L-Proline induced self-assembly of indolicidin derived palindromic tripeptide

Article abstract of DOI:10.1016/j.tetlet.2014.04.090

We describe the synthesis, crystal structure, and various microscopic studies of the palindromic tripeptide WPW derived from antimicrobial peptide indolicidin. The present study reveals that tripeptide 1 and 2 undergo self-assembly to form vesicular structures after prolonged incubation, thus giving an interesting insight into the contribution of l-proline and flanking tryptophan residues in the self-assembly process. These vesicles were also amenable to simple focused ion beam (FIB)-aided bisection and thus possible to mill these vesicles to create different shapes. The circular dichroism (CD) analysis indicates that incubation promotes and stabilizes the more favorable secondary structures for 1 and 2. Preliminary result shows that tripeptide 1 exhibits appreciable interaction with Tb³⁺ as determined by quenching in tryptophan fluorescence.

Full text of DOI:10.1016/j.tetlet.2014.04.090

Accepted Manuscript
L-Proline induced self-assembly of indolicidin derived palindromic tripeptide
Khashti Ballabh Joshi, Prabhpreet Singh
PII:
S0040-4039(14)00727-8
DOI:
http://dx.doi.org/10.1016/j.tetlet.2014.04.090
TETL 44552
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 March 2014
26 April 2014
27 April 2014
Please cite this article as: Joshi, K.B., Singh, P., L-Proline induced self-assembly of indolicidin derived palindromic
tripeptide, Tetrahedron Letters (2014), doi: http://dx.doi.org/10.1016/j.tetlet.2014.04.090
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L-Proline induced self-assembly of
indolicidin derived palindromic tripeptide
Leave this area blank for abstract info.
Khashti Ballabh Joshia* and Prabhpreet Singhb ∗
The crystal structure and microscopic studies of the palindromic tripeptide WPW gives interesting insight into contribution
of L-proline and flanking tryptophan residues in the self-assembly process.

1
Tetrahedron Letters
jo u rn al h ome p ag e : w ww .e lse vie r .c om
L-Proline induced self-assembly of indolicidin derived palindromic tripeptide
Khashti Ballabh Joshia* and Prabhpreet Singhb∗
a Department of Chemistry Dr. H.S.G. Central University Sagar-470003, (M.P.)-India
b Department of Chemistry, UGC Centre for Advance Studies-I, Guru Nanak Dev University Amritsar-143005 (P.B.)-India
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We describe the synthesis, crystal structure and various microscopic studies of the palindromic
tripeptide WPW derived from antimicrobial peptide indolicidin. The present study reveals that
tripeptide 1 and 2 undergo self-assembly to form vesicular structures after prolonged incubation,
thus gives interesting insight into contribution of L-proline and flanking tryptophan residues in
the self-assembly process. These vesicles were also amenable to simple focused ion beam (FIB)-
aided bisection and thus possible to mill these vesicles to create different shapes. The circular
dichroism (CD) analysis indicates that incubation promotes and stabilizes the more favourable
secondary structures for 1 and 2. Preliminary result shows that tripeptide 1 exhibit appreciable
interaction with Tb3+ as determined by quenching in tryptophan fluorescence.
Available online
Keywords:
Self-Assembly
Peptide
L-Proline
Tryptophan
Biofabrication
2014 Elsevier Ltd. All rights reserved.
Indolicidin (ILPWKWPWWPWRR-NH2), originally isolated
from bovine neutrophils, is a 13-mer cationic antimicrobial
peptide (AMPs) with a +3 overall charge, an amidated C-
terminus and belongs to cathelicidin family.1-3 This short peptide
is effective against a broad spectrum of organisms including
bacteria, fungi, protozoa and acts as an inhibitor/modulator for
various enzymes. The peptide kills the HIV-1 virus but also
exhibit cytotoxicity against human T-lymphocytes and
erythrocytes.4-5 Due to the presence of high content of tryptophan
residues (39%) interspersed with proline residues (23%)
throughout the sequence, it probably assumed a structure distinct
from the well-known α-helical and β-structured peptides.6
Moreover, the presence of high content of tryptophan is regarded
as an essential feature for exhibiting high antimicrobial activity,
because these Trp- residues have a natural liking for the
interfacial region of lipid bilayers.7
growth of fibrils on vesicular platform) on the peptide-peptide
interface.
Tryptophan is an interesting aromatic amino acid for determining
the 3D structure of protein and play pivotal role in self-assembly
of peptide. Like Phe-Phe dipeptide, Trp-Trp dipeptide is not able
to form any self-assembled structure in the solution state8a,12
,
however instantaneous vesicular morphology was observed for
tetrapeptide PWWP. The orientation of tryptophan moiety for π-π
interaction is highly dependent on two proline residues present at
N- and C-terminus of the tetra peptide. This prompted us to
investigate the self-assembly of palindromic tripeptide Trp-Pro-
Trp (WPW), derived from the antimicrobial peptide indolicidin
sequence, in solution and solid state. In this sequence the L-Pro
residue situated in between the two L-Trp residues.
HN
HN
The self-assembly and folding of protein, peptide building blocks
to form discrete nanostructured materials (via hydrogen bonding
and other non-covalent interactions), their chemical diversity,
facile synthesis and biocompatibility make these building blocks
very attractive for various bio-nanotechnological applications.
These protein and peptide based nanostructures can be further
used for patterning through fabrication, useful for micro and
nanofluidic sensing or to make responsive materials sensitive to
external stimuli for applications in drug delivery systems and
(bio)chemical sensors.8-10 Our group11-14 have earlier elucidated
that peptide building blocks could form diversity of aggregate
morphologies, such as spherical aggregates11, ditryptophan
transforms biotin fibers into compact spherical ball-like
O
O
O
H
N
H
N
N
N
O
N
H
O
H2N
OH
O
O
O
O
HN
HN
[2]
[1]
Figure 1: Chemical structure of protected tripeptide Boc-WPW-OEt (1) and
unprotected tripeptide NH2-WPW-COOH (2).
Herein we describe the solution phase synthesis, crystal structure
and various microscopic techniques for investigating the
vesicular morphology obtained from protected tripeptide Boc-
WPW-OEt (1) and unprotected tripeptide NH2-WPW-COOH (2)
structures12, nanofibrils13 and hybrid structure14 (templated
———
∗ Corresponding author. Tel.: +91-8427101534; e-mail: prabhpreet.chem@gndu.ac.in

