Biochemical Applications of Solid Supported Membranes on Gold Surfaces: Quartz Crystal Microbalance and Impedance Analysis

Planar Lipid Bilayers (BLMs) and their Applications H.T. Tien and A. Ottova-Leitmannova (Editors) 9 2003 Elsevier Science B.V. All rights reserved.

Chapter 36

Biochemical Applications of Solid Supported Membranes on Gold Surfaces: Quartz Crystal Microbalance and Impedance Analysis Andreas Janshoff, a Hans-Joachim Galla, b and Claudia Steinem c*

aInstitut fiir Physikalische Chemie, Johannes-Gutenberg Universit~it, JakobWelder-Weg 11, 55128 Mainz, Germany bInstitut ftir Biochemie, Westf~ilische Wilhelms-Universit~it, Wilhelm-KlemmStr. 2, 48149 MOnster, Germany CInstitut for Analytische Chemie, Chemo- und Biosensorik, Universit~it Regensburg, 93040 Regensburg, Germany 1. I N T R O D U C T I O N : S O L I D S U P P O R T E D M E M B R A N E S

Since their inception in 1985 by Tamm and McConnell [ 1], solid supported lipid bilayers have been widely used as model systems for cellular membranes [2]. They have been applied in fundamental and applied studies of lipid assemblies on surfaces, to study the structure of membranes and membrane dynamics, lipidreceptor-interactions and electrochemical properties of membranes [3-5]. Several attempts have been made to apply solid supported membranes (SSM) in biosensor devices [6]. Planar lipid membranes can be formed on various surfaces, i.e. glass, silicon, mica or metal surfaces such as platinum or gold. Surface attachment of the lipids is typically achieved following two different strategies, the deposition of Langmuir-monolayers or more easily by selfassembly techniques. The major advantage of this membrane type is their attachment to a solid support, resulting in long-term and high mechanical stability. They can be combined with all kinds of surface sensitive techniques and electrochemical methods provided that the support is a conducting surface such as metals, inorganic materials (indium-tin oxide) or conducting polymers. 2. SOLID SUPPORTED MEMBRANES ON GOLD SURFACES 2.1. Functionalization of gold surfaces

If one is interested in performing electrochemical measurements on supported lipid membranes it is most straightforward to use noble metal surfaces

992

Chapter 36

and in particular gold surfaces as they are inert and can be easily modified by sulfur-bearing reagents. The best characterized and most widely studied selfassembly systems of sulfur-bearing compounds are long-chain thiols, HS(CHz)nX [7, 8]. These compounds adsorb spontaneously from solution onto gold and for n > 10, they form densely packed defect-free monolayers that are stable against acids or bases. The choice of the functional group X determines the properties of the self-assembled organic surface. Hydrophobic alkanethiol monolayers (X = CH3) are the basis for the formation of a second lipid monolayer atop it. pH-dependent groups (X = C O O - o r X = NH3 +) confer hydrophilicity and charge to the surface allowing membrane attachment via electrostatic interactions. In this case, even short chain thiols (n = 2-3) have been successfully used [9]. A different approach is to use phospholipids or cholesterol, whose headgroups are modified by a hydrophilic thiol-terminated spacer [10, 11]. Based on these surface modifications lipid bilayers can be formed on gold surfaces. In the last couple of years more complex surfaces were created by coadsorbing two or more thiols with different X-functionalities or different chain lengths [12]. For patterning two or more different thiols are applied on a gold surface in a spatially defined manner using techniques such as microcontact printing and microwriting. The first thiol compound is spatially delivered on the surface while the second one fills up the residual areas. Micromachining and photolithography selectively remove thiols from a preformed monolayer and the resulting pattern is then filled by a second thiol species. 2.2. Formation of lipid membranes on gold surfaces Supported membranes might be classified according to their different architectures on the gold surface: (A) hybrid bilayers composed of a lipid monolayer on a hydrophobic support (B) lipid bilayers on a hydrophilic or charged surface, and (C) covalently anchored lipid bilayers on a hydrophilic surface. (A) For the formation of a hybrid bilayer composed of a lipid monolayer on a hydrophobic support, the gold surface is first functionalized by an alkanethiol, or a hydrophobic hairy-rod polymer [13]. Various techniques are available to deposit a second lipid monolayer atop the first hydrophobic layer. Apart from the Langmuir-Sch~ifer dip, where a lipid monolayer is transferred from the airwater interface to the hydrophobic solid support all techniques are based on the physisorption of lipids. Techniques comprise the deposition of a lipid monolayer from organic [14] or detergent solution [10], or from vesicle suspension [15], where the latter one leads to solvent and detergent free bilayers. These supported lipid layers are well suited for the investigation of reactions at membrane surfaces, i.e. the interaction of membrane-confined receptors anchored in one

Biochemical applications of solid supported membranes

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leaflet only with their ligands in solution, and association of peptides and peripheral proteins. (B) Functionalization of gold surfaces with charged molecules enables one to adsorb oppositely charged lipid membranes onto the surface. It is also possible to attach negatively charged lipid membranes onto negatively charged functionalized gold surfaces through calcium ion bridges. However, electrostatic attachment of lipid membranes makes them susceptible to increasing ion strength resulting in a detachment of the membrane from the surface [9]. (C) Membranes can be covalently attached to solid supports by using lipids with hydrophilic spacer groups terminated by a thiol or disulfide functionality. These tethered lipid membranes have been shown to act as fluid lipid bilayers possessing an aqueous phase separating the membrane and the support. This is advantageous with respect to a functional reconstitution of large membrane spanning proteins. The spacer acts as an elastic buffer, decoupling the lipid layer from the solid surface and generating a water layer in between [16, 17]. 3. PROTEIN M E D I A T E D ION TRANSPORT IN SOLID SUPPORTED MEMBRANES

The insertion of fully functional membrane spanning proteins is a major goal in the field of solid supported membranes with respect to biosensor applications. However, not all membrane systems are suited to host membrane proteins. Hybrid lipid bilayers on a hydrophobic support are in general not reasonable for the insertion of transmembrane proteins in a functionally active form. This is due to the rigid and often crystalline structure of those membranes preventing a proper insertion and folding of the protein component. Moreover, they are directly attached to the solid support thus the proper folding of the extramembraneous parts of membrane proteins is also abolished. However, tethered membranes acting as fluid lipid bilayers with a hydrophilic aqueous phase separating them from the support enable one to incorporate ionophores such as valinomycin, alamethicin and gramicidin [6, 1821]. Some examples are also given for a successful incorporation of complex proteins. For example, Salamon et al. [22] managed to insert rhodopsin and cytochrome c oxidase. Naumann et al. [23-25] incorporated the FoF1 ATPase from chloroplasts and cytochrome c oxidase in peptide-tethered lipid bilayers and Steinem et al. [26] inserted fully functional bacteriorhodopsin into lipid bilayers on gold surfaces. Our primary goal was to incorporate ion-transporting peptides and proteins in lipid membranes attached to gold surfaces and investigate the specific ion transport mediated by those components by impedance spectroscopy.

