pharmaceutics
Article
A Diphenylalanine Based Pentapeptide with Fibrillating
Self-Assembling Properties
Stefania-Claudia Jitaru 1 , Andrei Neamtu 2,3 , Gabi Drochioiu 1 , Laura Darie-Ion 1 , Iuliana Stoica 4 ,
Brindusa-Alina Petre 1,2, * and Vasile-Robert Gradinaru 1, *
1
2
3
4
*
Faculty of Chemistry, Alexandru Ioan Cuza University, 11 Carol I Bd., 700506 Iasi, Romania
TRANSCEND—Regional Institute of Oncology, 700483 Iasi, Romania
Department of Physiology, “Gr. T. Popa” University of Medicine and Pharmacy, 16 University,
700115 Iasi, Romania
“Petru Poni” Institute of Macromolecular Chemistry, 41-A Grigore Ghica Voda Alley, 700487 Iasi, Romania
Correspondence: brindusa.petre@uaic.ro (B.-A.P.); robert.gradinaru@uaic.ro (V.-R.G.)
Abstract: Peptides and their related compounds can self-assemble into diverse nanostructures of
different shapes and sizes in response to various stimuli such as pH, temperature or ionic strength.
Here we report the synthesis and characterization of a lysozyme derived pentapeptide and its ability
to build well-defined fibrillar structures. Lysozyme FESNF peptide fragment was synthesized by
solid phase peptide synthesis using the Fmoc/t-Bu strategy, purified by analytical high-performance
liquid chromatography (HPLC) and its molecular weight was confirmed by matrix-assisted laser
desorption/ionization mass spectrometry (MALDI–MS). Spectroscopic features of this pentapeptide
were investigated by UV-visible spectroscopy and fluorimetry showing the pattern of marginal phenylalanine residues within the peptide sequence. Self-assembling properties were determined using
atomic force microscopy (AFM), aggregation index and thioflavin T assay (ThT). FESNF generating
fibrillar structures observed by AFM and aggregation propensity were primarily influenced by pH
conditions. Moreover, the experimental data were confirmed by molecular dynamics simulation
studies. The obtained fibrils will be used next to explore their potential to act as support material for
medical and cosmetic application.
Citation: Jitaru, S.-C.; Neamtu, A.;
Drochioiu, G.; Darie-Ion, L.; Stoica, I.;
Petre, B.-A.; Gradinaru, V.-R. A
Keywords: pentapeptide; self-assembling; egg white lysozyme; MALDI-ToF; fluorescence;
aggregation; AFM
Diphenylalanine Based Pentapeptide
with Fibrillating Self-Assembling
Properties. Pharmaceutics 2023, 15,
371. https://doi.org/10.3390/
1. Introduction
pharmaceutics15020371
In the last few decades, self-assembling bioinspired molecules have gained significant
interest due to their simple structure, biocompatibility and biodegradability. In particular,
free or capped single amino acids or oligopeptides are renowned for these properties.
Inspired by nature, many peptides have been designed to obtain diverse self-assembled
structures based on well-known proteins. A wide range of applications were described so
far for peptide self-assembled structures, such as drug delivery, tissue engineering, cell
carriers biosensors and gene delivery [1–3].
Peptide biomimetics is an expanding research area that targets new synthetic materials
with similar or unrevealed functions as compared to those of the widely investigated
natural compounds.
The lysozyme is a low molecular weight enzyme that plays an important role in the
innate immune response [4]. Hen white lysozyme has the capacity to assembly into fibrils
under heating and acidic conditions [5]. Both fibrils and worm-like structures display
antimicrobial and antifungal activities [6]. Lysozyme-like amyloid networks were successfully used as scaffold material for tissue engineering [7]. Molecular dynamics studies also
support the lysozyme capacity to form beta-strands [8] and the self-assembly conformation
of the lysozyme was shown to be a calcium dependent process [9]. An amyloid core region
Academic Editor: Giancarlo Morelli
Received: 29 November 2022
Revised: 16 January 2023
Accepted: 18 January 2023
Published: 21 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Pharmaceutics 2023, 15, 371. https://doi.org/10.3390/pharmaceutics15020371
https://www.mdpi.com/journal/pharmaceutics
Pharmaceutics 2023, 15, 371
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GILQINSRW named K peptide was found to be able to form aggregates at pH 4 [10]. