J. Phys. Chem. B 2008, 112, 3193-3201
3193
Activity of Membrane Proteins Immobilized on Surfaces as a Function of Packing Density Marcel G. Friedrich,†,‡ Vinzenz U. Kirste,†,‡ Jiapeng Zhu,§ Robert B. Gennis,§ Wolfgang Knoll,† and Renate L. C. Naumann*,† Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany, and Department of Biochemistry, UniVersity of Illinois, 600 South Mathews Street, Urbana, Illinois 61801 ReceiVed: October 4, 2007; In Final Form: December 27, 2007
A systematic study of the influence of the packing density of proteins on their activity is performed with cytochrome c oxidase (CcO) from R. sphaeroides as an example. The protein was incorporated into a proteintethered bilayer lipid membrane and CcO was genetically engineered with a histidine-tag, attached to Subunit II, and then tethered by an interaction with functionalized thiol compounds bound to a gold electrode. The packing density was varied by diluting the functionalized thiol with a nonfunctionalized thiol that does not bind to the enzyme. After attaching the CcO to the gold surface, a lipid bilayer was formed to incorporate the tethered proteins. The reconstituted protein-lipid bilayer was characterized by surface enhanced infrared reflection absorption spectroscopy (SEIRAS), electrochemical impedance spectroscopy, surface plasmon resonance, and atomic force microscopy. The activity of the proteins within the reconstituted bilayer was probed by direct electrochemical electron injection and was shown to be very sensitive to the packing density of protein molecules. At low surface density of CcO, the bilayer did not effectively form, and protein aggregates were observed, whereas at very high surface density, very little lipid is able to intrude between the closely packed proteins. In both of these cases, redox activity, measured by the efficiency to accept electrons, is low. Redox activity of the enzyme is preserved in the biomimetic structure but only at a moderate surface coverage in which a continuous lipid bilayer is present and the proteins are not forced to aggregate. Electrostatic and other interaction forces between protein molecules are held responsible for these effects.
Introduction A central issue of studying proteins confined to surfaces is the preservation of their activity. This is particularly critical for membrane proteins since they require a lipid environment but are still subject to denaturation in the immediate vicinity of surfaces. To address this problem, biomimetic membrane systems have been developed such as solid-supported membranes (sBLMs),1 polymer-supported membranes,2 hybrid bilayer lipid membranes,3 and tethered bilayer lipid membranes (tBLMs).4,5 Advantages and disadvantages of these systems have been discussed in a recent review.6 All of them provide the lipid environment required for the function of the protein. Polymersupported membranes also provide a hydrophilic cushion separating the lipid bilayer from the metal or semiconductor support so as to overcome the strong hydrophobic interaction of the protein with the solid support which otherwise would lead to impairment of the function or even to the denaturation of the protein. The hydrophilic polymer seems to be the ideal substitute of the intermembrane space considering the tendency of lipid vesicles to spread on hydrophilic surfaces, such as glass or silicon to spontaneously form well ordered fluid lipid bilayers. Polymer-supported membranes have thus been used very successfully, mostly as a model of the cell surface.7 The use as a model system for charge-transfer processes, however, was less frequent. Many of these systems do not fulfill the requirement, the so-called giga-seal derived from patch-clamp measurements, * Corresponding author. E-mail:
[email protected]. † Max Plank Institute for Polymer Research. ‡ Marcel G. Friedrich and Vinzenz U. Kirste contributed equally to this work. § University of Illinois.