994

Chapter 36

3.1. The technique: Impedance spectroscopy Impedance spectroscopy (IS) is a versatile technique for investigating the electrical properties of a variety of different materials, which may be ionic, semiconducting or even insulating [27]. It provides information about both the materials' bulk phase (e.g. conductivity, dielectric constant) and their inner and outer interfaces (e.g. capacitance of the interfacial region and derived quantities). The method is based on measuring the frequency dependent impedance of the electrochemical system of interest followed by its analysis applying equivalent circuits modeling the electrical properties of the system. There are basically two different approaches to acquire impedance data that differ with respect to the excitation signal. The first and most common technique (continuous wave IS) is to measure the impedance in the frequency domain by applying a single sinusoidal voltage of small amplitude with a defined frequency to the system and recording the corresponding current. Applying a discrete set of different frequencies provides a frequency spectrum of the system's impedance. The second approach (Fourier Transform IS) makes use of a transient excitation signal that is applied to the electrochemical system. The system's response is monitored in the time domain and subsequently transformed to the frequency domain by Fourier Transformation providing the frequency dependent impedance of the system.

A

B ! Ag/AgCl-reference electrode

\L glass slide ~x~ with gold electrodes !~ 9 -

_1

C~,.,~

Rm

Pt-counter electrode : ;='~'~connection lead

11\,; 9 : il

Impedance analyzer

C.,2 measuring chamber (PTFE)

Cm

Fig. 1. A Equivalent circuit representing a solid supported lipid bilayer adjacent to an electrolyte solution. B Typical setup used for impedance analysis of solid supported lipid bilayers immobilized on gold surfaces. One interesting and important application of impedance spectroscopy in biochemistry and biophysics is the characterization of solid supported lipid bilayers on a conductive surface. Impedance analysis allows studying the formation process of solid supported bilayers and investigating their long-term-

Biochemical applications of solid supported membranes

995

stability and their stability towards experimental conditions such as temperature, pH, etc. [9]. By applying appropriate equivalent circuits membrane specific parameters can be readily extracted from impedance spectra (Fig. 1A). Moreover ion transport through these bilayers mediated by ion carriers or channel proteins is readily accessible by IS. A typical setup used for the investigation of solid supported membranes on gold surfaces is depicted in Fig. lB. It basically consists of a gold electrode serving as the working electrode and a counter electrode, which might be either a second gold electrode equally functionalized or a platinized platinum wire. Gold electrodes may be easily prepared by evaporating gold on a glass substrate through a mask. The glass substrate is mounted in a cell made of PTFE sealed with an O-ring. The electrodes are connected to an impedance analyzer connected to a personal computer. To apply an external d.c. potential a reference electrode is included such as an Ag/AgC1 electrode. Impedance analysis is ideally suited to determine the coverage of electrodes and the thickness of dielectric layers. Since alkanethiols form almost defectfree and insulating monolayers the resulting impedance spectra can be represented by a simple capacitance Cm in series to an Ohmic resistance R e representing the resistance of the electrolyte and the wire connections (Fig. 2).

A

B

1.0 r ,

o

Cm

10 5

0.8 0.6

Re

10 4

::L

0.4 10 3

0.2 0.0

.

2

i

,

i

,

i

.

i

,

,

.

,

.

|

.

|

4 6 8 l0 12 14 16 18 Number of C-Atoms

10 2 10 ~

10 i

10 z

10 3

10 4

10 5

f~ Hz

Fig. 2. A (O) Inverse capacitance as a function of alkanethiol chain length as determined from impedance analysis of chemisorbed thiols. (O) Calculated inverse capacitance taking the chain length (all-trans conformation) and a dielectric constant of 2 into account. The deviation is probably due to a small number of defects. B Impedance spectra together with the fitting results using the displayed equivalent circuit of an octanethiol monolayer (r-I, Cm = (2.4 + 0.2) ~tF/cm2) and a lipid bilayer composed of octanethiol and POPC (O, Cm = (1.0 + 0.2) ~F/cm2). Fitting the parameters of the equivalent circuit to the data results in characteristic values for the capacitance of the alkanethiol monolayer indicative of the formation of a complete insulating lipid layer. As the thickness of an alkanethiol monolayer increases its capacitance decreases assuming a simple plate condenser (Fig. 2A). These results indicate that the representation of an alkanethiol monolayer immobilized on a gold surface by a plate condenser is a

996

Chapter 36

good first approximation. Impedance analysis also allows one to follow the formation of a second phospholipid monolayer atop the first hydrophobic alkanethiol monolayer (Fig. 2B). 3.2. I m p e d a n c e analysis of ion transport m e d i a t e d by peptides and proteins

3.2.1. Gramicidin D mediated ion transport in solid supported membranes To garner information about the selective transport of ions through solid supported membranes we focused on the development of a solid supported membrane system on gold surfaces containing the channel-forming peptide gramicidin D [ 19]. Gramicidin D, an antibiotic synthesized by Bacillus brevis, is a linear pentadecapeptide forming a channel in lipid bilayers consisting of two antiparallel oriented monomers bound to each other by six hydrogen bonds. The resulting dimer has a length of 26 A sufficient to span the hydrophobic part of a membrane. The van-der-Waals size of the pore is 4 A in diameter and strongly selective for monovalent cations. The sequence of conductivity is determined to be H + > NH4 + > Cs + > Rb + > K + > Na + > Li +. Circuit I

Circuit II

R,,,

~w Rc

o

II C M PA

I

II

'"I

~

I

e

R,,~ Re

I1 II

I

I

~

II

CM A'

Cm

Cm

10 7

B

10 6

105 l0 4

103 10 7 10 6

105 104 103 10 ~

i0 ~ 10 ~ 102 10 3 104 10 5

f~ Hz

10 ~ 10'

102

103

104

105

106

f~ Hz

Fig. 3. Impedance spectra ([ZD of a gramicidin D (1 mol%) doped DODAB bilayer electrostatically immobilized on a MPA-monolayer in the absence (1"-I) and presence of 7.4 mM (O) and 21.8 mM (A) of the corresponding cation. A) LiC1, B) NaC1, C) KC1, D) CsC1. The continuous lines are results of the fitting procedure with equivalent circuit I (without alkali cations) and II (with alkali cations), respectively,"shown in the figure.

