Mar 16, 2016 - The label-free electrochemical platform presented here enables studies of ... proteins, transmission electron microscopy (TEM), scanning.
Article pubs.acs.org/ac
Electrochemical Platform for the Detection of Transmembrane Proteins Reconstituted into Liposomes Jan Vacek,*,† Martina Zatloukalova,† Jaroslava Geleticova,‡ Martin Kubala,‡ Martin Modriansky,† Ladislav Fekete,§ Josef Masek,∥ Frantisek Hubatka,∥ and Jaroslav Turanek∥ †
Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic ‡ Department of Biophysics, Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacky University, Slechtitelu 27, 783 71 Olomouc, Czech Republic § Institute of Physics, Academy of Sciences of the Czech Republic, Na Slovance 2, 18221 Prague, Czech Republic ∥ Department of Pharmacology and Immunotherapy, Veterinary Research Institute, v.v.i., Hudcova 70, 621 00 Brno, Czech Republic S Supporting Information *
ABSTRACT: The development of new methods and strategies for the investigation of membrane proteins is limited by poor solubility of these proteins in an aqueous environment and hindered by a number of other problems linked to the instability of the proteins outside lipid bilayers. Therefore, current research focuses on an analysis of membrane proteins incorporated into model lipid membrane, most frequently liposomes. In this work, we introduce a new electrochemical methodology for the analysis of transmembrane proteins reconstituted into a liposomal system. The proposed analytical approach is based on proteoliposomal sample adsorption on the surface of working electrodes followed by analysis of the anodic and cathodic signals of the reconstituted proteins. It works based on the fact that proteins are electroactive species, in contrast to the lipid components of the membranes under the given experimental conditions. Electroanalytical experiments were performed with two transmembrane proteins; the Na+/K+ATPase that contains transmembrane as well as large extramembraneous segments and the mitochondrial uncoupling protein 1, which is a transmembrane protein essentially lacking extramembraneous segments. Electrochemical analyses of proteoliposomes were compared with analyses of both proteins solubilized with detergents (C12E8 and octyl-PoE) and supported by the following complementary methods: microscopy techniques, protein activity testing, molecular model visualizations, and immunochemical identification of both proteins. The label-free electrochemical platform presented here enables studies of reconstituted transmembrane proteins at the nanomolar level. Our results may contribute to the development of new electrochemical sensors and microarray systems applicable within the field of poorly water-soluble proteins.
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outside the membranes can be altered or nonexistent.5 As a result, membrane model systems for specific MPs are introduced and optimized,6 such as lipid monolayers, supported lipid bilayers, liposomes, lipid nanodiscs, or more complex systems such as lipidic cubic phases.7,8 Electrochemical methods are promising tools for protein investigations. These methods have found broad applications in research focused on the function, structure and biomolecular interactions of nonconjugated water-soluble proteins in particular.9 A number of techniques based on electrochemical sensors and chip technologies were proposed for biomedical applications.9,10 Electrochemical methods are also useful for the analysis of the redox properties of the nonprotein moieties in
embrane protein genes represent approximately a third of all the genes identified so far in humans.1,2 Membrane proteins (MPs) have a variety of functions ranging from membrane transport to cell signal transduction,2 and many of them are molecular targets for drugs and low molecular ligands.3 Therefore, new analytical methods are under development to enable better characterization of MPs in terms of their structure and function. From the methodology point of view, MPs and their complex with lipids serve as the prime example of the most intricate biomacromolecular systems. In particularly with integral MPs, all methodology approaches are limited by the low solubility of MPs in aqueous solutions. One of the basic strategies is the solubilization of proteins in detergents or structure stabilization agents.4 The presence of surface active agents and stabilizers often interfere with the method of analysis. Moreover, the analysis of solubilized proteins may yield artifacts, because the structure and function of MPs © 2016 American Chemical Society
Received: February 15, 2016 Accepted: March 16, 2016 Published: March 16, 2016 4548
DOI: 10.1021/acs.analchem.6b00618 Anal. Chem. 2016, 88, 4548−4556
Article
Analytical Chemistry conjugated proteins,11 and because it is feasible to anchor membrane systems at the surface of electrodes, these methods can be used to analyze the function of membrane transporters,12,13 membrane permeability,14 or enzyme interactions with lipid systems.15 The methodology for the electrochemical monitoring of MPs, besides the examples listed, was introduced by our laboratory in 2012.16 The technique relies on the solubilization of MPs in nonionic detergents and adsorption of the detergent/MP complex to the surface of Hg-containing or carbon electrodes. The proposed methodology was utilized in the investigation of the interactions of low-molecular ligands with MPs and the structural changes in MPs.17 Similarly, an experimental protocol for monitoring the interactions of poorly water-soluble proteins with solid (metallic) surfaces was also introduced.18 This work aims at developing a new electrochemical platform for the analysis of MPs, which is based on chronopotentiometric and voltammetric techniques. Here we focus on two transmembrane proteins, Na+/K+ ATPase (NKA)19 and uncoupling protein 1 (UCP1)20 following their reconstitution into liposomes. The proteoliposomes formed may be adsorbed onto electrode surfaces and henceforth used for the sensitive analysis of MPs. Electrochemical data are complemented by several other methods, including functional analysis of both proteins, transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), and dynamic light scattering (DLS) of the liposomal systems, immunochemical identification of MPs, and molecular models of both proteins.
NKA Proteoliposomes Preparation. NKA from porcine kidney was isolated as described previously.21,22 Proteoliposomes were prepared by cosolubilization of lipids, protein, and detergent. NKA was added to the lipid/detergent mixture. Proteoliposomes were formed by the detergent removal method similarly as described above. Finally, the vesicle suspension was ultracentrifuged for 1 h at 100 000g at 4 °C, and the pellet was resuspended in 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 20 mM KCl, and 3 mM MgCl2. For other details see ref 23. NKA Activity Assay. Measurements of ATPase activity were performed according to ref 24 with some modifications. This method is based on the colored reaction of inorganic phosphate with ammonium molybdate, which is monitored as an absorbance change at 710 nm. For other details see the Supporting Information. Uncoupling Protein 1 (UCP1) Procedures. Isolation and Reconstitution of UCP1. UCP1 was purified from brown fat mitochondria obtained from Golden Syrian hamsters. Briefly, mitochondria amounting to 5 mg of total protein were mixed with poly(ethylene glycol) octyl ether (octyl-PoE) detergent with or without lipids (L-α-phosphatidylcholine and cardiolipin) and then incubated on hydroxyapatite (HPT) column as described previously.25 The HPT eluate containing no lipids was used for control experiments without further manipulation. The HPT eluate containing the protein/ detergent/lipid mixture was adjusted to the internal medium composition (84.4 mM TEA2SO4, 29 mM TEA-TES, 0.6 mM TEA-EGTA, pH 7.2; TEA, tetraethylammonium; TES, 2-[(2hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid; SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium, inner salt; EGTA, ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid). An equivalent HPT eluate containing the protein/detergent/lipid mixture was adjusted to the internal medium composition plus 2 mM SPQ and further incubated for 2.5 h in a Bio-Beads column to slowly remove the detergent and allow the formation of proteoliposomes. The final step involved the removal of the external probe with a Sephadex G-50 column. It should be stressed that with UCP1, the proteoliposomes are formed from the original protein/detergent/lipid mixture by the single-step removal of the detergent and not by insertion of the protein into preformed liposomes. Liposomes lacking the UCP1 protein were prepared by the exact same procedure. Activity Assay of UCP1. The activity of the reconstituted UCP1 was monitored as UCP1-mediated fatty acid anion uniport by the SPQ quenching method. UCP1-mediated uniport was induced by a valinomycin-clamped K+ gradient across the liposomal membrane in the presence of laurate. For other details see ref 25 and the Supporting Information. General Procedures. Microscopy. The morphology of proteoliposomes was characterized using TEM. Specimens for TEM analysis were prepared by drop-casting particles on carbon coated copper grids stained with phosphomolybdenic acid solution (2%) and dried at room temperature before observation. Bright field imaging was performed using TEM (Phillips 208 S, FEI, Czech Republic) operating at 80 kV. SEM and AFM images were obtained using a Hitachi SU 8010 (Hitachi High Technologies, Japan) and Dimension Icon (Bruker, Billerica, MA) microscopes, respectively. For SEM and AFM details, see the Supporting Information. Dynamic Light Scattering. The size distribution of liposomes and corresponding proteoliposomes was determined
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EXPERIMENTAL SECTION Reagents. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), octaethylene glycol monododecyl ether (C12E8), poly(ethylene glycol)octyl ether (octyl-PoE), and sodium dodecyl sulfate (SDS) were obtained from Sigma-Aldrich (St. Louis, MO). Primary antibodies, rabbit IgG anti Na+/K+ ATPase α (H-300), rabbit anti IgG Na+/K+ ATPase β1 (H115), goat polyclonal IgG anti UCP1 (M-17) and secondary antibodies, goat antirabbit IgG-HRP and rabbit antigoat IgGHRP were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Biobeads SM-2 were obtained from Bio-Rad, and amido black 10 B was obtained from Merck (Kenilworth, NJ). Buffer components and other chemicals for NKA and UCP1 isolation and characterization were all obtained from SigmaAldrich. All solutions were prepared using reverse-osmosis deionized water (Ultrapur, Watrex, CZ). Na+/K+ ATPase (NKA) Procedures. Liposomes Preparation. The desired amount of DPPC and DPPE lipids was dissolved in chloroform and dried to obtain a thin film on a glass round-bottom flask using a vacuum pump Heidolph 4000 (Heidolph, Germany). The mixed micelles were prepared by solubilization of the lipid film by 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 20 mM KCl, and 3 mM MgCl2 containing detergent C12E8 (70 mg/mL). Solubilization was performed at 40 °C (the temperature above the critical phase transition temperature of both lipids). Clear solution of lipid mixed micelles was formed within 10 min. Liposomes were prepared by the detergent removal method using the sorbent Biobeads (200 mg/mL) at intervals of 90, 45, and 15 min. Temperature was kept at 40 °C during the detergent removal process. The beads were removed by centrifugation for 5 min at 4 °C. This procedure yielded small vesicles
