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Self-assembly
Cyanine Dye Coupling Mediates Self-assembly of a pH Sensitive
Peptide into Novel 3D Architectures
Rita Fernandes, Suvrat Chowdhary, Natalia Mikula, Noureldin Saleh, Katerina Kanevche,
Hans v. Berlepsch, Naoki Hosogi, Joachim Heberle, Marcus Weber, Christoph Böttcher,* and
Beate Koksch*
Abstract: Synthetic multichromophore systems are of great importance in artificial light harvesting devices, organic
optoelectronics, tumor imaging and therapy. Here, we introduce a promising strategy for the construction of self-
assembled peptide templated dye stacks based on coupling of a de novo designed pH sensitive peptide with a cyanine
dye Cy5 at its N-terminus. Microscopic techniques, in particular cryogenic TEM (cryo-TEM) and cryo-electron
tomography technique (cryo-ET), reveal two types of highly ordered three-dimensional assembly structures on the
micrometer scale. Unbranched compact layered rods are observed at pH 7.4 and two-dimensional membrane-like
assemblies at pH 3.4, both species displaying spectral features of H-aggregates. Molecular dynamics simulations reveal
that the coupling of Cy5 moieties promotes the formation of both ultrastructures, whereas the protonation states of
acidic and basic amino acid side chains dictates their ultimate three-dimensional organization.
Introduction
The formation of supramolecular structures is an essential
prerequisite in natural systems for physiological and/or
structural function. Natural photosynthetic systems use
proteins as scaffolds for chromophores to facilitate a
coherent energy transfer for moving excitation energy. The
resulting delocalized excitons are spread in a wavelike
manner over the interacting dyes.[1] Just because of this
feature, molecular excitons have gained considerable inter-
est arising from their potential applications in artificial light
harvesting, organic optoelectronics,[2] nanoscale
computing,[3] and tumor imaging and therapy.[4] The realiza-
tion of devices using excitonic systems requires controlled
delocalization of excitons along specific structures. Cur-
rently, deoxyribonucleic acid (DNA)-based nanotechnology
has emerged as an effective and accessible method in the
design of scaffolded chromophore aggregate systems at sub-
nanometer scales according to simple design rules.[5] Cya-
nines with different lengths of the polymethine chain (PIC,
Cy3, Cy5) have been demonstrated to form strongly coupled
aggregates templated on the DNA strands similar to J- or
H-aggregates in terms of their molecular packing, spectro-
scopy, and energy transfer properties.[6] However, the
accuracy and the depth of the available structural data in
DNA nanotechnology have remained limited[7] compared to
the structural data that are routinely generated in the field
of protein design.[8] Therefore, spectroscopic methods sup-
ported by molecular modelling have been the work horses in
studies involving DNA.
In contrast to the remarkable progress achieved in recent
years in understanding the DNA-templated systems, there
have been very few systematic studies of peptides as
scaffolds.[4a,9] This is surprising since synthetic peptides offer
several advantages, namely precise control over amino acid
composition, chain length and numerous chemical function-
alities of amino acid side chains,[10] as well as straightforward
synthesis procedures.[11] In addition, peptides can be de-
signed to self-assemble in aqueous solvents, adopt a wide
variety of morphologies, such as fibers, tubes, vesicles, and
membranous or fibrillar networks and last but not least can
[*] R. Fernandes, S. Chowdhary, Prof. Dr. B. Koksch
Department of Chemistry and Biochemistry,
Freie Universität Berlin
Arnimallee 20, 14195 Berlin (Germany)
E-mail: beate.koksch@fu-berlin.de
N. Mikula, Dr. N. Saleh, Dr. M. Weber
Mathematics for Life and Materials Sciences,
Zuse Institute Berlin
Takustraße 7, 14195 Berlin (Germany)
K. Kanevche, Prof. Dr. J. Heberle
Department of Physics, Experimental Molecular Biophysics,
Freie Universität Berlin
Arnimallee 14, 14195 Berlin (Germany)
Dr. H. v. Berlepsch, Dr. C. Böttcher
Research Center for Electron Microscopy and Core Facility
BioSupraMol, Freie Universität Berlin
Fabeckstraße 36a, 14195 Berlin (Germany)
E-mail: christoph.boettcher@fzem.fu-berlin.de
N. Hosogi
Nakagami JEOL, Ltd
Akishima Tokyo 196 (Japan)
© 2022 The Authors. Angewandte Chemie published by Wiley-VCH
GmbH. This is an open access article under the terms of the
Creative Commons Attribution Non-Commercial NoDerivs License,
which permits use and distribution in any medium, provided the
original work is properly cited, the use is non-commercial and no
modifications or adaptations are made.