Biochemical applications of solid supported membranes

997

The peptide was reconstituted into large unilamellar vesicles (LUV) of dimethyldioctadecylammoniumbromide (DODAB), which were fused onto a negatively charged monolayer of 3-mercaptopropionic acid (MPA). Formation of solid supported bilayers was monitored by impedance spectroscopy. First, the specific capacitance of the MPA-monolayer was determined by fitting a series connection of a capacitance CMPA and a resistance Re to the data. Typical values for MPA-monolayers lie in the range of CMPA -- 9-10 pF/cm 2. Second, large unilamellar DODAB vesicles were added resulting in bilayer formation within an hour (Fig. 3 and 4). Selective cation transport through the incorporated gramicidin channels was followed by means of impedance spectroscopy. Altogether, the impact of four different monovalent cations in various concentrations on the bilayer was investigated. In Fig. 3 impedance spectra of a DODAB bilayer with 1 mol% gramicidin D in the absence and presence of LiC1, NaC1, KC1, and CsC1 in the bulk phase are presented.

A

B

C YRp,w R., o

GPA

Re I----I

---4t Cn

Fig. 4. A Electrochemical model of ion transport facilitated by gramicidin in a DODAB bilayer electrostatically adsorbed on a MPA monolayer on gold. The scheme shows processes that are involved in passive ion transport through a solid supported lipid bilayer taking into account aqueous diffusion and first order interfacial kinetics. Subscript w indicates the water and rn the membrane phase, km and kw denote the rate constants, Dm and Dw the diffusion constants of the ions in the membrane and water phase. The corresponding equivalent circuit consists of the electrolyte resistance Re, the membrane capacitance Cm in parallel to the Warburg impedance Crw and the membrane resistance Rm together with the phase transfer resistance Rpt and capacitance Cpt. The capacitance CMPA represents the membrane/solid interface, which is the capacitance of the preformed self-assembled monolayer of MPA. The phase transfer resistance Rpt may be merged with the membrane resistance Rm, since Cpt can be neglected. B Atomic force microscopy image of a DODAB bilayer electrostatically attached to a MPA-monolayer chemisorbed on gold. The height of the bilayer is 5.4 nm. Image size: 8 x 8 ~tm2.

998

Chapter 36

It is obvious that the presence of alkali cations alters the impedance of the electrochemical system considerably. For a quantitative analysis an equivalent circuit adapted from a work of de Levie was applied (Fig. 4A) [28, 29]. In a comprehensive theoretical study de Levie derived an equivalent circuit for the ion transport of membrane soluble ions through planar lipid membranes with five parameters that are related to ion transport, Cm, Rm, and Cpt, Rpt accounting for the phase transfer of the ions as well as O'w, the Warburg impedance representing the mass transport from the bulk solution to the electrode. This model had to be modified to account for the solid support, while neglecting the phase transfer of the ions. Moreover, a complete description of the electrochemical system requires an additional capacitance CMPA accounting for the self-assembled monolayer and the electrolyte resistance Re (Fig. 3, circuit II)). Impedance spectra were fitted according to this equivalent circuit keeping Cm and CMPA fixed during the fitting routine. Notably, it turned out that the values for Crware orders of magnitude larger than expected from theory. One has to take into account the dramatically reduced area as ion transport takes place merely through a gramicidin dimer. Plotting Rm vs. the inverse cations' bulk concentration results in a linear relation as supposed from theory (Fig. 5). The slope decreases in the following sequence: Li + > Na + > K + > Cs + corresponding to the known gramicidin sequence of conductivity. The increasing conductivity of the larger alkali-cations arises from the decreasing hydration enthalpy. 70 o

60 5o 4o ~-

E

30 o

A

20 10 o~ o.o

0.2

0.4

0.6

0.8

1.0

-!

c

/mM"

Fig. 5. Dependence of the resistance Rm on the reciprocal alkali cation concentration in the bulk phase. The plot shows the nearly linear relation between Rm and c1 dependent on the alkali cation; (O) LiC1, (l) NaC1, (A) KC1, (,) CsC1. Monovalent ions exhibiting ion radii larger than 347 pm are excluded from the gramicidin channel, which was demonstrated by addition of N(CH3)4C1 in a concentration range of 1-22 mM. No decrease in Rm was observed upon addition of tetramethylammonium ions being 347 pm large.

Biochemical applications of solid supported membranes

999

Even though the evaluation of impedance spectra with equivalent circuits is discussed controversially, the application of the modified semiempirical network derived by de Levie leads to cation concentration dependent Rm values confirming the selectivity sequence of alkali cations published for gramicidin as well as the exclusion size of the channel rationalizing the procedure. 3.2.2. Insertion of an ion carrier in solid supported membranes In contrast to gramicidin as a small membrane channel, which can be incorporated into gel-like lipid membranes without loosing channel activity, the functionality of an ion carrier requires high membrane fluidity to allow for diffusion of the carrier in the membrane phase. Several attempts have been made to improve the flexibility of the first chemisorbed monolayer. Lang et al. synthesized phospholipids based on phosphatidic acid linked to one, two or three ethoxy groups with a terminal thiol anchor allowing chemisorption of those lipids [10]. We synthesized a phospholipid based on phosphatidylethanolamine linked via succinic acid to four ethoxy groups (Scheme 1, compound 1) [20].

O

( Scheme

O~o S I H

[.~ [.,,,.t,O /

H N~ / ~ 0 . 0

0 /~.1C14H29 O P~o,~,~ O , , . ~ C 16H.~3 O -"

!

