Article pubs.acs.org/Langmuir
Creation of Cross-Linked Bilayer Membranes That Can Incorporate Membrane Proteins from Oligo-Asp-Based Peptide Gemini Surfactants Shuhei Koeda,† Katsunari Umezaki,† Ayumi Sumino,† Tomoyasu Noji,† Atsushi Ikeda,‡ Yasushi Yamamoto,† Takehisa Dewa,† Keijiro Taga,† Mamoru Nango,§ Toshiki Tanaka,† and Toshihisa Mizuno*,† †
Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, Aichi 466-8555, Japan Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan § Osaka City University Advanced Research Institute for Natural Science and Technology, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan ‡
ABSTRACT: We designed novel bilayer-forming amphiphiles based on the cyclic oligo-Asp-based peptide gemini (PG) surfactants cr-D2C12 and cr-D3C12, which consist of −Cys(Asp)nCys− (n = 2 or 3) as a core peptide and two Cys residues containing a dodecylamidomethyl group. Dynamic light scattering and transmission electron microscopy measurements revealed the formation of spherical bilayer membranes that could incorporate the light-harvesting antenna complex 2 (LH2) from Rhodopseudomonas acidophila. Furthermore, this proteoliposome-like conjugate could be assembled onto cationized glass and mica to form planar bilayer membranes incorporating LH2. Using atomic force microscopy, we observed LH2 protruding (ca. 1.2−1.5 nm) from flat terraces of the planar bilayer membranes formed from cr-D2C12 or cr-D3C12. Thus, our designed PG surfactants are a new class of bilayerforming amphiphiles that may be applied to the study of various membrane proteins.
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mide,11 the creation of the artificial amphiphiles to form bilayers in an aqueous environment has been extensively studied. Similar to natural phospholipids, most of these artificial amphiphiles have a fork structure consisting of two long alkyl chains and one polar headgroup,12 which facilitates lamella-type molecular assemblies. However, amphiphiles capable of incorporating membrane proteins in bilayer membranes are still relatively unavailable.13 Furthermore, successful observation of the membrane protein structure by scanning probe microscopy, such as atomic force microscopy (AFM), after incorporating membrane proteins in planar bilayer membranes has not been reported. Recently, we reported novel peptide-containing gemini-type (PG) surfactants consisting of a hydrophilic linear oligo-Asp peptide core [(Asp)n (n = 1−5)] and two peripheral dodecylamidomethyl-conjugated Cys residues (DnC12; n = 1− 5), which exhibited high micelle formation and a tendency to form bilayers.14 The successful formation of planar bilayer membranes onto cationized mica and spherical bilayers in a buffer solution was observed preferentially for D3C12 and D4C12, which have linear trimeric and tetrameric Asps as core
INTRODUCTION One-third of natural proteins expressed from open reading frames (ORFs) in chromosomal DNA are predicted to be membrane proteins1 involved in various biological events in cell membranes, such as energy production,2,3 ion transport,4 and signal transduction.5 However, in comparison to water-soluble proteins, handling of membrane proteins becomes difficult (solubilization in an aqueous buffer, isolation of target membrane protein, crystallization, etc.) with suppression of their denaturation, which not only limits structural analyses6 but also various applications, such as fermentation enzymes in chemical processes,7 biomarkers for clinical tests,8 therapeutic drugs,9,10 etc. In this context, the design of novel artificial amphiphiles for use in bilayer membranes that are able to incorporate membrane proteins, such as phospholipid liposomes, is important. Because artificial bilayer-forming amphiphiles can be subjected to various chemical modifications and provided with structural diversity through simple synthetic procedures, the design of artificial amphiphiles enabling the regulation of the topology of membrane proteins in bilayer membranes in vitro might be possible. Such attempts may alleviate some of the conventional reliance of membrane protein chemistry on natural phospholipids. After the report by Kunitake and Okahata on the formation of bilayer membranes by didodecyldimethylammonium bro© 2013 American Chemical Society
Received: April 27, 2013 Revised: August 7, 2013 Published: August 14, 2013 11695
dx.doi.org/10.1021/la401566h | Langmuir 2013, 29, 11695−11704
Langmuir
Article
