Angewandte
Chemie
DOI: 10.1002/anie.201205701
Artificial Membranes
Dynamic Interface Imprinting: High-Affinity Peptide Binding Sites
Assembled by Analyte-Induced Recruiting of Membrane Receptors**
Benjamin Gruber, Stefan Balk, Stefan Stadlbauer, and Burkhard Kçnig*
Molecular recognition between membrane-associated recep-
tors and external ligands as stimuli is important for many
biological processes.[1] The dynamic formation of domains and
the clustering of receptors in fluid membranes play a key role
for example, in signal transduction leading to highly specific
binding of competing multivalent ligands.[2]
and His, shows an affinity of lgK = 7.5 (see Table 1 entries 1
and 2, and the Supporting Information). Instead of covalently
connecting two receptor sites, as in Zn46, distinct binding sites
are now simply embedded in synthetic lipid bilayers. Recruit-
ing and self-organization by the peptide ligand allows the
divalent binding of both pSer and His residues of P1 at the
membrane interface.
Various model systems for biological membranes have
been developed to understand and mimic multivalent inter-
actions at interfaces[3] as well as for applications in delivery,
sensing, and catalysis.[4] The principle of template-guided
assembly has also been extensively exploited in molecular
imprinting,[5] but the noncovalent arrangement of binding
sites separated by precisely defined distances still remains
a challenge. We have investigated a concept that mimics
immunological synapses, where receptors are recruited at
a membrane interface and spatially organized by the binding
partner, and this orientation triggers a specific response.[6]
Our vesicles with membrane-embedded luminescent recep-
tors produce a characteristic optical response in the presence
of small peptides as external ligands. Depending on the
functional groups present in the peptide, suitable receptors
with complementary binding sites are recruited and arranged
in the fluid membrane, which triggers a fluorescence resonant
energy transfer (FRET) signal.
Table 1: Summary of the apparent binding constants for peptide P1 by
synthetic receptors in homogeneous solution (entries 1 and 2) and
embedded in vesicle membranes (HEPES buffer, pH 7.4, 258C).
Entry
Lipid
Receptor
mol%
lgK
1
2
3
4
5
6
7
8
–
–
Zn26
Zn46
Zn25
Zn25
Zn25
Zn22
Cu4
Zn22+Cu4
Zn22
–
–
1
10
1
1
1
1 (each)
1
1
4.8[a]
7.5[a]
5.9
8.1
8.6
6.2
5.0
6.3
5.5
5.8
8.8
DSPC
DSPC
DOPC
DSPC
DSPC
DSPC
DOPC
DOPC
DOPC
9
10
11
Cu4
Zn22+Cu4
1 (each)
[a] From Ref. [9].
We have previously reported on the recognition of small
biomolecules and phosphorylated proteins by multisite inter-
actions[7] at the lipid–water interface using synthetic vesicle
membranes with embedded artificial receptors based on
transition-metal complexes of 1,4,7,10-tetraazacyclododecane
(cyclen) and nitrilotriacetic acid (NTA). We now expand this
concept to the dynamic recruiting of synthetic receptors
allowing the multivalent recognition of phosphorylated
peptides. The selective recognition of O-phosphoserine
(pSer) and histidine (His) moieties in the model peptide P1
(see Scheme 1) by metal-complex binding sites was inves-
tigated in a previous study in homogeneous solution.[8] It was
found that in buffered aqueous solution the ZnII–cyclen
receptor Zn26 binds peptide P1 with lgK = 4.8, while dimer
Zn46, allowing a simultaneous two-prong interaction to pSer
First we embedded Zn25 (1 mol%) in vesicular lipid
bilayers made from 1,2-distearoyl-sn-glycero-3-phosphocho-
line (DSPC; Scheme 1) using previously reported procedures
(see the Supporting Information). The emission intensity of
peptide P1 increases significantly upon binding to the surface
receptors (see the Supporting Information) and a binding
constant of lgK = 5.9 (Table 1, entry 3) at ambient temper-
ature was determined. This value is in good agreement with
the recognition of pSer by a single Zn25 receptor[7a] and
reasonable, because DSPCꢀs high phase-transition temper-
ature (548C)[9] restricts diffusion and thus participation of
more than a single metal-complex binding site in the peptide
recognition (cf. Figure 1a). Binding affinities could only be
enhanced by drastically increasing the vesicle receptor
loading to 10 mol% (Table 1, entry 4) resulting in the
formation of tightly packed metal-complex patches.[4d] How-
ever, replacing the saturated DSPC lipid by unsaturated 1,2-
dioleoyl-sn-glycero-3-phosphocholine (DOPC), which has
a much lower transition temperature of À208C,[9] leads to
an increase in the binding affinity to peptide P1 by more than
two orders of magnitude (Table 1, entry 5). The embedded
receptor sites can now diffuse in the membrane at room
temperature allowing binding of one P1 by two Zn25 units
through a dynamic assembly process (Figure 1a). The particle
[*] Dipl.-Chem. B. Gruber, M. Sc. S. Balk, Dr. S. Stadlbauer,
Prof. Dr. B. Kçnig
Institut fꢀr Organische Chemie, Universitꢁt Regensburg
Universitꢁtsstrasse 31, 93053 Regensburg (Germany)
E-mail: Burkhard.koenig@chemie.uni-regensburg.de
[**] We thank the Deutsche Forschungsgemeinschaft (DFG) for finan-
cial support.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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