1. Structure of compound 1.

This very long hydrophilic spacer is terminated by a thiol group allowing the formation of self-assembled monolayers on gold, which were characterized by XPS and impedance spectroscopy. Starting with these monolayers, solid supported lipid bilayers with a second POPC monolayer were produced and valinomycin was inserted by adding it to the preformed bilayer [20]. Valinomycin is a cyclic depsipeptide consisting of three identical units. It mediates the transport of alkali and alkaline earth cations in organic films or solvents of low polarity and acts as an ion carrier in lipid membranes. It is known to selectively facilitate the transport of K + and Rb + over Li +, Na + or alkaline earth cations by forming a three-dimensional complex with the cations. Impedance analysis of monolayers formed from compound 1 reveals that the resistance of such monolayers is too low to be detected. However, fusion of POPC vesicles onto the hydrophobic functionalized monolayer results in an impedance spectrum that can be analyzed by the equivalent circuit shown in Fig. 5. The mean value obtained by six independent measurements of a lipid bilayer

Chapter 36

1000

composed of compound I and POPC in 10 mM Tris, 50 mM N(CH3)4C1, pH 7.0 amounts to Cm = (1.0 + 0.2) gF/cm 2 and Rm = (11000 _+ 1000) f~ cm 2. Compared to SSM composed of alkanethiols and POPC the membrane resistance is considerably smaller. This might be explained in terms of the larger flexibility of the self-assembled monolayer and bilayer resulting in less ordered films. Incorporation of the carrier into the preformed lipid bilayers was achieved by adding valinomycin, dissolved in dimethyl sulfoxide to the aqueous phase, which decreased the magnitude of impedance in a frequency range of 50-500 Hz, in which the membrane resistance is predominately determined. A

B i

~

m

k~

12 9

I

E 10

Rv

8 Re -----ff--"-F---~

E

6 4

C,,, Rm

circuit III

|

0

|

|

89

I

4

a

6

c v / IaM Fig. 6. A Equivalent circuit modeling ion transport via membrane carriers. ~-~ symbolizes the diffusion of the free uncharged carrier in the membrane with the diffusion constant Dm v and the diffusion and migration of the ion-carrier-complex in the a.c. field with the diffusion constant Din. ~ represents the interfacial reaction of the cation and the carrier which is assumed to be of first order with the rate constants kw and kin. The corresponding equivalent circuit consists of the capacitance of the gold electrode Cel, the membrane capacitance Cm, the membrane resistance Rm, the resistance due to the interfacial kinetics Rpt, the capacitance and resistance Cv and Rv due to the diffusion of the unloaded carrier in the membrane and the electrolyte resistance Re. The equivalent circuit III is the simplified model used in this study to analyze the obtained impedance spectra. B Dependence of the membrane conductance Gm on the valinomycin concentration in the bulk phase in the presence of 25 mM KC1. In order to evaluate the impedance spectra we applied the equivalent circuit shown in Fig. 6A (circuit III) to the experimental data, which was derived from

Biochemical applications of solid supported membranes

1001

adapting an equivalent circuit, based on the theory of de Levie (Fig. 6A) [28, 30]. The resulting conductance Gm increases almost linearly with increasing valinomycin concentrations in the bulk as expected from theory (Fig. 6B). However, the membrane capacitance Crn also increases upon addition of valinomycin. The insertion of the peptide presumably causes an increase in the dielectric constant of the lipid bilayer. To investigate the cation selectivity of valinomycin embedded in the solid in supported membrane the influence of different K +- and Na-concentrations + solution on Rm in the absence and in the presence of valinomycin was investigated. In order to determine the increase in conductance solely caused by valinomycin we assumed that the membrane resistance Rm~ which is elicited by defects in the membrane, is parallel to the resistance Rmv representing the transport of cations in the membrane as mediated by valinomycin. Hence, the conductance due to the transport of cations facilitated by valinomycin Gmv for each alkali cation concentration in solution can be evaluated using the following expression:

CmVCC+w) -- (cO' v CC+w)--cO' v CO))--(cO CC+w)-- cO

(1)

Gmv'~ is the conductance obtained from the impedance spectra in the presence of valinomycin and Gm~ in the absence of valinomycin. Fig. 7 shows the conductance Gmv versus different concentrations of potassium and sodium cations in solution. 2.0 1.5

~

1.0 0.5 0 .0~

,

0

I

5

10 C w

,

ZI

15

,

IY

!

20

+/mlM

Fig. 7. Plot of the conductance dependent on sodium (9 and potassium ions (e) in the aqueous phase. In agreement with theory, a linear relationship between the potassium ion concentration and the specific conductance was observed.

The membrane conductance increases significantly in the presence of potassium ions and only slightly in the presence of sodium ions. A linear relation between the cation concentration and the conductance Gmv is expected

1002

Chapter 36

from theory and confirmed by the experiments. The slope for potassium ions is about twelve times larger than that for sodium ions. This is in accordance to reported observations that valinomycin is highly selective for potassium.

3.2.3. Reconstitution of an anion channel of Clavibacter michiganense ssp. nebranskense in solid supported membranes Besides small antibiotic peptides it is even more interesting to insert proteins into SSM to study their channel activity. In this context, it is also conceivable to use SSM to sense the toxicity of a protein due to the protein's channel activity. Such a phytotoxin is produced by the Corynebacterium Clavibacter michiganense ssp. nebraskense causing Goss' wilt and blight in Zea mais [31, 32]. In general, members of the genus Clavibacter have been described to produce phytotoxins, which were classified as high molecular mass polysaccharides (Clavibacter michiganense ssp. michiganense) [33] or glycolipids (Clavibacter rahtayi) [34]. However, the presence of polysaccharides did not explain the discrete action on chloroplasts and their membranes. Schiirholz et al. [35, 36] were the first, who systematically searched for toxic activities excreted into the culture medium of Clavibacter michiganense ssp. nebraskense and identified a membrane-active component that forms anion selective channels in planar lipid bilayers. The relative molecular mass of the Clavibacter anion channel was determined to be 25 kDa by functional reconstitution of the channel protein from SDS gels. Voltage clamp measurements demonstrated the specific anion selectivity (C1- > F- > SCN- > I- > C204- > $042-) and that channel activity can be abolished by protease treatment and anion specific channel inhibitors such as indanyloxyacetic acid (IAA-94) and by OH-ions (pH >10). Voltage clamp experiments also revealed that single channel conductance increases exponentially with voltages up to 200 mV saturating at 250 mV and that the channels are closed at negative potential relative to the side of insertion. The average number of open channels also increases with the applied potential. According to Schtirholz et al. [36] the Clavibacter anion channel (CAC) inserts spontaneously into planar lipid membranes when culture fluid of this species is added to the aqueous phase. Our major objective was to study the CAC by incorporating the channel into solid supported lipid bilayers immobilized on gold surfaces and thoroughly characterize the electrical parameters of the systems by means of impedance spectroscopy. Two different membrane systems were investigated, one based on the electrostatic immobilization of DODAB membranes on a negatively charged MPAmonolayer and one based on a chemisorbed thiol lipid with a hydrophilic spacer and a second physisorbed phospholipid monolayer [37, 38]. Both systems led to similar results and as an example, the results obtained from the electrostatically immobilized DODAB membranes will be summarized here.