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Zitierweise: Angew. Chem. Int. Ed. 2022, 61, e202208647
Internationale Ausgabe: doi.org/10.1002/anie.202208647
Deutsche Ausgabe: doi.org/10.1002/ange.202208647
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be structurally well characterized by cryo-TEM at high
resolution.[8d–e]
A key aspect for the applicability of peptide-based
materials is the possibility to precisely control and tune the
formation of well-defined morphologies. This can be
achieved by rational design using known sequence-structure
relationships and/or by changing environmental cues such as
temperature and pH.[12] There are several examples of
stimuli-responsive supramolecular assemblies based on β-
sheet adopting peptides and, to a lesser extent, on α-helix
forming peptides.[13] In the latter case, examples of the
controlled assembly of fibers,[14] nanotubes,[15] and
hydrogels[16] by changes in pH and temperature have been
reported. Whereas important advancements toward struc-
tural elucidation at the atomic level have been made,[8d] the
design of primary sequences capable of inducing desired
molecular interactions that lead to highly organized assem-
bly processes and structures still remains challenging.
Here we present a simple, chemically accessible pH-
sensitive peptide combined with an N-terminal coupled Cy5
as chromophore. We studied its self-assembly behavior by a
combination of spectroscopic and microscopic, notably
cryogenic TEM (cryo-TEM), and cryo-electron tomography
technique (cryo-ET), as well as molecular dynamics simu-
lations. We were in particular interested in how the tune-
able peptide scaffold affects the packing characteristics of
chromophores and hence their optical features and, vice
versa, how the dye modifies the peptide packing architec-
ture. Compared to the neat peptide system a restructuring
of the peptide scaffold was expected upon covalent coupling
of an organic dye molecule, alone for steric and hydrophobic
reason. Moreover, in multichromophore systems the elec-
tronic interaction between chromophores acts as an addi-
tional driving force for self-aggregation. It is well docu-
mented for numerous dye molecules, in particular
cyanines,[17,18] that they are able to form mesoscopic
structures spontaneously from monomers in aqueous me-
dium. Therefore, a mutual influence of the self-aggregation
of the peptides, on the one hand, and of the dye molecules,
on the other hand, is expected.[19] An additional factor
considered for this study was the capability of the designed
peptide (STAP1) of responding to pH changes by forming
different supramolecular structures. The detailed analysis
revealed two different but highly ordered ultrastructures on
the micrometer scale depending on pH, both spectroscopi-
cally characterized by face-to-face stacking motifs of the Cy5
moieties namely H-dimers and higher H-aggregates, respec-
tively.
To our knowledge, the dye-peptide conjugate (STAP1)
described herein represents the first of its kind being able of
assembling into two distinct highly ordered structures based
on complementary effects of excitonic dye coupling and pH
triggered peptide interactions. Our current work focuses on
the detailed spectroscopic and structural characterization of
the obtained supramolecular assemblies.
Results and Discussion
Peptide Design, Self-Assembly and Optical Characterization
The peptide investigated here is based on sequences
previously published by our group,[20] and it follows the
design rules for the formation of α-helical coiled-coil
bundles.[21,22] This folding motif is characterized by the
repetition of seven amino acids referred to as the heptad
repeat, denoted abcdefgn, where positions aand dare
hydrophobic, positions eand gare occupied by charged
amino acids, and the remaining positions b,cand fare
generally polar residues.[23] As illustrated in Figure 1, the
stimuli-triggered aggregating peptide (STAP1) described
here consists of a regular distribution of amino acids in a
hpphppp pattern. Leucine residues drive self-assembly due
to hydrophobic effects and serine residues increase peptide
solubility. Eight lysines and eight glutamates were incorpo-
rated to form inter- and intrahelical salt bridges. Since
STAP1 has 16 ionizable side chain residues, it is highly
responsive to changes in pH. At the N-terminus, we coupled
a cationic pentamethine cyanine (Cy5). Cyanines are
excellent model dyes because of their well-documented
ability to form multichromophore assemblies by self-aggre-
gation in solution due to dispersive forces (π-π interaction).
In addition, the large extinction coefficient facilitates
spectroscopic detection.[19,24–26]
As a control peptide, STAP2 was synthesized with a free
N-terminus, i.e. lacking the dye. (HPLC data of both
peptides are given in Figures S1 and S2). Circular dichroism
(CD) spectroscopy was applied to probe for peptide
conformation and stability in solution. CD analysis revealed
the typical helical motif with the double minima around 208
and 222 nm for all pH conditions (Figure S3a) and almost
Figure 1. a) Helical wheel representation of the primary sequence of
STAP1 and STAP2. b) Linear representation of STAP1 showing the N-
terminal Cy5 dye indicated by a blue star; STAP2 contains no dye.
c) Cy5 structure: glutamic acid (blue circles), leucine (yellow circles),
lysine (red circles) and serine (grey circles), Cy5 (blue star).