2
sequence. The microscopic studies reveal that presence of
proline, in between tryptophan (WPW) delayed the process of
self-assembly in solution. The CD spectra of tripeptides after 5
days indicate that incubation promotes and stabilize the more
favorable secondary structures for 1 and 2. The vesicles obtained
via self-assembly of peptide 1 and 2 were also acquiescent to
focused ion beam (FIB)-aided milling to create different shapes.
Furthermore, the supramolecular recognition properties towards
heavy transition metal ions using tripeptide 1 were studied.
sample was aged for 2 days for peptide 1 and 5 days for
peptide 2. Time-dependent follow up confirmed that longer
incubation periods simply altered the dimensions of spherical
structures without affecting the gross morphology hence SEM
investigated data are well corresponded with optical microscopic
data.
Peptide 1 and 2 (figure 1) were synthesized by solution phase
peptide synthesis techniques involving protection/deprotection
and condensation methodologies using DCC/HOBT as coupling
reagent (see supplementary data for synthesis).
Tripeptide 1 crystallizes in monoclinic space group P1 21 1 (No.
19) (Figure 2). Crystallographic signature of 1 displayed the solid
state conformation, which perhaps is the basis of significant
ordered self-assembly in solution state. The values of most of the
torsional angles (φ1, ψ1, φ2 and ψ2) of the constituent amino acid
residues fall within the polyproline II (PP-II) structural region
(supplymentary data, CCDC No. 670518). The solid state
structure of 1 involved two strong intermolecular interactions
involving indolic -NH…O (carbonyl of Boc group) (2.23 Å) and
Pro-α-CH…π (indole ring) (2.63 Å) (Figure 2b). Owing to these
two interactions the molecule of 1 appeared in ring shape
architecture stabilized by pyrrolidine-aromatic interactions as
well as hydrogen bonding. We were unable to grow crystals for
2, even after repeated attempt.
Figure 3: Fluorescent optical micrograph (a-c) and SEM micrograph (d-f) of
1 mM (60% CH3OH:H2O solution) of tripeptide (1) and (2); (a) 2 days aged
sample of 1, (b) 5 days aged sample of 1 (arrow indicates almost same size of
vesicles), and (c) 5 days aged sample of 2 (d) 2 days aged sample of 1,
[inset] magnified image of vesicular morphology (e) 2 days aged sample of 1
in 100% methanol (f) 5 days aged sample of 2.
Further we studied the effect of solvent (60% to 100%
CH3OH in H2O) and concentration (0.5 - 3 mM) on the
morphology of the peptide 1 through SEM. We observed that as
the concentration of methanol increased (for one specified
concentration of peptide 1) the number of vesicles increased and
in 100% methanol, 1 resulted in extensive aggregation of
vesicular structure. However, fresh sample of 1 again failed to
show any resolvable vesicular structure in 100% methanol. Also,
there is no effect of concentration on the morphology of the
vesicular structure is observed, when we change the
concentration of compound 1 from 3 mM to 0.5 mM. The sizes
of the vesicles are about ~1 µm. These vesicular structures were
exclusively formed without any indication of other self-
assembled structure such as fibrils, filaments etc. Such vesicular
morphologies for 1 and 2 were preserved when we observed
through TEM and AFM.
Figure 2: (a) ORTEP view of crystal structure of tripeptide 1 and (b)
Diamond generated architecture of 1 showing strong Pro-α-CH...π interaction
and indolic N-H…O=C hydrogen bonding. tert-Boc group has been removed
for sake of clarity
In preliminary experiments we evaluated the time dependent
aggregative propensity of peptide 1 and 2 (1 mM in 60%
CH3OH:H2O) under fluorescence optical microscope. The fresh
and incubated samples (at different time intervals) of 1 labeled
with rhodamine B revealed that the fresh solution of 1 failed to
give any significant and noticeable self-assembly. However, in
case of incubated samples, we observed ordered vesicular
structure after 2 days of incubation at ambient temperature with a
diameter of 0.8-2 µM (Figure 3). A continued follow up
confirmed that 5 days incubation time period refine the
dimension of vesicular structures, however gross morphology
remained same and intact. In comparison, peptide 2 did not
reveal any vesicular morphology in fresh solution as well as after
2 days incubation of samples at ambient temperature. However,
we observed vesicular structures of peptide 2 after 5 days
incubation period.
Transmission electron micrograph (TEM) also revealed the
presence of intersperse, circular structures with an average
diameter of 1-2 µm for 1 and 2. Aged solutions of 1 were
subjected to AFM analysis at various incubation time points,
including a freshly prepared solution. The self-aggregation was
observed in the case of 2 and 5 days incubated sample when
probed by AFM on a freshly cleaved mica surface whereas, the
fresh sample of 1 does not reveal any resolvable structures. As
the self-assembly of 2 was poorly resolved under SEM we did
not further studied the self-assembly process of 2 under atomic
force microscopic events. The observed self-assembly was
independent of the surface used, as similar structure was also
observed on highly oriented pyrolytic graphite surfaces (HOPG),
thus suggesting that the self-organizing property as an inherent
property. Further, on incubation or changing the concentration of
the peptide does not lead to any deformity in the structure (Figure
4).
The presence of vesicular structure for 1 (1 mM in 60%
CH3OH:H2O) was further confirmed by scanning electron
microscope (SEM), where uniform vesicles (more or less like a
tennis ball), with a diameter of 1-2 µM, were evident when the