Biochemical applications of solid supported membranes

1003

Similar to the experiments performed by Schtirholz et al. [35, 36] preformed DODAB bilayers were incubated with the culture fluid containing CAC while applying a d.c. potential of 50 mV and impedance spectra were taken after 15 min. For data reduction, equivalent circuit II shown in Fig. 3 was used. From the obtained parameters we concluded that the anion channel is inserted into the SSM resulting in an increased conductance of the membrane. To confirm this hypothesis, an experiment was performed in which the protein channel was digested by adding a protease before adding the culture fluid to the lipid bilayer. Indeed, no significant increase in conductance was observed after treatment of the membrane with the culture fluid. For a more detailed analysis of channel activity, voltage dependent measurements were performed. The most characteristic feature of the CAC is the exponential increase in single channel conductance with a rising applied d.c. voltage [36]. We investigated changes in the electrical membrane parameters (Rm and Cm) with an increasing d.c. potential. It turned out that in the absence of CAC, Cm and Gm = Rm-1 do not alter with increasing d.c. potentials. However, after adding CAC to the lipid bilayer the membrane resistance Rm and the Warburg impedance crw decrease exponentially with increasing potential indicative of the formation of voltage dependent ion channels. In Fig. 8A the conductance Gm is plotted vs. the applied d.c. potential. To solely account for the change in conductance induced by CAC we defined the conductance of CAC (GcAc) as:

GCAC(V)- (Gm_l (V)- Gm_l,0)-(Gm_ 2(V)- Gm_2,0)

(2)

Gm-1 and Gm-2 are the conductances of DODAB bilayers with and without CAC dependent on the applied d.c. potential. Gm-l,0 and Gm-2,0are the conductances of the DODAB bilayers before addition of CAC to account for variations in Rm of different bilayer preparations. Each conductance was determined by fitting Rm to the corresponding impedance spectrum. Calculating GCAC according to Eq. (2) leads to the plot shown in Fig. 8B. To further support the idea that indeed CAC is incorporated into solid supported DODAB bilayers we investigated the influence of increasing anion concentrations, i.e. chloride, on the conductance of the lipid bilayer and increasing CAC concentration in the bulk. In both cases, an increase in chloride concentration and CAC concentration in the bulk phase, respectively leads to an increase in conductance. The conductance GcAc depends on the chlorideconcentration and the CAC-concentration, respectively in a linear fashion in the observed concentration range. Schiirholz et al. [35] demonstrated that the Clavibacter anion channel can be inactivated by chloride selective channel inhibitors. We decided to use diphenylamine-2-carboxylic acid (DPC)

1004

Chapter 36

previously shown by single channel analysis to act on the cis-side that corresponds to the aqueous phase facing side. GCAC was monitored with and without DPC dependent on the applied d.c. potential (Fig. 9).

10

::k

10

A

B c#3 =.

6

r"D~ 4

6

s

< 4

/

2

0 ,

u = u ~ q~ ~ 5r 50 1O0 150 200

0

I0

'

5

I

100

,

I

150

,

I

200

d.c. potential / mV

d.c. potential / mV

Fig. 8. A Conductance Gmdependent on the applied d.c. potential (D) in the absence and (O) presence of CAC. B Conductance GCACobtained according to Eq. (2) vs. the applied d.c. potential. The solid lines are the results of fitting an exponential function to the data. The addition of DPC results in a considerable decrease in GCAC indicative of a selective inhibition of channel activity. A remaining channel activity was detected due to non-blocked channels. This result rules out that the increase in membrane conductance is caused by non-specific defects, which are formed due to the incubation of the solid supported membrane in the culture fluid of

Clavibacter michiganense ssp. nebraskense.

10 r/3 zl. ~5 <

k3 0

'

I

50

,

I

100

,

I

150

~

I

200

d.c. potential / mV Fig. 9. Conductance GCACdependent on the applied d.c. potential. (D) represents the data obtained in the presence of CAC and (O) those in the presence of CAC together with 0.13 mM DPC. The overall chloride concentration was 4.6 mM. The solid lines are the results of fitting an exponential function to the data.

Biochemical applications of solid supported membranes

1005

The experiments outlined above clearly reveal that the presence and activity of the Clavibacter anion channel in the culture fluid of Clavibacter michiganense ssp. nebraskense can be monitored and also thoroughly characterized by using solid supported lipid bilayers electrostatically immobilized on planar gold electrodes. Besides biophysical characterization on a long-term stable membrane system the detection of toxic channel activity in bacterial fluids by means of solid supported membranes combined with impedance spectroscopy might also be a promising step for the development of new biosensor devices scanning a solution with unknown composition for toxic ingredients. 4. SPECIFIC MEMBRANES

PROTEIN

BINDING

TO

SOLID

SUPPORTED

Lipid membranes immobilized on a solid support are highly ordered and thus well suited to orient membrane confined receptor molecules such as receptor lipids or proteins on surface. The surface density of receptor molecules can be readily adjusted so that steric crowdence on the surface is minimized. Moreover, it is well established that a fully membrane covered solid substrate prevents nonspecific adsorption of proteins very efficiently. Hence, these membranes are frequently used to study membrane confined adsorption phenomena. In recent years, the quartz crystal microbalance technique evolved as a labelfree method to study the interaction of proteins with lipid membranes in a time-resolved manner [3 9]. 4.1. The technique: the quartz crystal microbalance The quartz crystal microbalance (QCM) technique in non-biological applications has been well known for many decades. The core component of the device is a thin quartz disk, which is sandwiched between two evaporated metal electrodes and commonly referred to as a thickness shear mode (TSM) resonator. As this quartz crystal is piezoelectric in nature, an oscillating potential difference between the surface electrodes leads to corresponding shear displacements of the quartz disk. This mechanical oscillation responds very sensitively to any changes that occur at the crystal surfaces. It was Sauerbrey [40], who first established in 1959 that the resonance frequency of such a quartz resonator alters linearly when a foreign mass is deposited on the quartz surface in air or vacuum (Eq. (3)):

- 2fZAm Af

--"

A~/pq/lq

= SlAm

(3)

1006

Chapter 3 6

where j ; denotes the fundamental resonant frequency, A the electrode area, pq the density of the quartz (pq = 2.648 g/cm 3) and/~q the piezoelectric stiffened shear modulus of the quartz (ktq = 2.947.10 ll dyne/cm2). Sf denotes the integral mass sensitivity, which amounts to 0.036 Hz.cmZ/ng. From these resonance frequency readings it was possible to detect mass deposition on the quartz surface in the sub-nanogram regime and accordingly the device was named a microbalance.