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independent of thermal history (denaturation) (Figures S3b–
d). STAP2 is assumed to be in a single α-helical conforma-
tion. Under acidic conditions, STAP2 self-assembled into
long and flexible fibers made by the lateral association of
thin fibrils, whereas under neutral conditions only ill-formed
nano-aggregates were observed (Figure S4).
The situation changes significantly for the dye-conju-
gated peptide STAP1. Its CD spectra at pH 3.4 and 7.4 show
again the double minima characteristic of an α-helical
conformation (Figures S5A and S5B), but in particular at
neutral pH the minimum at 222 nm is enhanced with respect
to STAP2. The thermal stability of STAP1 was investigated
by recording the ellipticity as a function of temperature
(Figures S5C–F). At pH 7.4 an initial denaturation experi-
ment resulted in a melting temperature (TM) of �47°C.
Upon returning to room temperature the samples revealed a
significant increase in the ellipticity and in a subsequent
denaturation experiment TMincreased to �73°C (Fig-
ure S5D). Similar annealing effects have been previously
described for the formation of highly ordered α-helical
fibers.[27] It is also noteworthy that the ratio of signal
intensity at 222 nm relative to that at 208 nm increased after
denaturation. Similarly distorted spectra have been occa-
sionally observed and attributed to scattering effects due to
the formation of large aggregates in solution.[28] In contrast,
at pH 3.4, STAP1 shows no defined thermal transition, i.e.,
the peptide is highly stable up to 95°C (Figure S5C).
Depending on the spatial arrangement of dye molecules
within the aggregate (Figure S6 shows a schematic view),
different optical spectra can be observed. Thus, the spectro-
scopic characteristics of the dye allow for further probing of
the aggregates’ structures by means of steady-state absorp-
tion and fluorescence spectroscopy as well as linear dichro-
ism (LD) spectroscopy. First, free Cy5 dye was characterized
by absorption spectroscopy (Figure S7). In MeOH the
typical spectrum of cyanine monomers with a main absorp-
tion band at approximately 640 nm (labelled in Figures 2
and S7 as “M”) and two shorter-wavelength vibrational sub-
bands was observed. In contrast, in buffered aqueous
solutions at different pH values a new blue-shifted absorp-
tion band of the free dye at around 590 nm (labelled in
Figure S7 as “D”) evolved, which we ascribe to H-dimers.
The high propensity of free Cy5 to form aggregates is most
clearly observed at pH 3.4 (black trace in Figure S7). These
spectroscopic studies were extended to Cy5 in the context of
the STAP1 peptide in buffers of different pH values. The
spectra obtained (Figure 2) indicate the appearance of
different classes of aggregates, for which the absorption
bands are labelled “H” for H-aggregates, largely observed at
pH 3.4, “D” for H-dimers of the type mentioned above, the
major species at pH 7.4, and “J” for J-aggregates, mainly
present at pH 11.[6a] The three prototypical spectra most
likely represent mixed populations and contain contribu-
tions from residual monomers, that is, non-aggregated
peptide. Moreover, the large width of the absorption band
together with the appearance of a shoulder at around
700 nm for the H-aggregates might indicate an additional
(Davydov) splitting of the absorption spectrum due to an
oblique geometric configuration of the involved Cy5
chromophores.[29] The spectral features are very close to
those of reported DNA-templated tetramer aggregates
(“DNA Holliday junction scaffolds” (HJ)).[5e,6e] We also
investigated in detail the fluorescence behavior of the
solutions. Figure S8 for example shows the results for
pH 7.4. The emission spectrum is mirror-symmetric to the
absorption spectrum and almost identical with the emission
spectrum of the free dye Cy5 (not shown). This finding is a
strong indication that this emission is due to residual
monomers, while the H-dimers (D) are obviously non-
fluorescent as generally expected for face-to-face stacked H-
aggregates.[29] A quite similar fluorescence behavior was
observed also for the H-aggregates (pH 3.4)[5e, 6e] and for
pH 11.
Altogether these findings provide evidence that residual
monomers (non-aggregated peptide) are ultimately respon-
sible for the fluorescence emission. The absence of a
resonant emission for the J-type aggregates formed at pH 11
is surprising, because these normally show strong
fluorescence. Information about the molecular packing
orientation of Cy5 moieties within the assemblies was
provided by polarized absorption spectra recorded on
samples oriented by the streaming field in a Couette flow
cell. Detected is the linear dichroism (LD) spectrum, which
is the difference in absorption of light parallel and
perpendicular to the orientation direction. A positive LD
signal indicates an orientation of the responsible transition
dipole moment of dye molecules parallel to the flow, i.e.
parallel to the long axis of corresponding aggregates, while a
negative signal points to an orientation perpendicular to the
flow. Exemplary spectra are presented in Figures S9 and
S10.
