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Incorporation of Membrane Proteins in Solid-Supported Lipid Layers.

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example DMPE (dimyristoylphosphatidyl ethanolamine),
through the NH, group. A thiolipid monolayer with peptide
spacer 3 is thus formed by further self-assembly. The formation
of this layer was followed by electrochemical techniques
such as cyclic voltammetry (CV) und impedance spectroscopy
(IS) as well as by surface plasmon resonance spectroscopy
(SPR). Table 1 shows the decrease in capacitance and the
Incorporation of Membrane Proteins in
Solid-Supported Lipid Layers
Renate Naumann," Alfred Jonczyk, Ralf Kopp,
Jan van Esch, Helmut Ringsdorf, Wolfgang Knoll,
and Peter Griiber
Solid-supported lipid layers['] are able to incorporate membrane proteins only if an aqueous layer separates the lipid layer
from the substrate. This can be achieved by transferring lipids to
hydrophilic surfaces either by Langmuir-Blodgett techniques['] or by the fusion of lipo~omes.[~l
Alternatively such
layers can be formed by self-assembly if the lipid is attached to
the substrate by a hydrophilic spacer group. For example. disulfide-functionalized amphiphilic copolymers (hydroxymethacrylates) form lipid layers on gold substrates separated by a
swellable aqueous layer.[4351 The same holds for thiolipids having hydrophilic oxyethylene spacers of defined length, which
were reported to form lipid mono- and bilayers''] capable of
incorporating intrinsic membrane proteins such as the 5HT,
receptor from calf
Lipid layers having peptide spacers
were first prepared on polymer beads. Bacteriorhodopsin incorporated in these layers was shown to operate as a proton
pump.[*] Lipid layers on gold with peptide spacers will be described in the present paper.
In analogy to previous work[*] the relatively hydrophobic
pentamer of alanine 1 (Fig. 1) was first functionalized with ter-
Table 1. Capacitance (measured by CV [6]) and layer thickness (measured by SPR
and calculated using an effective refractive index of 1.5) of the films.
CapdCltdnce/pF cm-
2- 3
0.15 -
Dr. R. Naumann. Dr. A. Jonczyk, R. Kopp
E. Merck
D-64271 Darmstadt (Germany)
Telefax: Int. code + (6151)71-0773
Dr. .I. van Esch. Prof. H. Ringsdorf
lnstitut fur Organische Chemie der Universitiit M a i m
Prof W. Knoll
Max-Plnnck-lnstitut fur Polymerforschung.
Main/ (Germany)
Prof. P. Griiber
Institut fur Biologie der Universit;it Stuttgart (Germany)
bilayer. no protein
bilayer with ATPase
increase in layer thickness indicated by CV and SPR due to the
formation of the lipid mono- and bilayers. The capacitance of
perfect lipid monolayers determined by CV was reported to be
as low as 0.5 pF cm-2.16. The comparatively high capacitance
found in our case indicates a lower degree of perfection. This
conclusion is also supported by the fact that the layer thickness
is much less than the calculated length of the molecule (Fig. I ) ,
which can be explained by the incomplete coupling of the peptide in situ.
The thickness of these imperfect monolayers increases when
they are exposed to a suspension of liposomes (Fig. 2). This
increase does not occur below the transition temperature of the
lipid and also when the peptide layer alone comes into contact
with the liposomes (not shown). The shift in the resonance angle
in SPR due to the formation of the mono- and bilayer with and
without incorporated ATPase is shown in Figure 3. The layer
thickness given in Table 1 is calculated from these shifts based
on an effective refractive index of 1.5. In view of the theoretical
thickness of 5 nm for a perfect lipid bilayer, the thickness of
4 nm is compatible with the formation of an imperfect bilayer.
The thickness increases to 8.5 nm when the monolayer is exposed to liposomes with incorporated ATPase. Compared to the
lateral extension of ATPase, which is also 8.5 nm," ' I and since
a single orientation of the protein is possible only owing to the
large F, part, this is a strong indication for the formation of a
bilayer with incorporated ATPase. Figure 4 reflects the relative
Fig. 1. Structures of peptides 1 and 2 as well as the thiolipid 3 with peptide spacer.