For the majority of bioanalytical applications it was, however, necessary to follow adsorption processes under physiological conditions most notably in a liquid environment. The development of high-gain oscillator circuits that can overcome the viscous damping of the shear oscillation under liquid loading eventually paved the way to apply the QCM technique to many biological problems. Nowadays QCM measurements are frequently used to follow a multitude of biomolecular recognition processes like, for instance, antigenantibody binding or ligand-receptor interactions [39, 41]. In these applications either the ligands or the receptors are immobilized on the quartz surface and the corresponding counterpart is offered from solution. From readings of the characteristic shear wave parameters like the resonance frequency, it is then possible to extract binding constants and also binding kinetics with outstanding time resolution. Recently, it was also demonstrated that the QCM provides invaluable data about the interaction of adherent cells with solid substrates. [39, 42-44].

A

B ~

Temperaturecontrolled Faradaycage ) ......................................] Pump

~ 0

"'i... TSM-resonator

I Oscillator circuit ...................[..........:.......... Frequency counter

Voltage supply Computer

+ag2o -dq/2

Fig. 10. A Schematic diagram of the experimental setup used for QCM measurements. The quartz resonator is mounted in a crystal holder, which itself is placed in a temperature controlled Faraday cage. The quartz resonator is connected as the frequency-controlling element to an electronic oscillator circuit that compensates damping losses and thereby drives the shear oscillation. Addition of analyte is performed using a syringe, while buffer flows continuously over the quartz plate. B Schematic representation of the shear wave in the quartz plate.

Biochemical applications of solid supported membranes

1007

Since the electrodes of the quartz plates are commonly made of gold, solid supported bilayers can be readily prepared on quartz plates. A typical setup used to monitor adsorption of proteins on solid supported membranes by means of the quartz crystal microbalance technique is depicted in Fig. 10. The core component is an AT-cut quartz disk with a fundamental frequency of 5 MHz (d = 14 mm). Gold electrodes are evaporated on both sides of the quartz crystal to allow excitation of the quartz oscillation and to serve as substrate for membrane immobilization (Fig. 11A). The quartz plate is mounted in a holder made of PTFE. The oscillator circuit capable of exciting the quartz crystal to its resonance frequency in a liquid environment has been developed in our laboratory and supports the resonance frequency of minimum impedance. The crystal holder is designed to minimize parasitic damping due to mounting losses and to prevent the occurrence of longitudinal waves. Crystal holder and oscillator circuit are placed in a temperature controlled Faraday cage, while the resonance frequency is recorded with a frequency counter outside the cage. A personal computer is used to control the measurement and record the data. Using this active oscillator mode without amplitude control only one parameter, the shift of the resonance frequency can be monitored. A time resolution of practically 0.1-1 s can be readily achieved with a frequency resolution of 0.2-0.5 Hz for a 5 MHz resonator. In Fig. 11B a typical time course of protein binding to a solid supported membrane obtained from frequency recording is shown. Upon specific binding of the protein to the surface the resonance frequency of the quartz plate decreases and levels off at the point of binding equilibrium. From the obtained data, the resonance frequency shift Af can be extracted.

A

B Re

Phospholipid ~ Octanethiol Gold

~

Cm N

-10

~-20 Quartz with evaporated gold electrodes ~--~i ~

.... i ~

-30

'

0

.

.

10

.

.

20

30

40

t / min Fig. 11. A Schematic drawing of a functionalized quartz plate. B Time course of the frequency shift of a 5 MHz quartz plate upon addition of a protein to a functionalized lipid membrane and specific adsorption of the protein to the membrane. The solid line is the result of fitting the parameters of Eq. (7) to the data.

1008

Chapter 36

4.2. Specific adsorption of proteins to solid supported membranes Protein-receptor interactions at lipid membranes, for example gangliosidetoxin interactions play an essential role in biological processes. The first contact of a protein, virus or bacterium with its receptor at a biological membrane initiates a variety of reactions within and at the cell membrane. Solid supported membranes immobilized in a highly ordered fashion on gold surfaces are well suited for studying adsorption processes by means of the quartz crystal microbalance technique. The quartz crystal microbalance in combination with solid supported membranes composed of an alkanethiol monolayer and a second lipid monolayer obtained by vesicle fusion allows the determination of thermodynamic and kinetic parameters of protein-receptor couples without labeling the protein. The success of bilayer preparation can be easily followed using impedance spectroscopy. Direct control of bilayer preparation guarantees high reproducibility of the adsorption processes and minimizes the amount of non-specific adsorption. 4.2.1. Specific binding of a lectin to solid supported membranes To provide an example for the suitability of this approach the interaction of peanut agglutinin (PNA) with gangliosides monitored by quartz crystal microbalance technique is described. The lectin PNA from Arachis hypogaea (M = 110 kDa) is composed of four identical subunits each with a molecular weight of 27-28 kDa exhibiting a specific binding site for carbohydrates of the composition fl- Galp- ( 1--+3)- GalNAc. ...0. .................................

30 N 20

.."

I

O* 0

,

I

I

1

2

,

I

3

~

I

4

Fig. 12. Dependence of the frequency shift on the GMl-content in the POPC monolayer. A PNA concentration of 2 laM was added and the resonance frequency recorded. The solid line indicates the minimal value that is necessary for a complete PNA-coverage on surface, while the dotted line represents a spline. To investigate the interaction of PNA with a specific receptor on surface a lipid monolayer physisorbed on an octanethiol monolayer is doped with

Biochemical applications of solid supported membranes

1009

different concentrations of the receptor lipid GM~ (Scheme 2) and the frequency shift Afof the quartz crystal is monitored upon addition of PNA [45]. Fig. 12 demonstrates that a dopant concentration of 1.5 mol % of the receptor lipid GM~ is sufficient to achieve maximum protein surface coverage. A calculation of the theoretical value of the minimum number of necessary GM~ molecules within the lipid matrix assuming a homogenous distribution of the receptor lipids and correct values for the geometry of the protein leads to a value of 1.5 mol %. A comparison of the theoretical value with the experimentally obtained one implies that the utmost monomeric protein coverage on surface has to be close to one. Similar maximum protein coverage using a lipid matrix doped with 2 mol % of the receptor lipid was corroborated by Ebato et al. [46] who investigated the streptavidin-biotin couple with the quartz crystal microbalance. GMj" Gal-GalNac-Gal-Glc-Cer I NANA

asialo-GM~" Gal-GalNac-Gal-Glc-Cer

GD~a:

GM3: Gal-Glc-Cer

Gal-GalNac-Gal-Glc-Cer I I NANA NANA

Gv,b: Gal-GalNac-Gal-Glc-Cer I I NANA NANA I NANA

I NANA

GD,b: Gal-GalNac-Gal-Glc-Cer I NANA I NANA

Scheme 2. Schematic representation of various gangliosides.

