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Electrochemical Platform for the Detection of Transmembrane Proteins Reconstituted into Liposomes Jan Vacek, Martina Zatloukalova, Jaroslava Geleticova, Martin Kubala, Martin Modriansky, Ladislav Fekete, Josef Mašek, Frantisek Hubatka, and Jaroslav Turánek Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00618 • Publication Date (Web): 16 Mar 2016 Downloaded from http://pubs.acs.org on March 17, 2016

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Analytical Chemistry

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,∥ 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

*

Corresponding author: Tel.: +420585632303; Fax: +420585632302; E-mail address:

[email protected]

1 ACS Paragon Plus Environment


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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.

Key words: electroanalysis; electrochemical sensors; membrane protein; protein-lipid interactions; detection surface; electrodes


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Introduction Membrane 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 outside the membranes can be altered or non-existent.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 non-conjugated 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 non-protein moieties in 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



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MPs, besides the examples listed, was introduced by our laboratory in 2012.16 The technique relies on the solubilization of MPs in non-ionic 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.

Experimental Reagents. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero3-phosphoethanolamine

(DPPE),

octaethylene

glycol

monododecyl

ether

(C12E8),

poly(ethylene glycol)octyl ether (octyl-PoE), sodium dodecyl sulfate (SDS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies: rabbit IgG anti Na+/K+ ATPase α (H-300), rabbit anti IgG Na+/K+ ATPase β1 (H-115), goat polyclonal IgG anti UCP1 (M-17) and secondary antibodies: goat anti-rabbit IgG-HRP and rabbit anti-goat IgGHRP were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Bio-beadsTM


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SM-2 were obtained from Bio-Rad, and amido black 10 B was obtained from Merck (Kenilworth, NJ, USA). Buffer components and other chemicals for NKA and UCP1 isolation and characterization were all obtained from Sigma-Aldrich. 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 containig 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 detergent removal method using the sorbent Bio-beads (200 mg/mL) at intervals of 90, 45 and 15 min. Temperature was kept at 40 °C during detergent removal process. The beads were removed by centrifugation for 5 min at 4 °C. This procedure yielded small vesicles < 200 nm.

NKA Proteoliposomes Preparation. NKA from porcine kidney was isolated as described previously.21,

22

Proteoliposomes were prepared by co-solubilization of lipids, protein and

detergent. NKA was added to the lipid/detergent mixture. Proteoliposomes were formed by the detergent removal method as described above. Finally, the vesicle suspension was ultracentrifuged for 1 h at 100,000 g at 4 °C, and the pellet was resuspended in 20 mM TrisHCl (pH 7.4), 140 mM NaCl, 20 mM KCl and 3 mM MgCl2. For other details see.23



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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 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 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 mixture was adjusted to the internal medium composition (84.4 mM TEA2SO4, 29 mM TEATES, 0.6 mM TEA-EGTA, pH 7.2)*. 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 hours 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 singlestep removal of the detergent and not by insertion of the protein into pre-formed liposomes. Liposomes lacking the UCP1 protein were prepared by the exact same procedure.

*

Abbrev. TEA – tetraethylammonium; TES – 2-[(2-hydroxy-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.


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Activity Assay of UCP1. The activity of the reconstituted UCP1 was monitored as UCP1mediated 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 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 Hitachi SU 8010 (Hitachi High Technologies, Japan) and Dimension Icon (Bruker, Billerica, Massachusetts, USA) microscopes, respectively. For SEM and AFM details see Supporting Information.

Dynamic light scattering. The size distribution of liposomes and corresponding proteoliposomes was determined by DLS using Zetasizer Nano ZS (Malvern, UK). Silica cuvette of 45-µl volume (Hellma, Germany) was used. The measurements were carried out at 25 ºC. The size distribution of the liposomes was expressed as number distribution of hydrodynamic diameter.