to separate the small current signal of the receptor from the leak current of the membrane. Highly insulating polymersupported membranes have been prepared, though, provided a very smooth surface of the hydrophilic polymer could be achieved.8 There are, however, many more examples taking advantage of the tethering strategy applied to polymers and more often to oligomeric hydrophilic spacers attached to the hydrophilic head group of a lipid molecule.4,5 Monolayers of such compounds provide the highly hydrophobic surface known to promote the spreading of vesicles and the formation of lipid bilayers while the tethering molecules provide an interstitial space separating the lipid from the surface by an aqueous reservoir.9 Polymeric tethers seem to be a greater advantage, compared with oligomeric ones, provided they form highly ordered monolayers. Short tether molecules, however, are particularly useful when redox proteins are to be addressed by bioelectronic coupling to the electrode. The tether molecule then serves a second purpose, namely, the electronic wiring of the redox centers of the protein to the electrode. This purpose would be very hard to achieve by a hydrophilic polymer. One of the most promising systems investigated in this context is the protein-tethered bilayer lipid membrane (ptBLM),10-12 designed for complex redox-active membrane proteins such as the cytochrome c oxidase (CcO) (Figure 1). It is based on an nitrilotriacetic acid (NTA) functionalized surface which, after chelation with Ni2+ ions, reversibly binds proteins genetically engineered with histidine (his)-tags. A lipid bilayer is then reconstituted in situ around the bound proteins. Among the benefits of this strategy, in addition to defining the orientation of the protein with respect to the surface is the possibility to systematically vary the surface concentration of the protein. One
10.1021/jp709717k CCC: $40.75 © 2008 American Chemical Society Published on Web 02/19/2008
3194 J. Phys. Chem. B, Vol. 112, No. 10, 2008
Friedrich et al.
Figure 1. Schematics of the reconstitution of the protein (cytochrome c oxidase with the his-tag attached to SU II) bound via the Ni complex to the NTA modified gold surface into a protein-tethered bilayer lipid membrane (ptBLM).
can, therefore, test the possibility that inter-protein interactions might affect the activity of the enzymes. The distance between single proteins could be an important parameter considering that electrostatic and other interaction forces between molecules could influence charge transfer and other kind of processes involved in protein function. The above-mentioned ptBLM system is well-designed for this purpose. The surface concentration of proteins can be easily varied by varying the concentration of the chelator nitrilotriacetic acid (NTA) molecules. Two opposite orientations of the protein were investigated, either with the cytochrome c binding site directed toward the electrode surface11,12 or pointing away from it,10 simply by engineering the his-tag on the C-terminus of Subunit (SU) II or Subunit I, respectively. Bioelectronic coupling of the protein was achieved but only when primary electron acceptor, the CuA center, was directed toward the electrode, that is, when the his-tag was attached to SU II.11,12 Indications of the significance the surface concentration as a critical parameter were obtained in previous investigations of the CcO engineered with the his-tag on the C-terminus of Subunit I10 and II.11,12 For example, electron transfer initiated by direct electrochemical electron injection into the protein, which can be used as a measure of the protein redox activity,11,12 was shown to be affected by different packing densities. The aim of the present investigation is a more detailed investigation of this effect. Materials and Experimental Methods Di-thio-bis(N-succinimidyl propionate) (DTSP), di-thio-bis(propionic acid) (DTP), N-(5-Amino-1-carboxypentyl) iminodiacetic acid (ANTA), dodecyl-β-D-maltoside (DDM), cytochrome c from bovine heart, glucose oxidase and catalase were purchased from Sigma and used as obtained; biobeads from BioRad Laboratories GmbH, Munich, Germany were washed with ethanol and deionized water from a MilliQ water purification system, 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DiPhy-