Maximum lengths: I : 18.2.& 2 . 15.9 A, 3: 42.1 A.
minal sulfur groups followed by the more hydrophilic peptide 2. These peptides have a tendency to form helical structures,[91particularly when
they are attached to gold substrates through sulfur
groups and organize themselves by self-assembly.llolOnce the peptide 2 was covalently linked to
the substrate, the free terminal COOH group was
activated in situ and coupled to a lipid, for
Fig. 2. Reflectivity R (arbitrary units) of the lipid monolayer as a function of time exposed to B
suspension ofliposomes without ATPase. at 20°C (curve A) and 30°C (curve B). and with incorporated ATPase at 30"C (curve C ) .
c (ATP) I rnrnol L-'
-1 0
Fig. 5. Square wave voltammogram (corrected for the blmk curve) of a lipid
). 6 (=). 30 (.-. - ) .
double layer with incorporated EF,F, in the presence of 1 (
75 (- - - -). and 8 9 m m o l L - ' ATP ( - - . . ) .Insert: Peak height as i'unction of the
concentration of ATP without (- - - -) and with EF,,F, (
Fig. 3 Surtace plasmon resonance spectrum of the lipid monolayer before and after
fusion with liposomes with (curve A) and without incorporated ATPase CF,F,
(curve H) X is [he angle for the kinetic measurements.
s s s s s s s
0.0 - 0 . 1 -0.2 -0.3 4 . 4 4 . 5 4 . 6 -0.7 4 . 8
s s s s s s s
Fig 4. Schematic represcntation of the fusion of liposomes with the lipid monolayci'and (brm;itim of the lipid double layer with incorporation of ATPase.
Further indications of the incorporation of ATPase into the
lipid layers are obtained by electrochemical techniques. ATPases CF,F, from chloroplasts and EFoF, from E. ~ o l i [ cata'~]
lyze the translocation of protons across the lipid membrane by
hydrolysis of adenosine triphosphate (ATP). This process is
possible only with an intact lipid layer and results in a pH
gradient of pH 7-8 on the outside and p H 4 on the inside of the
lipid." 21 Protons transported to the inside can be detected by the
current when they are discharged at the gold surface. This discharge occurs only at relatively high negative potentials. To
avoid damage to the lipid layers, fast pulse techniques such as
square wave voltammetry (SWV) and chronoamperometry
(CA) were employed. The SWV of a lipid layer with incorporated ATPase EF,]F, shows a peak at -0.7 V (Fig. 5 ) , which increases in height as a function of the concentration of ATP. This
indicates an increase in proton activity on the inside of the lipid
layer to pH 4, while the pH of the solution remains the same,
pH 7.4. A similar result was obtained for ATPase CFoF, from
chloroplasts (not shown). Figure 6 shows the decline of the
I ..................................
5 -~
c (DCCD) I rnrnol L-'
Fig. 6. Peak height in SWV of a lipid double layer with incorporated EF,,F, in the
presence of constant concentrations of 1 ( - - - -) and 6 miiiol L - ' ATP ( --), as a
function of the concentration of DCCD.
SWV peak at -0.7 V caused by a specific inhibitor of ATPase,
dicyclohexylcarbodiimide (DCCD)
SWV measurements are designed to show only a static picture
of proton translocation. Chronoamperometry. on the other
hand, can be expected to yield information on the kinetics of the
enzyme. In these measurements a current is recorded as a function of time while a potential pulse is applied to the electrode.
The result is shown in Figure 7 for a lipid bilayer incorporating
EF,F, in the presence of increasing concentrations of ATP. In
chronoamperometric measurements a double potential pulse
technique is applied; that means the first pulse is applied
at + 0.2 V followed by a second at -0.7 V. The positive pulse is
The synthesis was monitored continuously by U V spectroscopy at 310 nni. After
removal of the Fmoc groups the peptide was coupled with tritylmercaptopropionic
acid. The peptide and side chain protecting groups were cleaved with TFA (triflnoroacetic acid). Purification was achieved by gel filtration and HPLC. The purity and
identity of the peptide was determined by FAB-MS (fast atom bombardment mass
spectroscopy), HPLC and capillary electrophoresis.
c (ATP) I mmol L-'
1 Gold (0.94 cm2) was deposited onto clean glass slides by electrothermal evaporation
over a sublayer of 30nm of chromium at 300'C and a pressure of
lo-' mbar using a Leybold-Heraeus L 650 vapor deposition apparatus. The thickness of the gold layer was 400 nm.
