Article pubs.acs.org/Langmuir
Binding of a Monoclonal Antibody to the Phospholamban Cytoplasmic Domain Interferes with the Channel Activity of Phospholamban Reconstituted in a Tethered Bilayer Lipid Membrane Serena Smeazzetto,† Alessio Sacconi,† Adrian L. Schwan,‡ Giancarlo Margheri,§ and Francesco Tadini-Buoninsegni*,† †
Department of Chemistry “Ugo Schiff”, University of Florence, 50019 Sesto Fiorentino, Italy Department of Chemistry, University of Guelph, Guelph, ON Canada, N1G 2W1 § Institute for Complex Systems, National Research Council, 50019 Sesto Fiorentino, Italy ‡
S Supporting Information *
ABSTRACT: Phospholamban (PLN), a membrane protein present in the sarcoplasmic reticulum of cardiac myocytes, is a crucial regulator of cardiac function. It is known that PLN appears as a monomer and as a pentamer. However, the role of the PLN pentamer and its ability to generate an ion channel are a matter of debate. To address this issue we employed an experimental approach that combines electrochemical impedance spectroscopy and surface plasmon resonance measurements. In particular, we investigated the channel activity of wildtype PLN reconstituted in a tethered bilayer lipid membrane (tBLM) on a gold surface. Our results indicate that reconstituted PLN can generate ion-conducting channels in a tBLM. Experiments with a PLN monoclonal antibody support an oriented incorporation of PLN in the tBLM. We show that the binding of the antibody to the PLN cytoplasmic domain interferes with PLN channel activity.
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INTRODUCTION Phospholamban (PLN) is a 52 amino acid integral membrane protein present in the sarcoplasmic reticulum (SR) of cardiac myocytes.1 PLN is involved in the contractility of cardiac muscle by regulating SR Ca-ATPase (SERCA). The activity of SERCA is inhibited by unphosphorylated PLN whereas PLN phosphorylation releases SERCA inhibition and allows Ca2+ pumping.2 PLN is composed of a helical cytoplasmic domain at the N-terminus, a semiflexible loop, and a transmembrane domain at the C-terminus which consists of a single-span highly hydrophobic α-helix.1 PLN appears as a monomer (6 kDa) and as a pentamer (30 kDa), and the two forms are in equilibrium.2 In particular, the role of the PLN pentamer is still a matter of active debate.3−7 Some structural data support8,9 and others reject10 the hypothesis that the PLN pentamer can form an ion channel. It was shown that PLN, when incorporated into planar lipid bilayers,4,11,12 exhibits ion channel activity. The reconstituted PLN was found to have moderate selectivity between monovalent cations and no appreciable Ca2+ permeability.12 Moreover, experiments based on the polymeric nonelectrolytes method, which allows us to estimate the pore size,13−15 indicate that the radii for the narrowest part and the wider part of the PLN channel are 2.2 and 6.2 Å, respectively.4 These values are in agreement with the radii found in the NMR structure and by the molecular modeling of pentameric PLN.8,9,16,17 A recent study on the incorporation of phosphorylated PLN in mercurysupported biomimetic membranes shows that phosphorylated © XXXX American Chemical Society
PLN does not permeabilize lipid bilayers toward ions at physiological pH but exerts a permeabilizing action toward monovalent cations following a small decrease in pH.18 In this work we employed surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) to characterize wild-type PLN reconstituted in a tethered bilayer lipid membrane (tBLM). The tBLM is an experimental model of a biological membrane that is used to study channel-forming peptides and proteins14,19−24 because it combines high stability with excellent biomembrane-mimicking features.19 The tBLM consists of a lipid bilayer tethered to a gold surface via a hydrophilic spacer, which is terminated with a thiol or disulfide group that forms a covalent bond with the gold surface.25 By combining optical and electrochemical methods we were able to investigate the channel activity and conductive properties of PLN reconstituted in a tBLM.