Morphology of Aggregates
For an initial structural characterization of STAP1 buffered
at pH values of 3.4, 7.4 and 11 we used negative-staining
Figure 2. Normalized absorption spectra of STAP1 solutions at different
pH values: 3.4 (black line), 7.4 (blue line), 11 (green line). STAP1
concentration: �0.5 gL1. Overlaid for comparison is the absorption
spectrum of free Cy5 in MeOH (red line).
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transmission electron microscopy (TEM). The images reveal
the formation of highly ordered micrometer scale aggregates
at pH 3.4 and 7.4. While the aggregates at pH 3.4 were
spontaneously formed after buffer addition, defined aggre-
gates at pH 7.4 were observed only after thermal treatment.
The samples had to be heated to 95°C with subsequent slow
cooling (at a rate of �3°C min1) to ambient temperature.
Otherwise the solutions showed only ill-defined assemblies
(Table 1 and Figure S11). At pH 11, small disordered
assemblies, probably helical oligomers, were observed under
all conditions. The absence of any ordered mesoscopic
structure is the reason why we did not explore this case
more thoroughly and restricted ourselves to neutral and
acidic solvent conditions. Experiments with the control
peptide STAP2 showed formation of long, thin fibrils at
pH 3.4, whereas small micelle-like objects were found at
pH 7.4 (Figure S4).
Detailed structural characterization was performed by
means of AFM imaging and cryogenic TEM (cryo-TEM)
techniques, in particular cryo-electron tomography (cryo-
ET). Notably, the morphologies of mesoscopic aggregates
turned out to be highly ordered but completely different in
their ultrastructural organization, as described below.
Assembly Structure at pH 7.4
At pH 7.4, three-dimensional rod-like aggregates with
widths in the 90–300 nm range and lengths up to several
micrometers are observed (Figures 3a, c). At higher magnifi-
cation, layered striations perpendicular to the long axis of
aggregates at a repetitive distance of 4.4 nm are prominent
(Figures 3b, S12 and S13).
If the rods 3D reconstructed from cryo-ET data are
rotated about the long axis, additional striations parallel to
the rod axis with exactly the same repetitive distance
(4.4 nm) are observed (Figure 3d) and only once again by
subsequent 180°rotation. If the motif of Figure 3d is tilted
by 90°forward-facing (Figure 3e) the rods reveal parallel
strands again at a repetitive spacing of 4.4 nm, so that the
overall rod structure can be described by a stacked arrange-
ment of strands arranged in a tetragonal manner as
illustrated in the scheme of Figure 3h. The 4.4 nm spacing
distance here observed is in line with previously reported
values for striated fiber-forming coiled-coils[8d,30] but the
surprising new observation is, that the filaments here are not
oriented parallel to the rod long-axis, as was usually
observed, but in perpendicular direction. It must be noted
that a peptide arrangement similar to the one proposed for
the gigadalton coiled-coil fibers of Sharp et al.[8d] in which
the peptide strands are hexagonally packed within the fibers,
was initially also expected for the STAP1 system. However,
this arrangement was ultimately rejected for STAP1 as it
was not found to be consistent with the cryo-ET data
obtained here.
We applied near-field spectroscopy (nano-FTIR) to
obtain information on the STAP1 folding motif within these
aggregates (Figure S14). All recorded spectra are dominated
by two bands at 1659 cm1and 1551 cm1; these correspond
to the C=O stretching vibration (Amide I) and the combined
NH bending and CN stretching vibrations (Amide II),
respectively. The band position of the Amide I at 1659 cm1
indicates an α-helical folding motif.[31] Besides enabling
assigning secondary structure, nano-FTIR spectroscopy is
also sensitive to the orientation of particular molecular
vibrations. As previously shown,[31] highly oriented protein
layers show strong Amide I and negligible Amide II
absorption. The comparable intensity of both protein bands
in our case indicates that the α-helices are not homoge-
neously oriented, i.e., not all helices are oriented perpendic-
ularly to the surface.
Assembly Structures at pH 3.4
The aggregates at pH 3.4 adopt a morphology completely
distinct from that of rods observed at neutral pH, in both
shape and size. Although the ultrastructure is also remark-
ably well-defined, these structures are primarily two-dimen-
sional and thus membrane-like layers (Figure 4a). Due to
the large size these peptide membranes are also observed to
be folded or crinkled. At the edges, where a folded
membrane is orientated perpendicular to the image plane,
the profile of the membrane becomes visible revealing a
thickness of about 9 nm and a regular internal structural
organization (Figures 4b,c).
In several cases, the stacking of more than one
membrane layer is observed by cryo-ET due to a multi-
layered rolled-up type structure and accounts for the thick-
ness values up to 71 nm observed using AFM (Figure 4e).
The flat membrane layer itself comprises a hexagonal
pattern of very high regularity and (Figure 4a) even if multi-
layered the membranes show only this particular motif in
projection due to a perfect stack alignment. Its detailed
volume structure is discussed in the next section. By means
of nano-FTIR, two bands (Amide I and Amide II) at
1655 cm1and 1551 cm1are also observed, indicating that
STAP1 adopts an α-helical folding motif also within this
morphological type (Figure S14). A third prominent vibra-
tional band at 1732 cm1is apparent for some objects under
this acidic regime. This could be attributed to either the
Table 1: Effect of pH on STAP1 aggregation.
pH Morphology Spectral Type Preparation
3.4 membranes H-aggregate formed spontaneously
7.4 striated rods H-dimer formed after thermal treatment
11 amorphous J-aggregate independent of preparation
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C=O stretch from the citrate buffer or the protonation of
glutamic acid, with its pKavalue of around 4.[32]
We used additional image processing procedures (for
details see Supporting Information) for both types of
assemblies to gain more precise structural information from
the cryo-ET data for the fitting of consistent molecular
models.
Processing and Modelling of pH 7.4 Rod Structures
For the rod assemblies at pH 7.4, an averaging over
manifold side-view layers taken from the reconstructed 3D
volume was performed to obtain sum images of improved
signal-to-noise ratio (Figure 3f). Individual stack layers are
remarkably well resolved and oval repetitive density spots
along the stacks mark the position of individual forward-
facing strands. The same approach was applied for the motif
perpendicular to the stacked planes (top view) and results in
the sum image of Figure 3g. Both sum images provide more
details than does the raw tomography data (Figures 3d, e),
e.g. even small repetitive density variations in the structure
can be revealed and validate a structure resolution level of
at least 10 Å obtained under the chosen microscopic and
data processing conditions (see details in the Supporting
Information). In the top view orientation (Figure 3g), the
linear strands, which are the elementary building blocks of
the rod morphology, reveal repetitive motifs of low (grey)
and high (white) electron density along the strands at a 5 nm
repeat distance. As mentioned above, if oriented perpendic-
ular to the top view (Figure 3f), the forward-facing strands
appear oval with dimensions of �3 nm×1.5 nm. Upon
accepting that two molecular strands in a side-by-side
arrangement would reasonably fit the cross section, we
suspect an antiparallel arrangement of the peptide strands,
stabilized by π-π stacking of pairs of Cy5 dye molecules
Figure 3. Structural morphology of STAP1 at pH 7.4: a) Cryo-TEM overview showing a dense population of STAP1 rod assemblies. b) Negative
staining (1 % PTA at pH 7.4) TEM of STAP1 highlighting the layered supramolecular organisation and also indicating partial disintegration into
individual layers. c) Topographical AFM height images (integers indicate sample numbers) of STAP1 rod assemblies. d, e) Volume reconstruction
of a rod (detail) based on cryo-ET data: d) Side view and e) top view of d) at 90°forward facing. The red traces represent the repetitive 4.4 nm
spacing between striations. f) Sum image (detail) of a rod side view (note inverted contrast) obtained by averaging 5000 slice motifs along the
reconstructed rod volume where high density motifs at 4.4 nm distance indicate the orthogonal strand orientation. g) Sum image of rod top view
layers from (e) (perpendicular to f) obtained by averaging 5000 individual layers motifs along the reconstructed rod volume. In each linear strand
repetitive motifs of lower density at a 5 nm repeat distance (marked with the red semi-bracket) is evidenced. Adjacent strands are 4.4 nm apart at
which 5 nm low density motifs are shifted by 2.2 nm against each other. h) Schematic illustration of the spatial strand arrangement in a rod.
i) Proposed arrangement of STAP1 at pH 7.4 within a layer in agreement with experimental dimensions, electron density pattern (cf. (f), (g)) and
LD spectra. j) Starting geometry of STAP1 used for MD simulations consisting of three molecular layers as shown in i) in a spatial arrangement in
full agreement with dimensions and density data obtained by electron microscopy. k) and l) Fit of STAP1 after 10 ns MD simulations into the
density map of (f) and (g), respectively, using the start geometry shown in (j). The top view of the arrangement of STAP1 at pH 7.4 still fit the
repetitive low density areas along the striations of (g) to the location of Cy5 stacks (blue regions). The inset shows the stacked arrangement of the
Cy5 moiety in accordance with LD spectra. Conditions: 0.1 mM of STAP1 in 50 mM phosphate buffer, pH 7.4 and after a heating/cooling cycle (95
to 20 °C at 3 °C min1).
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(Figure 3i). This model would fit the dimensions and also
explain the regular low-density areas along the strands due
to the tilted orientation of the dye stack. A conceivable
parallel arrangement would create chemically different
environments at the end-caps, namely the dye stack at one
end, and two O-termini of leucines at the other. The
consequence would be an immediate further terminal
stacking interaction of the dye (formation of tetramers,
along or perpendicular to the strands long-axis), in other
words the formation of molecular bilayers as e.g. found for
individual strands at pH 3.4 (Figure 4f). This would even-
tually lead to 10 (2×5) nm long sections of high density in
the according density map along the strands, what we did
not observe. Also tetramers were not found by spectroscopy
as was e.g. the case with membranes at pH 3.4. Moreover,
the dominance of dimers (D) in the absorption spectrum of
these aggregates and the corresponding negative LD signal
(Figure S9), which points to an orientation of the respon-
sible transition dipole moment of Cy5 molecules perpendic-
ular to the long axis of aggregates support the proposed
molecular arrangement of Figure 3i. The distance between
(antiparallel) strands and layers is 4.4 nm and 5 nm between
the dye stacks along the antiparallel peptide arrangement.
Molecular dynamics (MD) simulations were performed
by extending the antiparallel starting geometry of Figure 3h
for STAP1 at pH 7.4: twelve peptide strands were arranged
in a plane and three such layers were stacked (Figures 3j and
S16a). After a simulation time of 10 ns only small changes
were observed, indicating that the suggested structure
represents a stable geometry at pH 7.4 (Figure S16b).
Notably, STAP1 does not tend to adopt a coiled-coil
conformation but rather single peptide chains remain
parallel arranged within each layer (Figure 3k). This is
obviously a consequence of π-π stacking of the Cy5 moieties
of two neighboring peptide strands in antiparallel orienta-
tion, preventing their free rotation around the long-axis
towards an expected coiled-coil formation. This two strand
structure is additionally stabilized by salt bridges between
Figure 4. Structural morphology of STAP1 at pH 3.4. a) Cryo-TEM of STAP1 highlighting a layered supramolecular organization with arrows pointing
to the membrane layer orientated parallel to the incident electron beam and revealing the membranes density profile. Bar is 50 nm. b) Top view
and c) side view orientation of the membrane (indicated by the scheme showing the geometrical orientation of the membrane and projection
direction highlighted by white and yellow arrows, respectively). The cryo-tomographic volume data are shown in Voltex representation using AMIRA
software (Thermo Fisher Scientific) with inverted contrast. Bar is 25 nm. d) Surface representation of c) revealing the left-handedness of double
super-helices. e) Topographical AFM height images (integers indicate sample number) give thickness values up to 71 nm indicating multi-layered
roll-up of membranes. f ) Proposed arrangement of STAP1 strands at pH 3.4 taken after 10 ns MD simulations where stacking between two Cy5
moieties (blue regions) leads to the longitudinal elongation and twisting towards a helical ultrastructure. Note the C-terminal connection of the
central peptide strands. g) Average of the hexagonal pattern of 60 motifs taken from areas of (a) where the membrane is orientated perpendicular
to the incident beam. h) Fit of two modelled double super-helical strands in the volume of the 3D reconstructed pattern (g) is the top view thereof,
which illustrates the formation of the underlying hexagonal motif. For original 3D super-helix data see Figure S15. The repetitive orientation of the
Cy5 pairs (blue) allows for their additional lateral stacking and eventually leads to the formation of assemblies with a two-dimensional geometry.
Conditions: 0.1 mM of STAP1 in 50 mM citrate buffer, pH 3.4.
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lysine and glutamate, which in this case are pointing towards
the core (Figure S16c). The designed heptad repeats allow
for the additional presentation of a hydrophobic edge of
leucine that further stabilizes a side-by-side arrangement of
the so formed “double strands” within the layers and
prevents the disintegration into individual strands. The exact
locating of the strands rotational position (Figure 3k) due to
the combined molecular interactions (π-π stacking, salt-
bridges, hydrophobic edge) leads to additional electrostatic
attractions between the layers, which are then responsible
for the compact tetragonal top to bottom layer stacking of
the strands. The potential energy surfaces gained after a
10 ns simulation time at pH 7.4 shows that the negative and
positive partial charges are optimally oriented in the layered
structure, with each layer being predominantly negatively
charged on one side and positively charged on the other,
thus providing directionality to the stacking of strand layers
by electrostatic interactions (Figure S16d). This finding is
supported by the observation that the use of the polyanionic
contrasting agent PTA for the TEM experiments, which is
known for its membrane disrupting tendency,[33] leads to the
apparent disintegration of the rods into individual layers
(Figures 3b and S12) probably by neutralizing positive layer
surface charges. We surmise that the layer stacking is the
dominant process of rod assembly formation over the linear
growth of the strands, which eventually results in a packing
of strands perpendicular to the rod long axis.
Processing and Modelling of pH 3.4 Membrane Structures
We then also used an averaging procedure of a single
membrane layer from the cryo-tomographic data in order to
reveal the structural features with better signal-to-noise ratio
in three dimensions. Figure 4g shows the representation of
the corresponding volume stack as a 2D projection image
providing a very detailed hexagonal structure pattern. The
most interesting result of this reconstruction was that the
hexagonal pattern is actually made up of paired (left-
handed) super-helical strands (Figure 4d). This becomes
directly evident (i) in the particular side-view orientation of
the membrane (Figure 4c) or (ii) when the volume recon-
struction of Figure 4g is tilted (Figure S15). It can be directly
observed that these strands comprise two super-helical
strands wrapped around each other (each helix strand
having the diameter of a single alpha-helical strand of
1.5 nm as presented in Figure 4f). Figure 4h shows the
processed hexagonal volume pattern superimposed with a
pair of modelled double super-helices, which fit the pattern
in longitudinal direction as well in the side-by-side align-
ment. The individual super-helical strands have a pitch of
34 nm and a width of 9 nm (thus fitting the thickness of a
single membrane layer) and they are phase shifted by
7.15 nm (75.7°) with respect to each other. Double super-
helices in the side-by-side arrangement are 17 nm apart but
again are phase-shifted by 90°. In this specific spatial
arrangement of linear helical assemblies, the generation of
the observed hexagonal pattern becomes evident. If projec-
tion images of the model data are calculated, the exact
density pattern as obtained from the experimental data is
reproduced.
MD simulations were initiated with the same starting
geometry as discussed for pH 7.4, (Figures 3h,i and S16),
except that the glutamic acid residues were fully protonated
and only 2 layers with 3 peptide strands each were used
(Figure S17). Interestingly, the 10 ns simulation revealed a
prominent trend in which the compact packing organization
observed for pH 7.4 was dissolved towards the formation of
individual strands with an apparent twisted conformation
(Figures 4f and S17). Strand elongation was a result of Cy5
contacts, which remained intact throughout the simulation,
and hydrogen bonding between the residues in the C-
terminal regions of adjacent peptide strands (Figures 4f and
S17c).
The prominent H-band of the absorption spectra (Fig-
ure 2) suggests that several dye molecules are electronically
coupled in the assemblies, in contrast to the strict dimer case
observed at pH 7.4. We therefore assume that the lateral
packing of strands is stabilized by additional lateral dye
interactions. Such an arrangement is realized by the helical
repeat of dye pairs (blue dots in Figure 4h), which occupy a
repetitive position along the strand axis and allow for an
alignment with the dye pairs of the neighboring strands. The
suggested assembly of four interacting chromophores ap-
pears to be a structural analogue to the tetramer config-
uration accessed by templating the dyes via a DNA HJ. The
spectroscopic characteristics in the present case are very
similar. The particular dye interaction would also explain
the formation of a two-dimensional structure. Due to the
extended two-dimensional geometry of the membranes a
uniform orientation in a flow field is not to be expected, so
that additional information from LD spectra (Figure S10)
about the dye orientation in the assemblies is not readily
available.
The offset of helices is puzzling, however, we observe
faint densities in the microscopic density maps, which might
suggest connections between super-helices like spokes of a
ladder. MD simulations indicate at least intermediate cross-
links between neighbored twisted strands (Figure S17b). We
can only speculate, if in addition to the linear formation of
super-helices individual peptides could form such intermedi-
ate connections and thus initiate the double super-helix
distance. The phase shift of �7 nm, however, correlates with
the length of an individual peptide molecule.
Conclusion
Within natural light-harvesting systems the surrounding
proteins align the chromophores into arrays, which are able
of transporting light energy from the antennae to the
reaction centers. In recent years considerable effort has
been put forth to construct artificial multichromophore
systems by applying short DNA-based oligonucleotides as
biologically compatible scaffold. Here, we present a molec-
ular construct (STAP1) of a Cy5 dye linked to a de novo
designed heptad repeat peptide known as one of nature’s
tools to achieve molecular recognition and efficient self-
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assembly, as an alternative to the DNA constructs. The
peptide-dye conjugate has the freedom to self-assemble into
a supramolecular structure in which not the strict rules of
coiled coil formation dictate folding but rather the compet-
ing intermolecular forces due to the involved π-systems of
dyes in combination with hydrophobic and charged amino
acid residues to generate new and fascinating
supramolecular structures.
The uniqueness of the particular peptide scaffold was
found in its ability (i) to tune the formation of two different
H-type aggregates and (ii) to establish a charge pattern of
the peptide scaffold so that two completely distinct but
exquisitely well-defined ultrastructures can be created on
changing the solvent pH from neutral to acidic. Under basic
pH conditions J-type aggregates are formed, but these
assemblies are only a few nanometers in size, disordered,
and, beyond that, non-fluorescent, which makes them less
attractive for applications.
AFM and cryogenic electron microscopy revealed that at
pH 7.4 rod-like objects with diameters in the 90–300 nm
range and lengths up to the micrometer scale are formed.
On contrary, flat membrane-like objects with an apparent
hexagonal ultrastructure are spontaneously formed at
pH 3.4. Based on cryogenic electron microscopic data, MD
simulation, and optical spectroscopy we concluded that the
electronic coupling between the Cy5 moieties contributes
essentially to the formation of the two mesoscopic peptide
architectures. The protonation states of lysines and glutamic
acids ultimately direct the eventual formation of either rods
or two-dimensional membranes.
At neutral pH, the antiparallel linear arrangement of
peptide sequences is triggered by chromophore stacking
causing the formation of antiparallel double strands by
lateral interactions of salt-bridges, whereas hydrophobic
leucine edges promote assembly towards two-dimensional
layers of the strands. The resulting rotational lock of the
double strands, which prevents coiled-coil formation, en-
ables the directional presentation of negative charges of
glutamic acid and positive charges of lysines on each side of
the strand layer and hence triggers the formation of the
observed layer stacks (rods). The arrangement of strands
perpendicular to the rod long axis in a tetrameric packing
mode is caused by this directed electrostatic stacking
process, which obviously dominates over the growth of
linear strands. This combination of effects prevents the
otherwise expected alignments of strands in a hexagonal
packing mode along a rod long axis, as has been reported in
several earlier publications.
The protonation of glutamic acid and lysines at pH 3.4,
however, eventually interrupts this electrostatically fixed
assembly. While maintaining the chromophore stacking the
interaction of hydrophobic leucine edges and positively
charged lysine residues leads to a drastic change of the
overall structure, as it has been supported by MD simu-
lation. There is a trend towards the formation of a super-
helical ultrastructure, which enables (i) an additional lateral
stacking of the dye chromophores and (ii) a presentation of
positive charges towards the surfaces of an extended
membrane-like layer. The acidification of the peptide thus
creates a kind of polymeric amphiphile (hydrophobic leucine
edge vs. hydrophilic (positively charged) lysine residues)
which eventually leads to the formation of an assembly
structure where the hydrophilic lysine residues are exposed
towards the aqueous phase. This effect might explain the
observed separation of individual two-dimensional mem-
brane layers due to the charge repulsion of surface exposed
lysines.
By using a peptide as template for dye aggregation, we
present a novel concept that is an alternative to the
established attachment of dyes onto DNA oligomers in
mimicking photosynthetic light-harvesting systems. The here
described dye-peptide conjugate is capable of forming two
novel distinct supramolecular assemblies which are structur-
ally triggered by the complementary interplay of mutual
chromophore coupling and pH induced changes in the
peptide charge pattern. This dye-peptide system impres-
sively demonstrates how the photo-physical properties of
formed aggregate types can be tuned without synthetic
modification, solely by changing the environmental condi-
tions. In general, the tuning of dye self-assembly by choosing
an appropriate chemistry of a peptide scaffold as well as
adapting suitable preparation and environmental conditions
opens an avenue for the generation of new biomaterials for
further photo-physical research and potential technological
applications.
Acknowledgements
We thank the European Union’s Seventh Framework
Program for research, technological development and dem-
onstration under the Marie Curie ITN Scheme (Fluor21
grant number FP7-PEOPLE-2013-ITN-607787) for financial
support. The work of Noureldin Saleh has been financed by
the DFG Cluster of Excellence MATH +. We would like to
acknowledge the assistance of the Core Facility BioSupra-
Mol supported by the DFG. We thank Drs. Allison A.
Berger and Dorian Mikolajczak for proof-reading and for
the discussions that improved the manuscript. Open Access
funding enabled and organized by Projekt DEAL.
Conflict of Interest
The authors declare no conflict of interest.
Data Availability Statement
The data that support the findings of this study are available
in the Supporting Information of this article.
Keywords: Chromophore Assembly ·Cyanines ·De Novo
Peptide ·Electron Microscopy ·H-Aggregates
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Manuscript received: June 13, 2022
Accepted manuscript online: September 26, 2022
Version of record online: October 26, 2022
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