PC) was provided by Avanti Polar Lipids. All other chemicals were of analytical grade. CcO from R. sphaeroides engineered with his-tag on SU II was expressed and purified according to Mitchell and Gennis.13 Preparation of the Samples. Template-stripped gold (TSG) electrodes14 were used for SPR and electrochemistry measurements, immersed for 120 min in a solution of DTSP and DTP in ratios from 0.2 to 1 (corresponding to 20:80, 40:60, 60:40, 80:20, 100:0 (w/w)) in dry dimethylsulphoxide (DMSO) (total 2 mg/mL). After rinsing, the slides were functionalized for 48 h in a 0.15 M solution of ANTA buffered to pH 9.8 by adding 0.5 M KCO3. Finally, the glass slides were immersed for 30 min in 40 mM NiSO4 in acetate buffer 50 mM, pH 5.5. The excess Ni was removed by thorough rinsing with the Ni2+-free acetate buffer. Immobilization of the protein to the Ni-chelated NTA surface was performed in a solution of 100 nM CcO in detergent-containing phosphate/DDM buffer (K2HPO4 0.1 M, KCl 0.05 M, pH 8, 0.1% DDM).11,12 Biobeads were added to the lipid-detergent-containing phosphate/DDM buffer, (DiPhyPC 0.05 mg/mL, K2HPO4 0.1 M, KCl 0.05 M, pH ) 8, 0.1% DDM) in order to remove the detergent and to form a lipid bilayer Electrochemical Measurements. Electrochemical measurements were performed using an Autolab instrument (PGSTAT302) equipped with an FRA2-module for impedance measurements, an ECD-module amplifier for low-currents, an ADC750 module for rapid scan measurements, and a SCANGEN module for analog potential scanning, as well as the frequency response analyzer (FRA) software provided by Eco Chemie, B.V. (Utrecht, The Netherlands). Measurements under anaerobic conditions were done in a buffer solution containing K2HPO4 0.1 M, KCl 0.05 M, pH 8, and the oxygen trap consisting of glucose (0.3% w/w), glucose oxidase (75 µg/mL), and catalase (12.5 µg/mL).15 This solution was flushed with Ar purged of oxygen by washing through the oxygen trap containing the buffer solution for 1 or 2 h prior to the measurements
Activity of Membrane Proteins
J. Phys. Chem. B, Vol. 112, No. 10, 2008 3195
Figure 2. Upper part: the molecules used for the mixed monolayer include dithiobis (N-succinimidyl propionate) (DTSP), dithiobis (propionic acid) (DTP). The dithio-groups split up into two Au-S bonds upon self-assembly; DTP becomes thio (N-succinimidyl propionate) (TSP); DTP becomes thio (propionic acid) (TP). Lower part: coupling of N-(5-amino-1-carboxypentyl) iminodiacetic acid (ANTA) to form the NTA modified (mixed) monolayer of TSP mixed with TP.
to ensure a completely deoxygenated solution. Impedance spectra were recorded in a frequency range of 50 kHz-3 mHz with an excitation amplitude of 10 mV. Data were subsequently analyzed by the complex nonlinear fitting algorithm supplied included with the data processing software ZVIEW (Version 2.6, Scribner Associates, Inc.) applied to the equivalent circuits. The CPEs in the RC circuits account for the heterogeneity of the mixed DTP/DTSP-NTA layer. All electrochemical measurements were taken in a threeelectrode configuration with TSG as the working electrode, a Ag/AgCl,KClsat reference, and a platinum wire as the counter electrode. All electrode potentials are quoted versus NHE. Surface Plasmon Resonance Spectroscopy (SPR). SPR was performed with apparatus using the Kretschmann configuration with a measuring cell designed for the combination of SPR with electrochemistry. The glass slide (LaSFN9 glass from Hellma Optik, Jena, refractive index n ) 1.85 at 633 nm) was optically matched to the base of a 90° glass prism (LaSFN9). Monochromatic light from a He/Ne Laser, (Uniphase, San Jose, CA, λ ) 632.8 nm) was directed through the prism and collected by a custom-made photodiode detector. Recording the change of reflectivity at a fixed angle in the linear regime of the SPR curve as a function of time yields the time course of protein binding and reconstitution. Surface Enhanced Infrared Reflection Absorption Spectroscopy (SEIRAS). SEIRAS was carried out with a Bruker VERTEX 70 FTIR spectrometer with a resolution of 4 cm-1. The mirror velocity was 60 kHz, and 100 scans were taken for each spectrum. A gold film was formed by chemical deposition onto the surface of the hemispherical silicon ATR crystal.16 The result is a rough gold layer (rms roughness of about 5 nm) as required for an enhancement of the IR absorption of the biomimetic membrane system immobilized on the gold film.