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EXPERIMENTAL SECTION
PLN Expression and Purification. The gene encoding human wild-type PLN was expressed as being fused with the maltose-binding protein. The fusion protein was first isolated and then cleaved overnight at room temperature with a TEV protease.26 Subsequently, Received: April 29, 2014 Revised: July 9, 2014
A
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Figure 1. EIS measurements on a DPTL monolayer and a tBLM without or with reconstituted PLN. Impedance spectra were obtained by plotting log(Z) (logarithm of impedance) versus log( f) (logarithm of frequency) (A) and the phase angle versus log(f) (B) for a DPTL monolayer (stars) and a tBLM before PLN addition (filled squares), after PLN addition (empty triangles), and after overnight incubation with PLN (empty circles). The solid lines represent fitting curves of the EIS spectra by the equivalent circuit model shown. The model parameters are explained in the text, and their values are reported in Table 1.
Table 1. Model Parameters Obtained by Fitting the Equivalent Circuit Shown in Figure 1 to the EIS Spectra of Figure 1A,Ba DPTL monolayer tBLM tBLM + PLN (overnight incubation)
CPE (μF·cm−2 s(α − 1))
α
CPEpores (μF·cm−2s(αpores − 1))
αpores
Rpores (kΩ·cm2)
0.71 ± 0.05 0.61 ± 0.05 0.63 ± 0.05
0.99 ± 0.02 0.98 ± 0.02 0.99 ± 0.02
1.37 ± 0.02 0.93 ± 0.01 3.82 ± 0.05
0.58 ± 0.02 0.51 ± 0.02 0.75 ± 0.02
169 ± 15 204 ± 15 35 ± 5
The error gives the deviation from the fit. The solution resistance Rsol was (61.2 ± 1.5) Ω·cm2. For the definition and physical meaning of the model parameters, see the text. a
described31 with an REStec (Resonant Sensor Technology) spectrometer by recording the reflectivity variations versus time at a fixed angle of incidence (57.2°), corresponding to the highest sensitivity of the SPR spectrum whose angular shifts are proportional to the thickness of the adsorbed layer. EIS measurements (bias potential 0.0 V, frequency range from 105 to 10−2 Hz with 10 mV amplitude) were carried out using a PGSTAT12 Autolab potentiostat/galvanostat (Eco Chemie) with a built-in frequency response analysis FRA2 module. All potentials were referred to a three-electrode system, with an Ag/AgCl/0.1 M KCl reference electrode, a Pt counter electrode, and the gold sensor as the working electrode. PLN Reconstitution in a tBLM. Twenty microliters of PLN (1 mg/mL in Milli-Q water) was added to the Teflon cell, close to the bilayer, using a Hamilton syringe (final concentration ∼0.02 mg/mL). EIS and SPR measurements were performed after the initial addition of PLN and after overnight (∼16 h) incubation of the tBLM with the PLN-containing solution. For experiments with the PLN antibody (AbPLN), monoclonal mouse antibody ab2865 (Abcam) was employed. Twenty microliters of AbPLN (1 mg/mL) was added to the Teflon cell (final concentration ∼0.02 mg/mL). AbPLN addition was monitored through SPR and EIS.