In order to determine the binding constant of a protein-receptor couple the frequency shift Afhas to be monitored dependent on the protein concentration Co in solution. Assuming that the binding sites on the surface are energetically equivalent and that there is a homogeneous distribution of the receptor lipids, the binding constant Ka can be obtained by fitting the parameters of a Langmuir adsorption isotherm (Eq. (4)) to the data:

|

-

Ka c~ l+KaC 0

(4)

The established binding constants present information about the required chemical structure of the receptor essential for an appropriate binding, as demonstrated by the adsorption of PNA to GM~ and asialo-GM1. While the binding constant of PNA to GM~ is Ka - (0.83 + 0.04)" 106 M-~, it is determined to be Ka = (6.5 + 0.3)" 10 6 M -1 for asialo-GM1, almost a factor of 10 larger. This

1010

Chapter 36

difference is attributed to the fact that N-acetylneuramic acid of GM~ sterically hinders PNA binding. This is an example of how the molecular structure of a receptor molecule can be illuminated by varying the receptor molecules embedded in the lipid membrane using the quartz crystal microbalance. Upon quantifying the inhibition of binding in solution this method is capable of clarifying carbohydrate structures that play a pivotal role for receptor function. Monitoring the frequency shift upon binding of PNA to GM~ in the presence of an inhibitor allows determining the binding constant KI of the inhibitor in solution [45]. A prerequisite for the determination of KI is an appropriate ratio between KI and Ka. If the binding constants have similar orders of magnitude, an exact determination of the binding constant KI is practicable since the frequency changes continuously with the inhibitor concentration in solution. If there are several magnitudes between KI and Ka, the protein binds either almost unaffectedly to the surface or not at all. 4.2.2. Interaction of bacterial toxins with membrane-embedded receptor molecules Cholera toxin, the enterotoxin of Cholera vibrio, is an 87 kDa protein composed of six subunits (ABs) in which the five identical B subunits form a pentagonal ring surrounding the A subunit. The B subunits harbor the binding sites for the cell surface receptor. Using solid supported membranes, binding of cholera toxin to GM~ containing POPC-membranes was quantified [47]. Adsorption isotherms of cholera toxin binding to POPC monolayers doped with either 10 mol% GM~ or 10 tool% asialo-GM1 were recorded and association constants were obtained by fitting Eq. (4) to the data. For GM~ doped lipid layers, a binding constant of Ka = (1.8 + 0.1)" 108 M -~ with a maximum frequency decrease of Afmax -- (l 1 ] ___ 2) H z w a s obtained, while for asialo-GM1 doped bilayers a significant lower binding constant of Ka - (1.0 + 0.1)" 107 M -~ with a maximum frequency decrease of Afmax-- (34 + 2) Hz was obtained (Table 1) [48]. The higher affinity of cholera toxin to GM~ clearly demonstrates the importance of the sialinic acid in the receptor structure. Simple electrostatic interaction driven by the negative charge of the sialinic acid could be excluded since no adsorption was detected on GM3 doped lipid layers. In a similar approach the receptor structure for tetanus toxin binding was investigated [47, 48]. Different gangliosides as receptor lipids serving as binding sites for tetanus toxin were analyzed. The exotoxin of Chlostridiuum tetanii is known as one of the most effective toxins from bacteria. It consists of two subunits, fragment B (M = 99 kDa) and C (52 kDa). Fragment C harbors the specific receptor binding site. Four different gangliosides GM3, GDla, GDlb and GTlb (Scheme 2) were incorporated into the outer leaflet of a solid supported lipid bilayer by fusing POPC vesicles doped with 10 mol% of the corresponding

Biochemical applications of solid supported membranes

1011

ganglioside on a hydrophobic octanethiol monolayer chemisorbed on the gold electrode of the quartz plate. Concentration dependent measurements allow the determination of binding constants applying Eq. (4). The results are summarized in Table 1.

Table 1 Bindings constants and maximum frequency shifts of the interaction of cholera toxin and the C-fragment of tetanus toxin to various gangliosides. Toxin

Ganglioside

Cholera toxin

GM3/ 10 mol% GM1 / 10 mol% asialo-GMl / 10 mol% GM3 / 10 mol% GDla / 10 mol% GDlb / 10 mol% Gylb / 10 mol%

C-fragment

Ka / M -I

- mfmax

/ HZ

w

1.8.108 1.0.107

111 34

2.4.106 3.0.106 1.7-106

28 99 66

4.2.3. Adsorption of Raf to solid supported membranes The Ras/Raf/MEK/ERK cascade plays a pivotal role in the regulation of cell growth and-differentiation. One major constituent of this cascade is Raf, a member of the serine/threonine protein kinase family that mediates signals from the cell surface to the nucleus via activation of the mitogen activated protein kinase [49-51 ]. CR1 RafRBD Raf-C1

CR2

CR3

Ser/Thr rich d o m a i n

Catalytic domain

NH

COOH _.3

CR1

B Maltose binding protein (MBP)

RafRBD Raf-Cl His-Tag / His6

NH

COOH 51

194

Fig. 13. A Schematic drawing of the structure of full-length c-Raf-1. The protein is composed of three conserved regions CR1, CR2 and CR3. CR1 comprises the Raf-Ras-binding domain (RafRBD) and the cysteine-rich domain termed Raf-C1. CR2 is a serine-threonine rich domain, which is a phosphorylation site. CR3 is the catalytic domain located near the Cterminus. B Schematic representation of the protein construct used in this study. CR1 including amino acids 51-193 comprising RafRBD and RafC1 is fused to maltose binding protein. A His-tag composed of six histidine residues is added at the C-terminus for a facilitated purification procedure of the protein construct.