The SEIRAS setup consists of an infrared light source, the hemispherical silicon-crystal, required for total internal reflection, and a Teflon cell with buffer solution. The attenuated total reflection (ATR) spectroscopy configuration allows for the excitation of surface plasmons at the rough surface, which is required for a sufficient enhancement of the IR signal. Atomic Force Microscopy (AFM). The AFM measurements were performed with a scanning probe microscope “Multimode Tuna TR” from Veeco, U.S.A. An AFM liquid cell was used, and the measurements were carried out in the tapping mode with a silicon nitride cantilever in a triangular shape. Results and Discussion Varying the Composition of the CcO Monolayer. To modify the concentration of CcO on the surface, the surface concentration of chelating NTA molecules was varied. This was done on the level of the first monolayer consisting of a thiol terminated active ester (DTSP) used for the functionalization with ANTA molecules. The structure of the molecules is given in Figure 2. DTSP was mixed with DTP in different ratios. DTP is the starting material for the synthesis of DTSP and has a similar structure. However, DTP is terminated by a COOH group which is unable to couple to ANTA. The di-thio groups split up into two Au-S bonds upon self-assembly. DTSP becomes thio-(N)-succinimidyl propionate, (TSP), and DTP is converted to thio-propionic acid (TP). ANTA reacts with TSP resulting in an NTA terminated surface mixed with COOH terminated TP molecules. After chelation with Ni, the NTA group is ready to form a stable complex with the poly-histidine tail of the CcO in order to anchor the enzyme to the surface. The binding and reconstitution of the CcO are illustrated in Figure 1. The composition of the mixed NTA-TP/TP monolayer measured by XPS closely corresponds to the mixing ratio of
3196 J. Phys. Chem. B, Vol. 112, No. 10, 2008
Friedrich et al.
Figure 3. XPS analysis of the surface concentration of the NTA modified (mixed) monolayer of TSP mixed with TP. Mixing ratios in solution are plotted versus the ratio of the elements oxygen and nitrogen vs sulfur. With ANTA-TP and TP containing 7 oxygens and 2 nitrogens and 2 oxygens and 0 nitrogens per 1 sulfur, respectively, the parameters for the two graphs were calculated for (Y ) A + B × X) and compared with the theoretical values in solution. oxygen
Figure 5. Peak height of the amide I band as a function of time during coupling of ANTA to the mixed TSP/TP monolayer (open and closed symbols are for the diluted and undiluted layer, respectively). Two parameter exponentials y ) a(1 - exp(-t/τ)) were fitted to the data obtaining the time constant τ ) 4.4 h for the 60% dilution experiment.
nitrogen
parameter
value
deviation
A theor. B theor.
1.5 2 6.2075 5
0.2 0.3
parameter A theor. B theor.
value
deviation
-0.059 0 1.97 2
0.248 0.37
DTSP/DTP in solution (Figure 3). A model calculation was carried out on the basis of the simplified assumption of a Langmuir isotherm for binding. From this calculation, a 0.6 mixing ratio of DTSP/DTP yields a mole ratio of approximately 0.5 on the surface (details of the calculation are given in Appendix). Coupling of ANTA to a monolayer of the same mixing ratio (0.6) of DTSP/DTP, monitored by SEIRAS as a function of time, illustrates that the dilution with DTP leads to a smaller amount of bound ANTA molecules. Figure 4 shows the actual difference spectra of ANTA molecules before and after binding. Figure 5 shows the peak height of the amide I band during the coupling of ANTA to TSP as a function of time (open and closed symbols are for the diluted (TSP/TP) and undiluted (TSP alone) layers, respectively). In any case,
Figure 4. SEIRAS (difference) spectra of the coupling of N-(5-amino1-carboxypentyl) iminodiacetic acid (ANTA) to the mixed TSP/TP monolayer. The active ester of the N-succinimidyl of DTP forms an amide (CO-NH) bond with the amino group of the ANTA molecule (see Figure 2).
Figure 6. 3D plot of the binding of CcO (100 nM) to the mixed NTA layer; bands increase at 1651 cm-1 (amide I, a), 1549 cm-1 (amide II, b), 1440 cm-1 (amide II, c) and 1279 cm-1. The his-tag of the CcO binds to the Ni-NTA chelating surface; see Figure 1.
ANTA coupling as monitored by SEIRAS takes up to 24 h. The time constant of the mixed layer is slightly smaller than that of the undiluted layer (time constants τ ) 4.4 vs 5.7 h). Binding of CcO to the diluted NTA layer thus formed (mixing ratio 0.6 in this case) was also monitored by SEIRAS (Figure 6). The bands at 1658 and 1549 cm-1 assigned previously17 to the amides I and II modes of the protein backbone vibrations of the CcO increase with time. The peak position of the amide I band at 1658 cm-1 is characteristic for the predominantly R-helical conformation of the CcO. Bands at 1436 and 1290 cm-1 also appear which have been assigned to conformational changes of the Ni-NTA. The peak areas of the amides I and II bands increase with time (τ ) 9.5 min, Figure 7) with binding kinetics similar to the undiluted layer.17 Probing CcO Binding and Reconstitution by SPR. The immobilization of the protein to the different NTA/Ni-modified gold surfaces and the formation of the lipid membrane around the CcO molecules by in situ dialysis was followed by a combination of SPR and electrochemical impedance spectroscopy (EIS) as described earlier.10,12 Reflectivity scans as a function of the angle of incidence (Figure 8, right panel) were used to determine the optical thickness before (0) and after (O) protein binding and reconstitution (3). SPR spectra were simulated on the basis of a fitting routine using the Fresnel
Activity of Membrane Proteins
Figure 7. Peak area of the peaks at 1658 and 1549 cm-1 displayed in Figure 6, plotted vs time. Two parameter exponentials y ) a(1 - exp(-t/τ)) were fitted to the data obtaining the time constant τ ) 9.6 min.
equations, using a four-layer model representing the prism glass, gold, DTSP-ANTA/protein, and lipid layers with dielectric constants of ) 3.14, -12.1 + i × 1.2, 2.1025, 2.25, respectively. However, since the exact value of the dielectric constant of the CcO is not known, precise information of the protein surface coverage cannot be obtained from SPR. Reflectivity at a fixed angle of incidence, shown in Figure 8, yields the time course of protein binding and reconstitution. The 0.6 mixing ratio is shown as an example. From this recording, the time course of immobilization was shown to be complete after 40 min, whereas in situ dialysis taking more than 20 h was usually done overnight. Probing CcO Binding and Reconstitution by EIS. Characteristic changes due to the formation of the layer-by-layer structure, as a function of the DTSP/DTP mixing ratio, were obtained by EIS spectra. Examples of a single set of spectra, before and after binding and reconstitution, are shown in Figure 9. These data are fitted to the equivalent circuits given in Figure 9c,d to yield the resistance and capacitance of the protein and the protein-lipid layer, respectively, as a function of mixing ratio. A statistical presentation of the collected data is displayed in Figures 10 and 11. For a low mixing ratio (0.2), the capacitance of the protein layer is 68 ( 6 µF cm-2 and decreases with increasing mixing ratio, with the largest drop between 0.4 and 0.6, finally reaching 9 ( 1 µF cm-2 with a layer made from pure DTSP (Figure 10). When the bilayer is reconstituted
J. Phys. Chem. B, Vol. 112, No. 10, 2008 3197 around the protein, the capacitance decreases for all mixing ratios except for the 100% DTSP, where an increase of the capacitance is only observed in some cases. (The error bars represent standard deviations of 7-10 sets of measurements). The resistance of the protein layers is already quite high at all mixing ratios (Figure 11). This is an indication of a random orientation of the enzymes before reconstitution. They may be more or less tilted with respect to the surface, thereby coming in close contact with each other to form a sealing layer to the bathing solution. This conclusion is based on the observation that the resistance of the protein layer decreases while conducting a series of potential sweeps, which could be due to reorientation and separation of the highly charged proteins. After reconstitution, the resistance increases in all cases, with the most significant change for the 0.6 mixing ratio. To better understand these changes, it has to be considered that the capacitance of a dielectric layer is determined by thickness and dielectric constant of the material. The capacitance (C) of a pure protein monolayer is estimated to be ∼6 µF cm-2 using C ) 0A/d with A as the area (1 cm2), d as the thickness (5 nm), as the dielectric constant of the dielectric layer ( ) 30, taken from an aqueous lysozym solution),18 and 0 as the permittivity of free space. Higher values of 13.1 ( 3.9 to 68 µF cm-2 as observed for different mixing ratios are likely due to the large amount of water molecules separating the protein molecules. The dielectric constant of the lipid is much smaller, ) 2.2, than that of either water ( ) 80) or protein. This explains the decreases of the capacitance upon reconstitution when water and detergent molecules are replaced by lipid bilayer patches. The lowest values reached are 7.3 ( 0.5 µF cm-2 for the 60% mixing ratio. This is in agreement with the estimate of more than 90% coverage of CcO molecules as deduced from the charge transferred by CV measurements due to the direct electron transfer to the CcO.12 Characterizing CcO Monolayers by AFM. Surface layers were exclusively prepared on template stripped gold (TSG) surfaces, due to their low roughness over large surface areas (rms < 0.2 nm). The roughness hardly changes when mixed NTA layers were prepared on this surface (Figure 12a). AFM images of CcO monolayers at mixing ratios varying from 0.001, 0.1, and 0.6 to 1 were then recorded (Figure 12b-e). Clear differences were obtained between the pure NTA layer and the protein layers, even for the very low mixing ratios 0.001 and 0.1. For the 0.001 ratio, structures with lateral dimensions in the range of 50-80 nm and a height of up to 15 nm are seen. For the 0.1 ratio, such structures are even larger, 50-180 nm
Figure 8. Kinetic trace of an SPR spectrum recorded during CcO binding (1) and reconstitution (2) at a constant angle of incidence (55°) and transferred into an optical thickness by using the Fresnel equation. The inset shows the reflectivity scans as a function of the angle of incidence before (0) and after (O) CcO binding and reconstitution (∆); solid lines are the fitted curves.
3198 J. Phys. Chem. B, Vol. 112, No. 10, 2008
Friedrich et al.
Figure 9. Impedance spectra, frequency normalized admittance (a) and Bode plots (b) of the Ni-ANTA modified surface before (1) after binding of CcO before (2) and after reconstitution of the protein (3). Dotted lines represent experimental data; solid lines show the curves fitted to the equivalent circuit (d) except for the Ni-ANTA modified surface which was fitted to the circuit (c).
Figure 10. Capacitances of the membrane obtained from EIS before (shaded bars) and after (full bars) in situ dialysis as a function of the mixing ratio DTSP vs DTP. The capacitance generally decreases during dialysis. The biggest change is at a ratio of 60%, at the same time reaching reasonably low values.
in the lateral dimension and up to 25 nm high. This is not compatible with dimensions of single enzymes obtained from the X-ray crystallography (4.5 nm × 7.0 nm for the in-plane dimension and 9 nm for the height of CcO from R. sphaeroides).19 These structures indicate the formation of aggregates, possibly due either to nonspecific protein binding (not NTAdependent) or to agglomeration of NTA-tethered proteins due to the flexibility of the immobilized enzymes without the stabilizing environment of a membrane. In the case of the 0.6 mixing ratio, the structures are smaller with 10-20 nm laterally and ∼10 nm in height. This is consistent with a monolayer of proteins with the dimensions according to X-ray crystallography. In this case, the arrangement of the enzymes could be stabilized by the high surface coverage. For the NTA layer formed on a 100% TSP layer, the structures are still smaller, mostly 10 nm in the lateral dimension and )
Insertion of eqs 1 and 2 into eq 4 and taking the numerical values for the parameters leads to / C0,DTSP PDTSP ≈ 0.67 / PDTP C 0,DTP
(5)
Activity of Membrane Proteins
J. Phys. Chem. B, Vol. 112, No. 10, 2008 3201
/ With the two simple relations C0,DTP ) C/0‚xDTP ) C/0‚(1 xDTSP) and PDTP ) P‚yDTP ) P‚(1 - yDTSP), where x and y are the ratio of DTSP and DTP at the surface, we finally get:
xDTSP yDTSP ≈ 0.67‚ 1 - 0.32‚xDTSP
(6)
For a mixing ratio in solution of 60% DTSP and 40% DTP, eq 6 yields 49.75 mol % DTSP and 50.25 mol % DTP on the surface. In this model calculation, the saturation of adsorbed species on the surface has not been taken into account considering that this effect should have an equal influence on the adsorption of both molecules. Moreover, the concentration of DTSP and DTP in solution changes only in the range of 0.01% during the absorption process, as calculated easily from the absolute initial concentration of 2 mg/mL, the area of the gold surface of approximately 15 cm2, and the volume of solution of approximately 5 mL DMSO. Therefore, the effect of a concentration change in solution was also neglected. References and Notes (1) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105. (2) Sackmann, E.; Tanaka, M. Trends Biotechnol. 2000, 18, 58. (3) Cullison, J. K.; Hawkridge, F. M.; Nakashima, N.; Yoshikawa, Sh. Langmuir 1994, 10, 877.
(4) Guidelli, R.; Aloisi, G.; Becucci, L.; Dolfi, A.; Moncelli, M. R.; Buoninsegni, F. T. J. Electroanal. Chem. 2001, 504, 1. (5) Knoll, W.; Morigaki, K.; Naumann, R.; Sacca, B.; Schiller, S.; Sinner, E. K. In Ultrathin Electrochemical Chemo- and Biosensors, Technology and Performance; Mirsky, V. M., Ed.; Springer-Verlag: Berlin, 2004; p 239. (6) Tanaka, M.; Sackmann, E. Nature 2005, 437, 656. (7) Sackmann, E. Science 1996, 271, 43. (8) Hillebrandt, H.; Wiegand, G.; Tanaka, M.; Sackmann, E. Langmuir 1999, 15, 8451. (9) Schiller, S. M.; Naumann, R.; Lovejoy, K.; Kunz, H.; Knoll, W. Angew. Chem., Int. Ed. Engl. 2003, 42, 208. (10) Giess, F.; Friedrich, M. G.; Heberle, J.; Naumann, R. L.; Knoll, W. Biophys. J. 2004, 87, 3213. (11) Friedrich, M. G.; Giess, F.; Naumann, R.; Knoll, W.; Ataka, K.; Heberle, J.; Hrabakova, J.; Murgida, D. H.; Hildebrandt, P. Chem. Commun. 2004, 2376. (12) Friedrich, M. G.; Robertson, J. W. F.; Walz, D.; Knoll, W.; Naumann. R. L. C. Biophys. J., in press. (13) Mitchell, D. M.; Gennis, R. B. FEBS Lett. 1995, 368, 148. (14) Naumann, R.; Schiller, S.; Giess, F.; Grohe, B.; Hartmann, K.; Ka¨rcher, I.; Ko¨per, I.; Lubben, J.; Vasilev, K.; Knoll, W. Langmuir 2003, 19, 5435. (15) Vanderkooi, J. M.; Maniara, G.; Green, T. J.; Wilson, D. F. J. Biol. Chem. 1987, 262, 5476. (16) Miyake, H.; Osawa, S.; Ye, M. Electrochem. Commun. 2002, 4, 973. (17) Ataka, K.; Giess., F.; Knoll, W.; Naumann, R.; Haber-Pohlmeier, S.; Richter, B.; Heberle, J. J. Am. Chem. Soc. 2004, 126, 16199. (18) Smith, P. E.; Brunne, R. M.; Mark, A. E.; Van Gunsteren, W. F. J. Phys. Chem. 1993, 97, 2009. (19) Svensson-Ek, M.; Abramson, J.; Larsson, G.; Tornroth, S. J. Mol. Biol. 2002, 321, 329. (20) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; John Wiley & Sons: New York, 2001. (21) Tanford, C. Physical Chemistry of Macromolecules; John Wiley & Sons: New York, 1963; p 345.