3
Figure 4: (a) TEM micrograph of protected tripeptide 1; (b) TEM micrograph
of deprotected tripeptide 2 vesicles in 5 days incubated solution; (c) AFM
image of tripeptide 1 vesicles in 5 days incubated solution and (d) 3D view of
tripeptide 1 vesicles on mica surface
The experiment with focused-ion-beam/high resolution
scanning electron microscope (FIB-HRSEM) also revealed
vesicular structures with an average diameter of 1-2 µm (Figure
5), which suggests that these spherical structures do not result as
drying artifacts.15 We attempted to see the interior core of the
spherical ball-like structures with the help of FIB milling
experiments.16-18 The microsphere from 1 was bisected by the
help of ion beam followed by electron beam imaging at 52º
angle, thus revealing interior part of the spherical structure. The
reasonable structural integrity of these structures enables them to
withstand ion currents for a short time period (Figure 5) compare
to WPWWPW sequence.11b These microspheres were also
amenable to simple FIB-aided bisection and thus possible to mill
these microspheres to create different shapes.

4
In the control experiments, we observed that the protected and
de-protected dipeptides of Trp-Pro (WP) and Pro-Trp (PW)
sequence were unable to show any kind of defined aggregates
(data not shown). These results demonstrated that the position of
tryptophan and proline in a truncated sequence playing a crucial
role in self-assembly process. Truncated PWWP and WPWWPW
sequence11, where two tryptophans are present in between
proline, results in instantaneous formation of vesicular structures
in fresh solution whereas; the presence of proline in between the
tryptophan (WPW) delayed the process of self-assembly in
solution.
assembled vesicles formation process, involving tryptophan
rich peptide model systems. Such spherical morphologies were
preserved when experiments were performed on the copper
surface (SEM), on highly oriented pyrolytic graphite surface
(HOPG) or under wet conditions (AFM), focused-ion-beam/high
resolution scanning electron microscope (FIB-HRSEM).
Acknowledgements
We thank Prof. Sandeep Verma, Department of Chemistry,
IIT Kanpur, for his constant support and suggestions. Financial
support from UGC and IIT-Kanpur is also gratefully
acknowledged.
Careful use of proline and aromatic amino acids systems will
not only help us to understand its role as a stabilizing feature in
aggregation, but also aid in de novo design of self-assembled
structures. Such kinds of nanomaterials have wide applications in
drug design/delivery for observation of the biological process at
nanoscale and further they can exhibit specific biomolecular or
supramolecular recognition properties using multifunctional
peptides.
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Figure 8. Fluorescence quenching of tripeptide 1 (10 µM) by various metal
ions (10 µM) in methanol/water (1:1) (1:1 v/v, 50 mM HEPES buffer, pH
7.0); λex =280 nm and λem =320 nm.
In conclusion, this study gives interesting insight into
contributions of proline and indole aromatic side chains of amino
acid residues in providing driving force and stability to self-