Dynamic interface imprinting: High-affinity peptide binding sites assembled by analyte-induced recruiting of membrane receptors

Article abstract of DOI:10.1002/anie.201205701

Come together: Dynamic molecular recognition events at biological membrane receptors play a key role in cell signaling. Artificial membranes have been prepared with embedded synthetic receptors which dynamically arrange and selectively respond to external stimuli, such as, small peptide ligands. Copyright

Full text of DOI:10.1002/anie.201205701

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
Homepage: http://www-oc.chemie.uni-regensburg.de/koenig/
[**] We thank the Deutsche Forschungsgemeinschaft (DFG) for finan-
cial support.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201205701.
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Scheme 1. Structures of synthetic receptors and ligands (counterions omitted).
sizes determined by dynamic light scattering were unchanged
(Supporting Information, Figure S11), which confirms the
binding of the peptide to receptors of one vesicle (intra-
membrane binding mode) and excludes peptide cross-linking
of different vesicles (intermembrane binding).
shows a significantly higher affinity for peptide P1 in the
nanomolar range (lgK = 8.8, Table 1, entry 11 and Fig-
ure 1b,c). We explain this by the divalent binding of P1 by
a heterodimeric Zn2– and Cu–receptor assembly in the fluid
lipid bilayer (cf. Figure 1b,c) resulting from the peptide-
induced self-assembly of the membrane receptors. The Jobꢀs
plot analysis (Figure S19) supports this conclusion.
For bioanalytical applications, the labeling of the peptide
analyte must be avoided. Therefore amphiphilic receptor
binding sites that signal the presence of the target analyte by
induced FRET were developed: the ZnII–DPA complex Zn21
is labeled with carboxyfluorescein (FAM) and the CuII–NTA
complex Cu3 is labeled with rhodamine (TMR). FRET
techniques are widely used in molecular biology to measure
distances within biomolecules or to explore membrane
structures.[12] We use the FRET signal, which is induced by
the close proximity of the membrane-embedded luminescent
binding sites to signal the presence of peptide P2, which in
contrast to P1, does not carry a fluorescent label. Divalent
binding of P2 to the membrane-embedded binding sites
recruits Zn21 and Cu3 into close proximity and therefore
within the Fçrster distance of 5.5 nm,[13] resulting in a FRET
signal as the optical output (Figure 2a).[14]
The vesicles responded to the presence of the target
peptide P2 with a significant FRET signature. Formation of
a Zn21–P2–Cu3 ternary complex leads to energy transfer from
the donor label FAM (lem = 490 nm) to the acceptor label
TMR (lem = 580 nm; Figure 2b and Figure S22 in the Sup-
porting Information). However, an excess of the target
peptide is necessary to reach the maximum energy transfer.
Control experiments with monovalent ligands (PP, PH, and P0;
Scheme 1) did not change the observed emission spectra
significantly (Figure S23). We exclude intervesicular FRET
through the cross-linking of distinct vesicles as the particle
size in solution remains unchanged in the presence of P2
(Figures S24 and 25). Additionally, vesicles were purified by
size-exclusion chromatography before the binding experi-
ments to remove traces of amphiphilic receptors and micellar
aggregates from the solution, and the concentrations of the
embedded receptors were verified by analysis of the Zn and
The experiments show that analyte-induced recruiting of
binding sites in a fluid membrane leads to higher binding
affinities as a result of multipoint interactions. Next, vesicle
membranes with two different embedded receptors, which
bind selectively either pSer or His of P1, were investigated.
The synthesis of the amphiphilic CuII–NTA complex Cu4 for
the recognition of histidine was reported previously,[7a] the
synthesis of the amphiphilic ZnII–DPA complex Zn22 (DPA =
3,5-bis[(bispyridin-2-ylmethylamino)methyl]-4-hydroxy-
phenyl (DPA) complex Zn22 for the selective recognition of
phosphate[10] is described in the Supporting Information.
Stable metal-complex-doped DOPC and DSPC vesicle mem-
branes were prepared using Zn22 or Cu4 and a mixture of
both (1 mol% each). For DSPC membranes with one type of
receptor embedded, the emission titrations with P1 revealed
monovalent binding through coordination to either Zn–DPA
or Cu–NTA,[11] with binding constants of lgK = 5.0 and 6.2,
respectively (Table 1, entries 6 and 7). Jobꢀs plots with P1 as
the limiting reagent confirm the 1:1 stoichiometry of the
binding event (Figure S18). Combining both receptors in gel-
phase membranes of DSPC vesicles (Figure 1b,c) does not
increase the binding affinity for peptide P1 (lgK = 6.3;
Table 1, entries 6–8). The binding isotherm for the receptor
vesicles bearing both complexes resembles the average of the
binding properties of the individual receptor vesicles (see the
Supporting Information for data). This is explained by the
random, but fixed arrangement of the metal-complex recep-
tors embedded in the lipid bilayer, which prevents the
formation of a ternary Zn22–P1–Cu4 complex (see the
Supporting Information for Jobꢀs plots). DOPC membranes,
in contrast, show completely different behavior: Whereas
vesicles with either Zn22 or Cu4 as the embedded receptors
give similar binding isotherms and affinity constants (lgK =
5.5 and 5.8, respectively; Table 1, entries 9 and 10), the liquid-
crystalline membrane containing both receptor binding sites
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Figure 2. a) Schematic representation of the recruiting of labeled
membrane receptors by a peptide ligand resulting in a FRET signal.
b) Fluorescence spectra of DOPC vesicles containing labeled receptors
Zn21+Cu3 (0.5 mol% each) in the absence (red) and presence
(green) of peptide P2. Relative FRET emission ratio (I580 nm/I520 nm) of P2
and monovalent PH and PP as control (inset).
the FAM and TMR labels in the absence of the peptide ligand
(Figure S20), which might be explained by the partial
formation of Zn21/Cu3 heterodimers. Upon addition of any
mono- or divalent ligand no change in emission was detected,
as the gel-phase state of the DSPC membranes at room
temperature limits the diffusion of the embedded metal
complexes (Figure S22).
In conclusion, we have demonstrated the specific recog-
nition of peptides by dynamic interface imprinting of
synthetic binding sites embedded in vesicle membranes. The
amphiphilic metal-complex binding sites are recruited in the
membrane by the target peptide, resulting in multivalent
interactions and nanomolar binding affinity. The use of
amphiphilic binding sites with FRET pair chromophores
leads to the self-assembly of a specific analyte epitope at the
lipid–water interface and a FRET signal indicating the
presence of the target analyte. The experimental data prove
that dynamic binding-site recruitment in fluid membranes by
analytes (dynamic interface imprinting, DII) leads to the
formation of high-affinity epitopes. The process allows the
specific detection of functional groups in peptides with
nanomolar affinity. To further improve the sensitivity and
selectivity of functionalized luminescent vesicles in chemical
bioanalysis the number and nature of simultaneously embed-
ded binding sites may be increased, including peptides or
nucleotides as recognition moieties. The combined use of
several chromophores in the membrane results in a specific
spectroscopic output depending on their spatial arrangement.
Such functionalized luminescent vesicles may replace anti-
body-based assays in some applications.
Figure 1. a) Schematic representation of receptor assembly in vesicle
membranes: Fixed receptors in gel-phase DSPC membranes versus
analyte-induced clustering in fluid DOPC membranes. b,c) Emission
titrations of P1 versus DSPC (b) and DOPC membranes (c) containing
Zn21, Cu3, or both receptors.
Cu content using ICP-AES (see the Supporting Informa-
tion).[15]
In additional control experiments, DOPC and DSPC
vesicles with only one of the two binding sites were prepared
and used for emission titrations with increasing amounts of
corresponding ligands (pSer/PP for Zn21 and His/PH for Cu3).
As expected, no significant change in the optical properties of
the fluorescent receptor vesicles was observed (Figure S21).
DSPC vesicles with both co-embedded receptors Zn21 and
Cu3 (0.1 mol% each) show a small energy transfer between
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Received: July 17, 2012
Published online: && &&, &&&&
Keywords: fluorescence · imprinting · metal complexes ·
.
molecular recognition · vesicle membranes
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[14] In the absence of the target analyte peptide no FRETemission is
detected, as the average distance between the two chromophores
in the fluid membrane is too large (Figure S20).
[15] As indicated by these measurements, vesicle preparation and
purification results in up to 25% loss of embedded amphiphiles.
All apparent binding constants reported here, however, were not
corrected to reflect this decreased receptor concentration. As
a result all lgK values are considered “minimum” affinities.
4
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Artificial Membranes
Come together: Dynamic molecular rec-
ognition events at biological membrane
receptors play a key role in cell signaling.
Artificial membranes have been prepared
with embedded synthetic receptors which
dynamically arrange and selectively
respond to external stimuli like small
peptide ligands.
B. Gruber, S. Balk, S. Stadlbauer,
B. Kçnig*
&&&& — &&&&
Dynamic Interface Imprinting: High-
Affinity Peptide Binding Sites Assembled
by Analyte-Induced Recruiting of
Membrane Receptors
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!