Western Blot Analyses and Determination of Protein Concentration. Protein samples were mixed and incubated for 5 min at 95 °C with loading buffer (containing 0.125 M Tris-HCl (pH 6.9), 4% SDS, 0.2 M dithiotreitol, 20% glycerol and 0.02% bromphenol blue) and then electrophoresed through 10% SDS-polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride membrane by electroblotting, and the membranes were blocked



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with 5% milk in 100 mM Tris (pH 7.4), 0.9 % NaCl, 0.05% Tween 20 for 2 h at room temperature, followed by incubation with appropriate primary antibodies overnight. Primary antibodies were visualized with goat anti-rabbit or rabbit anti-goat horseradish peroxidase conjugated secondary antibodies using a chemiluminescent reaction. The intensity of bands was determined by densitometric analysis using ImageJ software (ver. 1.4). Kaplan method was used for the determination of protein concentration.26 For details see Supporting Information.

Electrochemical Measurement. The solubilized MPs and (proteo)liposomal systems were analyzed using ex situ (adsorptive transfer) voltammetry and chronopotentiometry. Three types of working electrodes were used: an HMDE (hanging mercury drop electrode; area 0.4 mm2), a silver solid amalgam electrode, Ag-SAE (Hg/Ag ratio 70/30 (w/w), area 0.8 mm2) and a basal-plane pyrolytic graphite working electrode, PGE (9 mm2, source of PG: Momentive Performance Materials, USA). The electrodes were first dipped into a 5-µL aliquot of the studied sample. After an accumulation period (tA, time of accumulation), the electrodes were washed with deionized water and placed in an electrochemical cell containing pure

supporting

electrolyte.

Square

wave

voltammetry

(SWV),

constant-current

chronopotentiometric stripping analysis (CPSA) or alternating-current voltammetry (ACV) were performed at room temperature with a µAutolab III analyzer (Metrohm Autolab, NL) in a three-electrode setup with Ag/AgCl/3 M KCl electrode as a reference and a platinum wire as the auxiliary electrode. After each electrochemical experiment, the working electrode surface was regenerated according to the previously developed procedures.16, 18, 27

Molecular Models. Under the conditions used in the electrochemical experiments, NKA should be presumably found in its E1 conformation. The X-ray-determined high-resolution


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structure of NKA in the E1 conformation

28

was taken directly from the RCSB Protein Data

Bank (4HQJ). For UCP1, no experimentally-determined high-resolution structure has been reported so far, and so we prepared a homology model, see Scheme S1 in Supporting Information. The figures were prepared in PyMOL (Molecular Graphics System Version 1.6.0.0 Schrödinger, LLC) using a Conolly type surface.

Results and Discussion In this work we focused on the electrochemical characterization of two transmembrane proteins, NKA and UCP1, analyzed after reconstitution into liposomes. These results on the electrochemical behavior of proteoliposomal systems are presented here for the first time. The main portion of the experimental work was completed using NKA as the model protein, since it is an MP whose structure, function and physico-chemical properties have been described in detail.19

Preparation and Quality Evaluation of Samples. Three types of samples were tested throughout the study, a schematic representation is given on top of Figure 1. NKA was first solubilized in 4.5 mM non-ionic detergent C12E8,29, 30 that same sample was then stripped of the detergent and the protein was reconstituted into liposomes prepared from DPPC and DPPE lipids.23 The result of this protocol are proteoliposomes whose morphology and average size (< 200 nm) were verified by the negative staining TEM method

31

and DLS before

launching into the electrochemical analyses, see Figure 2A,C and Table S1 in Supporting Information. Because NKA is a protein consisting of two subunits, analyses confirming the presence of both subunits in the proteoliposomes were performed. We used immunodetection 32

with a polyclonal antibody H-300 against the 551-850 aa of the α1 subunit and antibody H-

115 against the 41-155 aa of the β1 subunit. SDS-PAGE and Western blots unequivocally



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confirmed the validity and structural integrity of the proteoliposomal samples (Figure 1A, inset). Evaluation of a functional protein was performed prior to any electrochemical analyses 33

whereby the detergent-solubilized as well as the reconstituted version are referred to as

functional (Figure 1A). For this purpose the enzymatic activity of NKA, i.e. the ability to hydrolyze ATP, was measured. For reconstituted NKA, the activity was higher than that in a sample prepared by simple solubilization in a detergent. Our results confirm that the protein integrated into a suitable lipid bilayer organizes itself into an optimal conformation, from the functional point of view, as the stabilizing effects elicited by the membrane lead to a higher activity than the detergent-solubilized protein. Liposomes and medium alone served as the control samples for electrochemical analyses, all of which, including adsorption of samples onto the electrodes, utilized the same medium, i.e. 50 mM Tris-HCl (pH 7.4) + 140 mM NaCl + 20 mM KCl + 3 mM MgCl2. The protein conformation in this particular environment was E1, and we used this conformation in our molecular model that served for results interpretation (see below). The activity of the reconstituted UCP1 protein was verified by the SPQ quenching method. Rates achieved in the presence of 20 µM laurate were comparable to those reported previously

25

and were inhibited by 1 mM guanosine diphosphate (GDP). The GDP binding

site within the UCP1 protein is only accessible from one side, under physiological conditions from the intermembrane space. The inhibition reached approximately 50%, thereby reflecting the approximately equal orientation of the UCP1 protein in the proteoliposomes, a phenomenon that is well known and described previously 25 (Figure 1B). The presence of the UCP1 protein in the proteoliposomes was verified by SDS-PAGE and Western blotting (Figure 1B, inset). Liposomes prepared by the same method, but lacking the UCP1 protein, displayed negligible activity regardless of the presence or absence of GDP in the assay,


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apparently reflecting only proton leak. Solubilized protein, i.e. the protein/lipid/detergent mixture, exhibited no UCP1 activity. This is consistent with the transporter characteristics of UCP1 protein that requires full assembly within a lipid membrane for its activity. In the absence of a membrane, no UCP1-mediated transport is observable.

Sample Adsorption and Electrode Preparation. Functional and integral samples prepared according to the protocols described above were adsorbed onto the electrode surface for 30 seconds, unless stated otherwise, followed by washing of the excess unadsorbed sample with distilled water. Electrodes prepared in this fashion, i.e. the adsorptive transfer technique, were used for electrochemical analyses.34 Such an approach is suitable to minimize the overuse of a sample whose volume was 5 µL for a single analysis, which is an important prerequisite, especially in MPs sensing where the isolated amount of functional proteins is often limited. Proteins and lipid systems alike are surface-active substances that adsorb easily onto working electrode surfaces, e.g. previously reported for liposomes’ interaction with Hg and carbon

37

35, 36

materials. Here, the basal-plane pyrolytic graphite electrode (PGE) and Hg-

containing electrodes were used for the investigation of anodic and cathodic processes, respectively. Apart from classical HMDE, Ag-SAE was applied for cathodic sensing because of the high applicability of amalgam materials in the development and construction of sensors for proteins and DNA.38, 39 The formation of adsorbed layers of proteoliposomes was monitored by SEM and AFM with solid electrode materials and by ACV with a Hg-electrode (see below). Two types of model substrates were used for microscopy experiments: a model mica slide and highly oriented pyrolytic graphite (HOPG). These model materials were selected because their surface is highly homogeneous compared to the actual electrode materials, i.e. the basal-plane pyrolytic graphite and silver amalgam used in this study. All experiments were performed



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with NKA proteoliposomes using aqueous adsorption media. The adsorbability of proteoliposomes was first confirmed by an SEM approach (Figure 2D,E) followed by detailed AFM characterization (Figure 2F-I). During the adsorption process, proteoliposomes interact with the surfaces as well as with each other, at full surface coverage, leading to microdomain formation and separate islands consisting of proteoliposomes on the surface of the electrode under investigation. This phenomenon was exemplified using HOPG and mica surfaces (Figure 2F,G, left). In addition, we also visualized single proteoliposomes (localized individually) at the same surfaces that undergo structure deformation, the spreading effect (Figure 2F,G, middle). A similar effect was also observed at the surfaces of Ag-SAE (Figure 2H) and PGE (Figure 2I). This observation is in good agreement with other authors.35-37 Spreading effects were subsequently investigated using profilometric analysis, where the average diameters (n=12) of NKA proteoliposomes in the adsorbed state are 114.9±12.1 nm (for Ag-SAE) and 113.5±11.6 nm (for PGE). High-resolution imaging was performed with HOPG substrates, AFM micrographs acquired in various experimental modes can be found in Figures S1 and S2 in Supporting Information.

Electrochemistry of Na+/K+ ATPase Proteoliposomes. The layer of proteoliposomes adsorbed onto the surface of the Hg-electrode was subjected to chronopotentiometric stripping (CPS) at constant current, and the results were compared to the results acquired with solubilized NKA. 16, 40

The proposed approach is based on the electrochemical inactivity of lipid components

under the given experimental conditions. The electrochemical signals are only produced by solubilized or reconstituted proteins. The cathodic signal, peak H, was observed in the CPS analysis, 41, 42 which is ascribed to electrocatalytically active amino acid (aa) residues predominantly localized at the protein surface, i.e. aa residues accessible to the surface of the working electrode. Peak H is related to


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the basic aa residues Lys, Arg and His as well as Cys residues.43-46 In NKA we found 85 Lys, 61 Arg, 17 His and 30 Cys residues in total, but only some of them participated in the electrocatalytic reaction. The localization of electrocatalytically active amino acid residues on the surface of NKA is highlighted in surface molecular models in Scheme 1. For proteoliposomes and solubilized NKA, peak H was observed at a potential of around –1.87 V. The height of peak H was lower in proteoliposomal samples than in the solubilized protein, an effect primarily linked to the competition between liposomal lipid components and protein for the active surface of the working electrode. Pure liposomes did not provide any peak H, as determined using both HMDE and Ag-SAE (Figure 3A). In the next phase of experimental work square-wave voltammetry (SWV) was applied for the analysis of anodic currents in the investigated samples. For this purpose, the Y&W peak of NKA at a potential of +0.79 V was monitored in proteoliposomal samples. This peak can be generally ascribed to the anodic reaction of Tyr and Trp residues.47, 48 In total 45 Tyr and 16 Trp are found in the structure of NKA. For surface visualization of Tyr and Trp residues that were most likely involved in the anodic reaction, see the surface models in Scheme 1B. Similarly to CPSA results, with SWV the relevant electrochemical signal was only observed for the solubilized protein or NKA proteoliposomes. Neither liposomes nor pure Tris-HCl buffer (for composition see above) produced any electrochemical responses (Figure 3B). The interfacial behavior of adsorbed proteoliposomes at the HMDE was investigated by ACV (Figure 3B, inset). The experimental setup requires an out-of-phase configuration that is sensitive to adsorption/desorption processes.49 The p.z.c. (potential of zero charge) of the electrode can be found at around –0.6 V, where neither the adsorbed layer of proteoliposomes, liposomes nor the solubilized protein underwent desorption during the cathodic scan at either a positively or negatively charged surface. The desorption processes



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can only be found at strictly negative potentials at around –2 V. The above could contribute to the reproducibility of our developed electrochemical method. Finally, the dependence of the heights of cathodic peak H and anodic peak Y&W on the concentration of NKA was plotted (Figure 4). The concentration of the isolated protein was adjusted using a complementary technique developed for the purposes of protein trace analysis in the presence of a high excess of lipids.26 Calibration curves were constructed in the concentration range 100-1500 nM (Figure 4A) and 0.5-10 µM (Figure 4B). For CPSA, the linear concentration range was observed to be approx. from 100 to 500 nM with R2 = 0.98 in the proteoliposomal sample and with R2 = 0.97 in the solubilized NKA sample. For SWV, a linear range was maintained from 0.5 to 6 µM for both proteoliposomes (R2 = 0.98) and for the solubilized protein (R2 = 0.99). Both cathodic and anodic approaches enabled a nanomolar/micromolar concentration of reconstituted NKA to be analyzed. This indicates that the developed electrochemical sensing platform presented here is useful for the quantitative analysis of proteoliposomes and mixed protein samples with a high lipid content. Details on the quantitative NKA analysis can be found in Table S2 (see Supporting Information).

Method Applicability. The applicability of our methodology was evaluated by the analysis of another protein, UCP1.20 UCP1 was used for the same electrochemical analyses as NKA, including a determination of its activity (Figure 1B) as described above, TEM and DLS liposome characterization (Figure 2B,C and Table S1 in Supporting Information), and an immunochemical analysis (inset in Figure 1B) of its proteoliposomes.25 UCP1 is more hydrophobic than NKA, and thus a more complex solubilizing mixture was used for its solubilization, i.e. poly(ethylene glycol)octyl ether (octyl-PoE), L-α-phosphatidylcholine and cardiolipin. Reconstitution was performed by a similar procedure as with NKA, by using a liposomal model prepared from the same components as were used for the solubilization


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step.25 The buffered medium for UCP1 isolation, preparation of proteoliposomes as well as the adsorption step were as follows: 84.4 mM TEA2SO4, 29 mM TEA-TES, 0.6 mM TEAEGTA (pH 7.2). The peak H potential for UCP1 proteoliposomes and the solubilized sample was –1.93 V and –1.91 V, respectively. The oxidation peak Y&W was determined at +0.79 V (for the proteoliposomes) and at +0.80 V (for the solubilized sample), see panels A and B in Figure 5. The electrochemical signals in UCP1 proteoliposomes are lower than those for solubilized protein (Figure 5), which resembles the results with NKA. In cathodic CPS scans of solubilized UCP1, one can observe not only peak H but also peak S

50-52

at a potential of

around –0.6 V. This peak is ascribed to a reduction of the Hg-S bond, which is formed between Cys-containing proteins and the Hg-electrode surface. Monitoring of peak S could be used in further studies devoted to interactions and the oxidative modification of Cys residues in UCP1. For this purpose, we previously introduced a methodology that enabled us to perform analyses of the water-soluble recombinant large cytoplasmic loop (C45) of NKA.16 The fact that peak S is not observable for NKA could be connected to specific interferences of NKA with the detergent C12E8

16

(for the solubilized sample) and most likely also to

competition with the protein for access to the active electrode surface in the presence of coadsorbing lipids (for the reconstituted sample). All the above indicates that peak S monitoring in MPs will be strictly dependent on the choice of solubilizing agents and lipid systems, and also the accessibility of Cys residues of the investigated protein to the surface of the working electrodes. These statements are in good agreement with our previously published observations.16 The total electroactive aa distribution in UCP1 is as follows: 9 Tyr and 2 Trp (for anodic peak Y&W) and 7 Cys, 16 Lys, 12 Arg and 4 His (for cathodic peak H). To better understand which aa residues are most likely involved in electrochemical reactions, see the



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surface models in Scheme S2 (see Supporting Information). Not only for UCP1, but also for NKA and other MPs, it is most likely that only the surface parts of MPs that did not interact with lipid membranes, and partially also with detergents, will be involved in the monitored electrochemical reactions. As in NKA, quantitative analysis results for solubilized and reconstituted UCP1 are summarized for both peak H and Y&W in Figure 5, panels C,D and Table S2 (see Supporting Information). The quantitative results acquired with transmembrane NKA and UCP1 confirm that the developed electrochemical platform will be applicable for investigating the redox and electrocatalytic behavior of various transmembrane MPs and their sensitive electrochemical analysis in an excess of lipid membranes in general.

Conclusions The focus of this manuscript is the first electrochemical characterization of proteoliposomes, i.e. generally well defined complexes of liposomes with MPs. The method relies on the oxidation/reduction of proteoliposomes containing Na+/K+ ATPase or UCP1. Proteoliposomes are analyzed in an adsorbed state. The lipid components are electrochemically inactive under our experimental conditions on both Hg-containing as well as carbon electrodes, and hence only the reconstituted proteins are subject to the electrochemical reaction(s). To our knowledge, only the electrochemical analysis of water-soluble fragments of MPs 22, 53 or MPs solubilized with the aid of amphiphilic solubilizing agents16-18, 40 have been reported so far. Taking this into account, the proposed method is a unique approach for investigating redox changes and the analysis of trace amounts of MPs in their native environment of lipid membranes. We think that this method will be applicable in future research on the intermolecular interactions of MPs with the membraneous systems alone and with ligands, e.g. drugs. Moreover, technologies based on the analysis of MPs using amalgam


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electrochemical sensors

9, 38

could be utilized for the construction of simple detectors taking

advantage of chip or microfluidic platforms.

Acknowledgements. The authors are indebted to dr. Bohdan Josypcuk (J. Heyrovsky Institute of Physical Chemistry of the ASCR, CZ) for preparation of the Ag-SAE, to Jakub Holan MSc. (Measurement Technic Moravia Ltd., CZ) for technical assistance with AFM, to dr. Martin Jaburek (Institute of Physiology of the ASCR, CZ) for kindly providing samples of brown fat mitochondria, to dr. Roman Kouril (Palacky University, CZ) for technical assistance with TEM, and to Ben Watson-Jones MEng. for language correction. This work was supported by the Czech Science Foundation (14-08032S, J.V.), by the MEYS / COST Action EU-ROS, BM1203 (LD14033, J.V.), by the Institutional Funding from Palacky University (IGA_LF_2016_012, J.V, M.Z. and M.M.), by the Project Centre of Excellence for Nanotoxicology CENATOX GAP503/12/G147 (J.T.), by the project MZE0002716202 of the Czech Ministry of Agriculture (J.T.), by the grant No. LO1204 (Sustainable development of research in the Centre of the Region Haná) from the National Program of Sustainability I, MEYS (J.G., M.K.) and by the MEYS project SAFMAT LM2015088 and LO1409 (AFM and other technical facilities under supervision of dr. Irena Kratochvilova at Institute of Physics of the ASCR, CZ). Technical facilities of Central European Institute of Technology (Masaryk University, Brno, CZ) are gratefully acknowledged, project LM2011020 by dr. Petr Skladal.

Supporting Information Experimental details: NKA and UCP1 activity analysis, AFM and SEM imaging and Kaplan method for the determination of protein concentration. Results: UCP1 homology model with electroactive aa residues localization on the surface of UCP1, size and polydispersity of liposomal samples by DLS, quantitative analysis of reconstituted and solubilized NKA and 17 ACS Paragon Plus Environment


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UCP1, AFM imaging of NKA proteoliposomes adsorbed onto HOPG surface and 3D micrographs of NKA proteoliposomes adsorbed onto HOPG surface (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.


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Figure Legends

Scheme 1. Electroactive amino acid residues on the surface of Na+/K+ ATPase, (A) Lys (orange), Arg (red), His (green), and (B) Tyr (blue), Trp (magenta), Cys (yellow). The left and right images are mutually rotated by 180° along the vertical axis for each panel. The horizontal lines approximately delineate the membrane.

Figure 1. Activity of native (full columns) and inhibited (unfilled columns) Na+/K+ ATPase – NKA (A) and uncoupling protein 1 – UCP1 (B) after reconstitution into liposomes (n=3). Insets: Western blots demonstrating α and β subunits of NKA (panel A) and UCP1 monomer (panel B) in following samples: 1) negative control (bovine serum albumin), 2) solubilized protein, 3) protein reconstituted into liposomes, and 4) pure buffer. NKA and UCP1 were inhibited with 1 mM ouabain and 1 mM GDP. There is a disproportional scale of proteins and membranes in the top panel.