Preparation of the lipid monolayers: After the glass substrates were cleaned in a
solution of 1 g ofpotassium bichromate in 100 mL ofsulfuric acid (98 Yo),they were
-1 00 IlpA
incubated in a solution of the peptide in TFA (1 mgmL-') for 96 h. The substrates
were then rinsed with TFA. dimethyl formamide (DMF). and dichloromethane
(CH,CI,). The terminal COOH groups of the peptide were activated with
20 p L m L - ' diisopropyl carbodiimide (DICD) and coupled in a solution of
1 m g m L - ' dimyristoyl phosphatidyl ethanolamine (DMPE) In a mixture of DMF,
-300 *
CH,CI,, and LiCl(20:40:0.4). After addition of 10 pL m L - ' N-ethyl diisopropylI
;iniiiie. the substrates were incubated for a further 96 h. Activation and coupling
! 4
0.12 were repeated using a fresh coupling mixture. The substrates were then rinsed with
D M F and CH,CI, and dried in a stream of nitrogen.
Preparation of the lipid double layer: Liposomes were prepared from phophatidylFig. 7. Chronoainperometric measurements of the lipid double layer with incorpocholine and phosphatidic acid (1 :20). The ATPases CF,F, and EF,F, were incorporated EF,F, as a function of the concentration of ATP (1 -90 mmolL-I). Insert:
rated into these liposomes by using biobeads 1181. Substrates with the lipid monoStationary current as function of ATP concentration without ATPase (- - - -). with
layers were incubated in suspensions of these liposomes at 30 'C; concentrations of
CF,,F, (=), and with EF,F, (-).
t h e l i p i d a n d t h e p r o t e i n w e r e 1 . 4 m g m L - ' a n d 3 0 ~ 1 g m L ~ ' o f C F , C FandEF,F,.
CV. SWV. and CA measurements were carried out at 30-C in a buffer solution of
K,SO, (0.1 m o l L - ' ) , tricine (0.0005 m o l L - ' ) , Na,HPO, (0.0005m o l L - ' ) , and
conducive to the activation of the enzyme," whereas the negaMgSO, (0.0002 mol L - ' ) at pH 7.4. in a cell equipped with a silver rod as counter
tive pulse causes discharge of protons. During this latter proelectrode and an AgjAgCl (sat. KCI) reference electrode, and with an Autolab
cess, a decrease in the capacitive current is first observed. This
instrument (ECO Cheinie) using GPESi software. ATP was added from a stock
solution of adcnosine-5'-triphosphoric acid disodium salt (0.25 mol L- ' adjusted
is followed by a stationary cathodic current, indicating the activwith NaOH to pH 7.4). Potential scan rate and range of potentials used in CV were
ity of protons, which shows saturation behavior with respect to
0.1 V s - ' and 0.3 to -0.2 V. respectively. Frequency and range of potentials in SWV
the concentration of ATP (insert, Fig 7). This stationary current
were 80 HL and 0.3 to -0.8 V. Potential pulses applied in CA were 0.2 and -0.7 V.
is about two orders of magnitude higher than the current calcuDuration of potential pulses was 0.07 s and duration of current sampling was
0.0001 1 s.
lated from the kinetics of ATPases EFoFl from E.
CFoFi from chloroplasts[' 21 (Table 2). Relative enzyme kinet-
SPRS study was carried out in Kretschinann configuration [191 with a HeNe Laser
i633 nm).
Received: March 18. 1995
Revised version: May 23. 1995 [Z78101E]
German version: A n g w . Climi. 1995, 107. 2168-2171
Table 2. Stationary current I due lo the discharge of protons at the gold electrode.
compared to the current calculated from the amount of protons translocated by the
calculated [a]
Keywords: ATPase electrochemistry * enzymes * lipid layers
membrane proteins
[a] From G = 1.1 x 10'" (enzymes per surface area. calculated from the concentration of enzymes in the liposomes and assuming a perfect lipid layer): activity:
100 ATPs-' (EFOFl) und 10-20 ATPsC' (CFOFI). at 4H':ATP.
ics, however, are reasonably well reflected. Hence the activity of
the enzymes appears to be preserved. This is possible only if the
enzymes are properly arranged in space and above all if a lipid
layer separates the inner aqueous layer from the outer solution.
Only under these conditions is a p H gradient conceivable as long
as the p H of the solution remains unchanged. Nonspecific adsorption of the enzyme is, therefore, highly unlikely. The relatively high current in the CA experiment could be due to an
acceleration of proton translocation in the electric field. This will
be investigated in further studies. The results so far are taken as
a first indication that the enzymes in the lipid layer have comparable similar activities to those in liposomes, and that protein
denaturation is apparently prevented by the peptide spacer.
Exprimenla[ Procedure
Preparation of peptides: 1 and 2 were obtained by solid-phase peptide synthesis in
a continuous flow synthesizer 1161 using Fmoc (9-tluorenyl-methoxycarbonyl)strategy [I71 with acid-labile protection of the side chain on an acid-labile Wang resin.
VCH Verlug.sgese/l.scliuft mbH, 0-69451 W&hrini, I Y Y 5
[ I ] A. Uhnan, An Inrroducriun to Ultruthin Orgunir Films: From Lunxmuir Blodget1 lo Sd/~As.semhlj~,
Academic Press, Boston, 1991.
[2] G. Puu, 1. Gustafson, P:A. Ohlsson, G. Olofsson. A. Sellstrom in Progress in
Memhrune 7itchnologj (Eds.: J C. Gomez-Fernander, D. Chapman. L. Racker). Birkhiuser. Basel, 1991, p. 279.
131 M. Kuhner. R. Tampe. E. Sackmann. BIophi~xJ . 1994. 67. 217.
[4] J. Spinke. J. Ymg, H. Wolf, M. Liley. H. Ringsdorf. W. Knoll, B k ~ p h j J~ .1992.
63. 1667.
[51 C . Erdelen, L. Hiussling. R. Naumann, H. Ringsdorf, H. Wolf, J. Yang. M.
Liley, J. Spinke, W. Knoll, Lungmuir 1994. 10, 1246.
[6] H. Lang. C. Duschl, H. Vogel, Langmuir 1994, If),197.
[7] H. Lang. B. Koenig. H. Vogel. WO-B93/21528, 1992.
[XI U. Rothe. H. Aurich. Biolech. Appl. Biochrm. 1988. 11. 18-25,
191 J. D. Lear. 2. R. Wassermann. W. D. DeGrado. ScImce 1988. 240, 1179.
1101 E. P. Enriquer. E. T. Samuelski. Muter. R ~ s Soc.
Synip. Pro<. 1992. 255,
[ I I ] E. J. Boekema. P. Fromme. P. Griiber, B w . Bnnwn-Ges. Phys. C h r . 1988. Y2.
[t2] G. Schmidt. P. Griber, Z. Nulurfarsch. C 1987. 42, 231.
[I31 Y. Moriyama, A. Iwamoto, H . Hanada, M. Maeda, M. Futai. J Bin/. Clleni.
1991. 266. 22141
[I41 R. McCarthy. E. C. Racker, .I Bid. C l i m . 1967, 242, 3435.
[I51 U. Junesch. P. Grlber. Biochm7. Bioph.vs. Actu 1987, 893. 275.
1161 A. Jonczyk. 1. Meienhofer in Pepridcs Proc. Xrh A m . P q t . Swnp.. 1983, pp.
[I71 C. D. Chang. A . M . Felix, M H. Timinez,. J. Meienhofer, Int. J. P e p . Pror.
Rcs. 1980. / S . 485.
1181 P. Richard. J.-L. Rigaud, P. Grlber, Eur. J. Biuchtvn. 1990. 193. 921
(191 E. Kretschmann. Z. P h w . 1971. 241. 313.
0570-0833/Y5/34IH-205x B I(I.W+ ,2510
Angew. C'hcm. Inr. Ed. EngI. 1995, 34. N o . I H
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