1012

Chapter 36

A critical step in the activation of Raf is its interaction with membraneanchored Ras, a small GTPase, via its Raf-Ras binding domain (RafRBD). In its active GTP-bound state, Ras recruits Raf to the plasma membrane in vivo, which is the first step in Raf-activation [52, 53]. However, Ras-interaction alone is not sufficient to activate the Raf-kinase, other events such as Rafphosphorylation may also be required [51 ]. Though there is little known about the mechanism of Raf-activation, the structure of Raf is well resolved. Three isoforms of Raf can be distinguished in mammals; A-Raf, B-Raf and C-Raf-1, the latter being the best studied one [54]. C-Raf-1 consists of a N-terminal non-catalytic region and a C-terminal kinase domain (Fig. 13A). If the N-terminal region is missing (v-Raf oncoprotein) the kinase is constitutively active indicating that the N-terminal part locks the kinase in an inactive conformation and is thus responsible for its regulation [55]. The non-catalytic N-terminus of Raf is composed of two regions (CR1 and CR2) that are highly conserved between different members of the Raf family. The first conserved region (CR1) consists of two modules that are referred to as RafRBD (amino acids 51-131) and a C 1-type cysteine rich domain (Raf-C1, amino acids 139-184). While Ras binding to RafRBD is well understood, the role of the RafC 1 cysteine-rich domain has remained elusive. The objective of our study was to develop a quantitative in vitro assay for the determination of thermodynamic and kinetic data of the Raf-C1 interaction with lipid bilayers employing solid supported bilayers in conjunction with the quartz crystal microbalance technique. With this assay it was possible to investigate the influence of variations in the lipid bilayer composition on the binding behavior of Raf-C 1 [56]. For all experiments, a protein construct composed of amino acids 51-194 including the RafRBD and Raf-C1 fused to a maltose binding protein (MBP) at the N-terminus to improve its solubility and a His-Tag containing six histidine residues at the C-terminus (Fig. 13B) was used and termed MBP-Raf-C1. Lipid bilayers composed of a first chemisorbed octanethiol monolayer and a second phospholipid monolayer subsequently fused onto the first one were prepared on the gold surface of a quartz plate for binding experiments. Time resolved frequency shifts were monitored upon addition of the protein. For a quantitative analysis of the kinetics of protein binding we assumed that the rate-limiting step is the adsorption of the protein on the surface, while diffusion-limiting steps are neglected and that all individual protein binding sites are independent of each other, i.e. no cooperativity occurs. The binding kinetics can then be ascribed by Eq. (5):

o,t,_

co

f

K d + c o ~,

r

~\X

\

z'JJ

(5)

Biochemical applications of solid supported membranes

1013

where ~ t ) is the surface coverage at any given time, Kd the dissociation constant of the monomolecular reaction and co the protein concentration of the bulk. r is defined as the lifetime (Eq. (6)):

r(Co)_

1 (6)

konr 0 + koff .

with kon , the rate constant of association and koff, the rate constant of dissociation. Since the resonance frequency shift Af is proportional to the amount of adsorbed material, Eq. (5) can be rewritten as (Eq. (7))"

(1-exp/-

(7)

where Afe is the equilibrium frequency shift for a given bulk protein concentration Co. By fitting the parameters of Eq. (7) to the data the equilibrium frequency shift and the lifetime r can be obtained.

60

1500 lK

N 1000

40 ~

, 20

500 0

5

10

15

co / gM

20

25

0

5

10

15

20

25

co / pM

Fig. 14. A Adsorption isotherm of MBP-Raf-C1 (m) and MBP (O). A lipid bilayer immobilized on a 5 MHz quartz plate composed of DMPC/DMPS (7:3) was used for each experiment. Af~ and r were obtained from fitting those parameters to the time course of the resonance frequency shift after addition of the corresponding amount of protein using Eq. (7). By assuming a Langmuir adsorption isotherm the dissociation constant Ka and the maximum frequency shift Afmaxwere extracted. B r vs. co plot. The rate constants of association and dissociation of MBP-Raf-C1 binding were determined by fitting Eq. (6) to the data. The values are summarized in Table 2. To obtain dissociation constants Kd and rate constants of association kon and dissociation koff, protein concentration dependent measurements were performed

1014

Chapter 36

on lipid bilayers composed of octanethiol and DMPC/DMPS (7:3). The concentration of MBP-Raf-C1 was varied between 0-16 gM and the equilibrium resonance frequency Afe and r were extracted. The results are shown in Fig. 14 as Afe vs. Co (A) and v vs. Co plots (B). By varying the phosphatidylserine content we addressed the question, whether the DMPS content and hence the effective negative surface charge density affects the thermodynamic and kinetic parameters of Raf-C1 binding to the bilayer. We monitored binding isotherms and concentration dependent lifetimes for bilayers containing 10 mol%, 30 tool% and 100 mol% DMPS. The thermodynamic and kinetic data are summarized in Table 2. Table 2

Thermodynamic and kinetic data of adsorption of MBP-Raf-C1 to various lipid bilayers immobilized on gold surfaces of 5 MHz quartz plates. ~DMPS/ mol% 10 30 100

-Afmax

/ Hz Ko / 10-7 M kon/ 103 (M s)l

19 + 3 50 + 3 96 + 6

koff/ 10-4 S-1 Kd*/ 10.7 M

1.5 + 0.2 2.4 + 0.1

1.4 + 0.1 2.0 + 0.1

5.5 + 0.9 10.5 + 0.3

3.9 + 0.7 5.4 + 0.3

8.3 + 0.3

1.8 + 0.2

10 + 1

5.8 + 0.8

* Kd is obtained from the kinetic constants of adsorption and desorption. The most significant difference between the three bilayer systems under investigation is the increase in A/max with increasing DMPS content. The dissociation constants only slightly increase with increasing DMPS content and no considerable change in the rate constants of association and dissociation were detected. In summary, solid supported membranes allow one to quantify thermodynamics and kinetics of lipid-protein interactions. 5. CONCLUSIONS Solid supported membranes on gold surfaces have been shown to be well suited to investigate protein mediated ion transport through membranes, and ligandreceptor interactions at the membrane surface delivering thermodynamic and kinetic data for different types of lipid-protein couples. Impedance spectroscopy mainly monitors conductance variations, whereas the quartz crystal microbalance allows to follow biomolecular recognition events at the membrane surface. Together with new developments in the design of nanoscaled biofunctionalized lipid arrays [57, 58] solid supported membranes will open up a new avenue within the fields of bioanalytics and nanobiotechnology including chip technology. Moreover, the possible applications will expand to more

Biochemical applications of solid supported membranes

1015

complex systems using whole cells and their mimics as sensor devices [59]. Indeed, both techniques already allow studying cellular processes like cellsurface adhesion, cell-cell-interaction and even signal transduction processes if morphological cell changes are involved [43, 44]. REFERENCES

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