Figure 2. Characterization of liposomal and proteoliposomal samples by microscopic and dynamic light scattering techniques. TEM images of (A) NKA and (B) UCP1 proteoliposomes. (C) DLS distribution diagram of NKA and UCP1 liposomes and proteoliposomes involved in this study. SEM imaging of NKA proteoliposomes (white spheres) adsorbed onto HOPG surface in deceleration (D) and backscattered (E) modes. 3D visualization of (F) HOPG and (G) mica slide covered with NKA proteoliposomes, detailed view of single proteoliposomes in adsorbed state (white arrows), and bare surfaces are shown on left, middle and right, respectively. AFM micrographs and profilometry results of NKA proteoliposomes (white arrows) adsorbed onto Ag-SAE (H) and basal-plane PGE (I) surfaces. For micrographs in middle of panels F and G, the signal of the topography is overlaid with 21 ACS Paragon Plus Environment


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non-calibrated Jung modulus signal which represents the material contrast; darker areas are softer (3D modulus).

Figure 3. Chronopotentiograms (A) and SW voltammograms (B) of Na+/K+ ATPase (NKA) in Britton-Robinson buffer (pH 6.5). Protein concentration: 500 nM (for A and Inset in panel B), 250 nM (for Inset in panel A) and 10 µM (for B). Ex situ CPSA (panel A): working electrode HMDE (Ag-SAE for Inset), time of accumulation 30 s, stripping current –30 µA (– 60 µA for Inset), Einit 0 V, Eend –2 V. Ex situ SWV (panel B): working electrode PGE, time of accumulation 60 s, frequency 20 Hz, Einit 0 V, Eend +1.3 V. Ex situ out-of-phase ACV (inset in panel B): time of accumulation 30 s, frequency 66.2 Hz, amplitude 5 mV, phase angle 90°, Einit 0 V, Eend –2 V.

Figure 4. Dependence of protein signal height on concentration of solubilized and reconstituted Na+/K+ ATPase (NKA) using ex situ CPSA (A) and SWV (B). For this purpose, cathodic peak H (c.f. A) and anodic peak Y&W (c.f. B) were monitored after the accumulation step tA = 30 s. Britton-Robinson buffer (pH 6.5) was used for both A and B as the supporting electrolyte. CPSA: working electrode HMDE, stripping current –30 µA, Einit 0 V, Eend –2 V. SWV: working electrode PGE, frequency 20 Hz, Einit 0 V, Eend +1.3 V.

Figure 5. Chronopotentiograms (A) and SW voltammograms (B) of 500 nM (for A) and 10 µM (for B) uncoupling protein 1 (UCP1) in Britton-Robinson buffer (pH 6.5). Dependence of peak H (C) and Y&W (D) heights on concentration of solubilized and reconstituted UCP1. Insets: zoomed area from –1 to –0.4 V (for panel A) and zoomed Y-axis (for panel C). Ex situ CPSA: working electrode HMDE, time of accumulation 30 s, stripping current –30 µA, Einit 0


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V, Eend –2 V. Ex situ SWV: working electrode PGE, time of accumulation 60 s, frequency 20 Hz, Einit 0 V, Eend +1.2 V.



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Scheme and Figures Scheme 1.

Electrochemical Sensing of Proteoliposomes ACS Paragon Plus Environment

1

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Figure 1.


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Figure 2.


3

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Figure 3.


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Figure 4.


5

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Figure 5.


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Graphical Abstract:

Electrochemistry of Reconstituted Membrane Proteins Anode

Cathode

Ads.

MP

Ads.

Red. Liposome

MP

Ox. Liposome

Lys, Arg, His and Cys-electrocatalysis

Tyr and Trp-oxidation

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