(hydroxymethyl)aminomethane (Tris) were purchased from Wako Pure Chemical Industries, Ltd. (Japan). Unless otherwise stated, other chemicals and reagents were obtained commercially and used without further purification. Synthesis of the PG Surfactant. Synthesis of each PG surfactant was performed on a rink-amide AM resin using commercially available Fmoc-protected amino acids, Fmoc−Cys(C12)−OH,1,2 and standard condensation reagents (HOBT/HBTU/DIEA). The N terminus of the PG surfactant was end-capped with Ac2O. cr-D2C12: HRMS (EITOF, [M + H]+) calcd. for C50H87N9O13S4Na, 1172.5198; found, 1172.5211. cr-D3C12: HRMS (EI-TOF, [M + Na]+) calcd. for C54H92N10O16S4Na, 1287.5468; found, 1287.5165. cr-D4C12: HRMS (EI-TOF, [M + H]+) calcd. for C58H97N11O19S4Na, 1402.5737; found, 1402.5438. Characterization of S−S Bond Formation by the 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) Test. Following the method to determine the concentration of −SH groups using DTNB, we examined whether free −SH groups of the PG surfactant remained. The PG surfactant (1 mM) was dissolved in 50 mM carbonate buffer (pH 10), and 10 mM DTNB was added and gently mixed for 30 min. A500, corresponding to the production of 4-nitorothiophenol, was analyzed using an ultraviolet−visible (UV−vis) spectrometer. Critical Aggregation Concentration (cac) Determination for the Cyclic PG Surfactant Using 8-Anilinonaphtharene-1sulfonic acid (ANS). Fluorescence spectral changes of ANS before and after incorporation into micelle- or bilayer-like aggregates of PG surfactants were used to evaluate the cac. From the double linearfitting analysis for F472, we evaluated the cac values of PG surfactants. Preparation of Liposome-Like Spherical Bilayers of Cyclic PG Surfactants Using the Emulsion Method. To a suspension of cr-DnC12 (n = 2−4) (∼1 mmol) in paraffin (500 μL), 30 μL of 50 mM carbonate buffer (pH 10), including 150 mM D-sucrose and 350 mM D-glucose, was added, and this mixture was sonicated using a probetype sonicator (100 W) for 20 min. The resulting emulsified suspension was carefully stacked on 500 μL of 50 mM carbonate buffer (pH 10) solution, including 500 mM D-glucose, in a 1.5 mL plastic tube, and it was centrifuged (21500g) for 20 min at 4 °C. The spherical single bilayers were finally produced in an aqueous phase. DLS Measurements of PG Surfactant Assemblies. Using a plastic cuvette with a path of 1 cm, we observed the DLS profiles of crDnC12 (n = 2−4) assemblies in the presence and absence of LH2 using a Zetasizer Nano ZS (Malvern Instruments, Ltd.). Observation of Assembly of Cyclic PG Surfactants by TEM. TEM images of PG surfactant aggregates were obtained using a JEM2500 SE microscope (JEOL, Japan). The solutions of the PG surfactant assemblies were cast onto a carbon-coated Cu grid, stained with 2% sodium phosphotungstate, and dried in vacuo. The accelerating voltage for these TEM observations was 100 kV. Fluorescence Recovery after Photobleaching (FRAP) Measurements for the Planar Bilayer Membrane of PG Surfactants onto Poly(lysine)-Coated Coverslips. First, to make the coverslip surface cationic, a poly(lysine) solution (Mn ∼ 75 000 Da, 100 mg/mL in Milli-Q) was mounted as a droplet onto the chemically cleaned coverslip for 30 min. After excess amounts of poly(lysine) were removed by washing in Milli-Q water, a 20 mM Tris-HCl (pH 8) solution of cyclic PG surfactant, including 1 mol % 1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-lissaminerhodamine B sulfonyl (NRh-DOPE), was applied to the poly(lysine)-coated coverslip and incubated for 30 min at room temperature. Then, excess reagents were extensively rinsed with 20 mM Tris-HCl (pH 8) to remove unabsorbed cyclic PG surfactants. FRAP measurements were performed using an objective-type TIRF microscope, TE-2000U (Nikon), equipped with an oil-immersion objective lens (Plan Apo 100H; numerical aperture = 1.45, Nikon), a laser with λ = 532 nm (25 mW, Crystalaser), a cooled charge-coupled device (CCD) camera (ORCA-ER, Hamamatsu), and AquaCosmos imaging analysis software. The filter set consisting of a 595 nm dichroic mirror and a 600−660 nm band-pass barrier filter was used. Preparation of Proteoliposome-Like Spherical Bilayers of PG Surfactants, Including LH2. PG surfactant (1 mg, ca. 1 μmol)
peptides, respectively. However, probably because of their flexible molecular structure, especially that derived from alkyl chains from the hydrophilic flexible peptide backbone, mixing of the prepared bilayer membranes with membrane proteins resulted in simple destruction of bilayer assemblies and a failure to prepare proteoliposome-like PG surfactant/membrane protein assemblies. Therefore, in this study, we designed new oligo-Asp-based PG surfactants with intramolecular crosslinking by introducing one S−S bond at the core peptide moiety. The PG surfactant with −Cys(Asp)2Cys− as a core peptide was named cr-D2C12; similarly, those containing −Cys(Asp)3Cys− and −Cys(Asp)4Cys− were named as crD3C12 and cr-D4C12, respectively (Figure 1). The −SH groups
Figure 1. Chemical structures of a series of PG surfactants, having linear and cyclic oligo-Asp peptides as core peptides.