PLN was purified using a C18 reverse-phase column. Details of PLN expression and purification are given in ref 4. DPTL Monolayer Formation. 2,3-Di-O-phytanyl-sn-glycerol-1tetraethylene glycol-DL -α-lipoic acid ester lipid (DPTL) was synthesized as previously described.27 EIS and SPR measurements were performed using a gold sensor obtained by depositing a 50 nm gold layer on a glass slide by highvacuum electron beam evaporation. The gold sensor was incubated overnight in 0.2 mg/mL DPTL in ethanol to form a covalently linked DPTL monolayer on the gold surface by self-assembly.28,29 For a DPTL monolayer self-assembled at a gold electrode surface, a packing density of ∼2.2 × 10−10 mol·cm−2 and an area per molecule of ∼85 Å2 were determined by chronocoulometric measurements.28 The gold sensor was then rinsed with ethanol to remove unbound DPTL molecules. The sensor was finally dried under a stream of N2 and was oil-matched to the base of a 45° roof LaSFN9 glass prism (Hellma Optik;, refractive index n = 1.85 at λ = 633 nm). By means of an Oring tight seal, the DPTL-coated gold sensor was mounted in a Teflon cell for in situ EIS and SPR analysis using the Kretschmann configuration.30 tBLM Formation. Vesicles were prepared using 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) (Avanti Polar Lipids). To obtain vesicles homogeneous in size, the vesicle suspension (1 mg/mL in 0.1 M KCl) was extruded through a polycarbonate filter (100-nmdiameter pores, Millipore) using a Hamilton extruder. The suspension of DOPC vesicles was added to the DPTL-coated gold sensor in the Teflon cell. Vesicles unrolled on the DPTL monolayer to form a DPTL/DOPC bilayer (tBLM).21 SPR and EIS Combined in Situ. All measurements were performed in the Teflon cell containing 0.1 M KCl. The DPTL monolayer and the tBLM were characterized by SPR and EIS combined in situ. The Teflon cell was placed on a rotating stage in a θ−2θ configuration. SPR measurements were performed as
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RESULTS AND DISCUSSION In this study we investigated small membrane protein PLN in a suitable biomimetic membrane, tBLM, by combining SPR and EIS. Because of the presence of a hydrophilic layer between the gold surface and the lipid bilayer, tBLMs allow the reconstitution of membrane proteins, e.g., ion channels,14,21,24,25 into the bilayer. An advantageous feature of a tBLM is its much higher stability with respect to the traditional black lipid membrane, whose inherent fragility precludes longB
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the DOPC vesicle suspension is nearly parallel, confirming the correct assembly of a DOPC monolayer on top of the DPTL film to form the complete tBLM. The measured angular shift between the DPTL and tBLM spectra is 1.0°, which, considering a refractive index of 1.52 for the DOPC monolayer,20 leads to an overall tBLM thickness of 8.6 ± 0.3 nm, in agreement with data previously reported.20 PLN Reconstitution in tBLMs. We then investigated PLN reconstitution in tethered bilayers by EIS and SPR measurements. As shown in Figure 1A,B, the impedance spectra of the tBLM before and after PLN addition are practically the same. A significant change in the impedance and phase angle versus frequency curves was, however, observed after overnight incubation of the tBLM with PLN (Figure 1A,B). Indeed, a remarkable decrease in the Rpores value (from 204 to 35 KΩ· cm2) could be determined after overnight incubation of the tBLM with PLN (Table 1). We also notice an increase in CPEpores after overnight incubation as well as a shift of the αpores value toward 1 (Table 1). We propose that the remarkable decrease in Rpores may be correlated with the formation of ionconducting channels following PLN incorporation into the tBLM. Also, the increase in the CPEpores and αpores values may be attributed to the presence of PLN-generated conducting pores in the tBLM. On the other hand, we observe that, after overnight incubation with PLN, the SPR spectrum of the tBLM does not show any significant modification (compare dashed and solid lines in Figure 2), suggesting that PLN reconstitution does not alter the stability and integrity of the tBLM. In fact, a disruption of the tBLM would introduce strong disuniformities in its refractive index that are expected to produce a welldetectable left shift of the resonance angle. In summary, our data suggest that under the present experimental conditions PLN can be reconstituted in a tBLM and generate channels that are permeable to small monovalent cations such as K+, as previously observed.4,12 Experiments with PLN Monoclonal Antibody. PLN reconstitution in the tBLM was further investigated by exploiting the affinity of PLN for its specific monoclonal