent agreement with helix crosssectional areas in protein crystals. These results further support the idea that, upon lateral compression, all helices orient normal to the bilayer interface. As mentioned above, lipids seem to facilitate the normal-tothe-interface orientation of the peptides, as judged by the difference in area per peptide as a function of compression in Figures 6 and 8. Therefore, the pure peptide isotherm in Figure 8 cannot be used to directly assess peptide orientation within the lipid monolayer. We believe, however, that the peptide isotherm in Figure 8, with its predictable characteristics, supports the general idea that pressure-area isotherms can indeed be used to infer peptide orientation in monolayers at the air/water interface. Thus, the pressure-area isotherms and the OCD data suggest that, upon compression, all peptides orient normal to the monolayer plane. The transmembrane orientation is retained after the bilayer is completed via vesicle fusion, as proven by the OCD spectrum in Figure 5. Orienting the peptides at the air-water interface may offer a means to achieve unidirectional orientation of the helices. In particular, we expect that the presence of a water-soluble domain at either the N- or the C-terminus will orient the TM helices unidirectionally such that all water-soluble domains face the

Langmuir, Vol. 22, No. 3, 2006 1253

aqueous medium. These soluble domains could be water-soluble charged carriers fused to the TM domain for expression in E. coli or chemically coupled charged polymers. A labile linkage of the water-soluble domain to the TM helix will add additional versatility. In the case of the isolated TM domains, directionality would likely be possible simply via sequence modifications of the two termini. Such modifications are not expected to affect the lateral interactions of TM helices, which are driven primarily by van der Waals interactions.6,46 Adding several charged amino acids to one of the termini, while substituting the charged residues at the other terminus with neutral hydrophilic amino acids may be enough to ensure the unidirectionality of the helices. Future work would test this hypothesis and explore in detail how termini modifications would impact bilayer architecture.

Conclusion We describe a directed-assembly approach for producing surface-supported bilayers with transmembrane helices. The bilayer is assembled from a peptide/lipid monolayer using Langmuir-Blodgett deposition, followed by vesicle fusion. The novelty in this approach is the incorporation of the peptides into the LB monolayer at the air/water interface, allowing for precise control over peptide concentration and orientation. Helix OCD spectra, measured here for the first time in surface-supported bilayers, prove that the orientation of the peptide is transmembrane. The peptides exhibit lateral mobility, and their directionality could, eventually, be controlled in the monolayer at the initial assembly stage. The described directed-assembly approach can be used to develop versatile bilayer platforms to study membrane proteins interactions in the native bilayer environment. Acknowledgment. We thank Xue Han, Dr. Takeo Iwamoto, and Dr. John Tomich for peptide synthesis. This work was supported by NSF MCB 0315663 and Whitaker RG01-0370 to K.H. LA051933H (46) MacKenzie, K. R.; Prestegard, J. H.; Engelman, D. M. Science 1997, 276, 131-133. (47) Albertorio, F.; Diaz, A. J.; Yang, T. L.; Chapa, V. A.; Kataoka, S.; Castellana, E. T.; Cremer, P. S. Langmuir 2005, 21, 7476-7482. (48) Weng, K. C.; Stalgren, J. J. R.; Duval, D. J.; Risbud, S. H.; Frank, C. W. Langmuir 2004, 20, 7232-7239. (49) Salafsky, J.; Groves, J. T.; Boxer, S. G. Biochemistry 1996, 35, 1477314781. (50) Chan, P. Y.; Lawrence, M. B.; Dustin, M. L.; Ferguson, L. M.; Golan, D. E.; Springer, T. A. J. Cell Biol. 1991, 115, 245-255.