of Cys residues for all samples were oxidized to generate the intramolecular S−S bond, which might have restricted the conformation of alkyl chains and, thus, facilitated their bilayer formation. Furthermore, partial exchange of the S−S bond from the intramolecular to intermolecular may further stabilize the bilayer assembly, which might allow for the incorporation of membrane proteins while retaining the original bilayer morphology. As a control membrane protein in this study, we used the light-harvesting complex 2 (LH2) from Rhodopseudomonas acidophila (10050),15 because its successful incorporation into the bilayer membranes of phospholipids has been reported.16 Assemblage of the reaction center (RC) complex, which function as a charge separator using excitation energy funneled from LH2 and the light-harvesting complex 1 (LH1) in the photosynthetic bacteria, along with the aid of chemically modified artificial planar bilayer membranes on an electrode, construction of efficient light-energy conversion semi-synthetic devices might be expected. Using transmission electron microscopy (TEM), dynamic light scattering (DLS), and AFM measurements, we characterized the bilayer formation and incorporation of membrane proteins of our designed cross-linked PG surfactants, cr-DnC12 (n = 2−4).
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EXPERIMENTAL SECTION
Materials. All N-(9-fluorenyloxycarbonyl) (Fmoc)-protected Lamino acids, 1-hydroxybenzotriazole (HOBT), 2-(1H-benzotriazol-1yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), rinkamide AM resin (200−400 mesh), N,N-diisopropylethylamine (DIEA), piperidine, trifluoroacetic acid (TFA), and N-methylpyrrolidone (NMP) were purchased from Merck Biosciences, Novabiochem (Switzerland), and Watanabe Chemical Industries (Japan). Dichloromethane (DCM) and methanol (MeOH) were purchased from Kanto Chemical Co., Inc. (Japan). 4,4′-Bipyridyl, iodomethane, 3-bromopropionic acid, 2-(N-morpholino)ethanesulfonic acid (MES), and tris11696
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and dansyl-conjugated D3C12 (0.1 mg, ca. 0.1 μmol) were first dissolved in 100 μL of 50 mM NaOH, and then 400 μL of 50 mM Tris-HCl buffer (pH 8) was added to set the pH of the solution to 8. At this stage, most the of cyclic PG surfactant, i.e., cr-D2C12 and crD3C12, formed spherical bilayers, as confirmed from the DLS analyses. After the addition of 500 μL of 20 mM Tris-HCl buffer (pH 8), containing 1.56 wt % n-octyl-β-D-glucopyranoside (β-OG), the PG surfactant solution was mixed with the 1 mL of LH2 solution in 20 mM Tris-HCl buffer (pH 8), including 0.78 wt % β-OG, and incubated for 1 h at 4 °C. The molar ratio of LH2 and cr-D2C12 or crD3C12 was set to approximately 1/100 (LH2/cr-D2C12 or cr-D3C12). To remove β-OG and form a proteoliposome-like conjugate, dialysis against 20 mM Tris-HCl buffer (pH 8) was applied for the above solution several times at 4 °C. Analysis of the Proteoliposome-Like Conjugate Composed of LH2 and Cyclic PG Surfactant Using Sucrose Density Gradient Centrifugation. A sucrose density gradient from 40 (bottom) to 20 (top) wt % in 20 mM Tris-HCl (pH 8) was prepared in a centrifuge tube, and 1 mL of a solution of the proteoliposome-like spherical bilayers of PG surfactants, including LH2, was first placed at the top layer. Ultracentrifugation (235000g, Himac CP70MX, Hitachi Kohki) was applied to this sample for 16 h. To observe the separation profiles of each analysis, we divided the sucrose layers in 10 fractions (1 mL each) from top to bottom after ultracentrifugation. Steady-State Fluorescence Spectroscopy. LH2 samples were subjected to steady-state fluorescence spectroscopy. The concentration of LH2 in all sample solutions was adjusted to an optical density (OD) of 0.1 in the B850 absorption band. Steady-state fluorescence spectra were obtained using a spectrometer equipped with a CCD detector (Spec-10:100BR/LN, Roper Scientific), monochromators (SP-150 M for excitation and SP-306 for emission, Acton Research Co.), and a lamp house (tungsten halogen light source, TS-428DC, Acton Research Co.), with excitation at 800 nm at an exposure time of 10 s. All of the data were obtained at room temperature. AFM Observation of the Planar Bilayer Membrane of PG Surfactants in the Presence and Absence of LH2 onto a Poly(lysine)-Coated Mica. AFM observations of the planar bilayer membrane of PG surfactants and those containing LH2 were conducted in 20 mM Tris-HCl (pH 8) containing 300 mM KCl (if needed) at ambient temperature. Imaging was conducted using a Picoplus5500 (molecular imaging) in the acoustic AC mode (AAC mode) and a standard silicon probe BL-AC40TS (resonant frequency of 110 kHz in air, tip radius of