antibody (AbPLN) and by monitoring via SPR the AbPLN− PLN binding reaction. Following overnight incubation of the tBLM with PLN, AbPLN was added to the cell, and the subsequent reflectivity changes expected from AbPLN−PLN binding were recorded. Assuming for the antibody layer a refractive index of 1.5,35 the reflectivity changes can be easily converted into a thickness increase in the AbPLN layer on top of the tBLM with reconstituted PLN. A typical result, obtained after two AbPLN additions and a final rinsing with 0.1 M KCl, is shown in Figure 3. In particular, a remarkable increase in the thickness of the AbPLN layer was observed after the first AbPLN addition, indicating that AbPLN can interact and bind to the tBLM incorporating PLN. A smaller thickness increase was also detected following a second AbPLN addition. However, as the cell was rinsed with the KCl solution, the thickness of the AbPLN layer decreased to a stable value of 1.7 ± 0.2 nm, very close to the value observed after the first AbPLN addition. Thus, we may conclude that the first AbPLN addition is sufficient to saturate almost all AbPLN binding sites available on the tBLM surface. We also verified the formation of the AbPLN layer on top of the tBLM by recording the final SPR spectrum following the second AbPLN addition and the rinsing step with the KCl solution, represented by the dotted line in Figure 2. A well-resolved angle shift of 0.37° with respect to the
term experiments. Thanks to the high stability of tBLMs, SPR measurements can be conveniently performed and are useful in analyzing molecular interactions on a surface. In particular, SPR allows using label-free antibodies for the detection of a protein target, thus avoiding the use of molecular labels that can compromise protein function.32 Characterization of the DPTL Monolayer and the tBLM. From the electrical point of view, the DPTL monolayer and the tBLM can be described by the equivalent circuit model14,33,34 shown in Figure 1. The model parameters obtained by fitting the equivalent circuit to the EIS spectra (Figures 1A,B) are summarized in Table 1. In the equivalent circuit model (Figure 1), the constant phase element CPE accounts for the monolayer or bilayer capacitance as well as the Helmholtz capacitance of an interface between the gold electrode and the electrolyte solution. (For a detailed characterization of the equivalent circuit model, see refs 14, 33, and 34.) A conduction pathway parallel to CPE consists of a resistance Rpores and a constant phase element CPEpores and accounts for the conductance of either defects in the monolayer or bilayer or PLN-generated channels in the tBLM. The DPTL monolayer and the tBLM are characterized by CPE values (Table 1) that are in good agreement with those reported in the literature,14,22,33 thus confirming the proper formation and stability of the tBLM. For both the DPTL monolayer and the tBLM, the value of the CPE exponent (α) is very close to 1 (Table 1), suggesting a nearly ideal capacitive behavior of the monolayer and lipid bilayer.14,33 Moreover, a tBLM with a small defect density exhibits a CPEpores exponent (αpores) that is close to 0.5 (Table 1: αpores = 0.51 for the tBLM), while an increasing density of defects/pores shifts this parameter toward 1.14,33 Also, the rather high Rpores value (204 kΩ·cm2) for the tBLM is consistent with an electrically insulating bilayer with a low defect density. The SPR spectra for the DPTL monolayer and the tBLM are reported in Figure 2. By comparing the DPTL monolayer trace (dashed−dotted line) and the tBLM trace (solid line), we verified the correct membrane assembly. Indeed, the shift of the SPR spectra following the incubation of the DPTL-coated gold sensor with
Figure 2. SPR spectra of a DPTL monolayer (dashed−dotted line), tBLM before PLN addition (solid line), tBLM after overnight (∼16 h) incubation with PLN (dashed line), and tBLM with reconstituted PLN after AbPLN binding and final washing with 0.1 M KCl (dotted line). C
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channels in the tBLM following PLN incorporation. Interestingly, in the presence of AbPLN, Rpores increases again to a value very close to that determined prior to PLN addition. This result suggests that the PLN-generated conducting channels in the tBLM can be occluded following AbPLN binding. As AbPLN specifically binds to the PLN cytoplasmic domain, our EIS data suggest an oriented incorporation of PLN in the tBLM, that is, with the transmembrane domain immersed in the bilayer and the cytoplasmic domain exposed to the solution containing AbPLN. We propose that the binding of AbPLN to the PLN cytoplasmic domain blocks the ion-conducting channel, thus decreasing the bilayer permeability.
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CONCLUSIONS We combined optical and electrochemical measurements to characterize PLN reconstitution and the PLN ability to form conducting channels in a tBLM. The high stability of tBLMs allows SPR analysis to be performed, which is convenient for investigating molecular interactions on a surface. Our data indicate that reconstituted PLN can generate conducting channels in the tBLM. Moreover, EIS experiments in the presence of a PLN monoclonal antibody indicate an oriented incorporation of PLN into the tBLM. Our experimental approach can be useful in studying the interaction of PLN with molecules that are able to bind to the PLN cytoplasmic domain and may act as potential blockers of the PLN-generated conducting channel.
Figure 3. SPR measurements in the presence of the PLN antibody. Thickness increase versus time curves for AbPLN interaction with a tBLM incorporating PLN (tBLM + PLN) and a tBLM containing no PLN (tBLM, control experiment). The arrows indicate the first (AbPLN1) and second (AbPLN2) additions of monoclonal antibody AbPLN and the final washing step with 0.1 M KCl.
spectrum of the tBLM incorporating PLN (dashed line in Figure 2) is visible, consistent with an AbPLN layer of 1.7 nm thickness, as verified by the best fitting of the final spectrum. As a control experiment, we confirmed that no interaction occurs between AbPLN and a tBLM incorporating no PLN (Figure 3), indicating that AbPLN specifically recognizes PLN reconstituted in the tBLM, as expected. Since the AbPLN used in this work is specific to the PLN cytoplasmic domain, AbPLN can bind reconstituted PLN with the cytoplasmic side facing the bulk solution. We then performed EIS measurements on tBLMs incorporating PLN in the presence of AbPLN. EIS spectra were recorded for a tBLM containing no PLN and following PLN reconstitution in the absence or presence of AbPLN at applied electrode potentials of −0.1, 0, and 0.1 V (versus Ag/AgCl/0.1 M KCl) (Figures S1−S3 in Supporting Information). Table 2
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EIS spectra for a tBLM containing no PLN and following PLN reconstitution in the absence or presence of AbPLN at applied electrode potentials of −0.1, 0, and 0.1 V (versus Ag/AgCl/0.1 M KCl). The EIS spectra were fitted using the equivalent circuit model shown in Figure 1, and the model parameters are reported in the corresponding table. This material is available free of charge via the Internet at http://pubs.acs.org.
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Table 2. Rpores at Applied Electrode Potentials of −0.1, 0, and 0.1 V (versus Ag/AgCl/0.1 M KCl) for a tBLM Containing No PLN and Following PLN Reconstitution in the Absence or Presence of AbPLNa
AUTHOR INFORMATION
Corresponding Author
*Phone: +39-055-4573239. Fax: +39-055-4573142. E-mail: francesco.tadini@unifi.it. Notes
The authors declare no competing financial interest.
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Rpores (kΩ·cm2) tBLM tBLM + PLN (overnight incubation) tBLM + PLN + AbPLN
ASSOCIATED CONTENT
S Supporting Information *
−0.1 V
0V
0.1 V
193 ± 15 28 ± 5
204 ± 15 35 ± 5
208 ± 15 42 ± 5
201 ± 15
187 ± 10
191 ± 15
ACKNOWLEDGMENTS We wish to thank Dr. Paolo Marsili for his help in the manufacturing of the SPR gold sensors. Financial support from the Italian Ministry of Education, University and Research (PON01_00937) and Ente Cassa di Risparmio di Firenze is gratefully acknowledged.
The Rpores values were obtained by fitting the equivalent circuit model shown in Figure 1 to the EIS spectra of Figures S1−S3. The error gives the deviation from the fit. a
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reports the resistance values (Rpores) obtained by fitting the equivalent circuit model (shown in Figure 1) to the EIS spectra of Figures S1−S3 (Tables S1−S3 in Supporting Information). It is worth noticing that Rpores for the tBLM containing no PLN does not vary significantly between +0.1 V and −0.1 V. On the other hand, we point out that after PLN incubation Rpores remarkably decreases at all potentials with respect to the value obtained before PLN addition. As mentioned above, the Rpores decrease can be explained by the presence of conducting
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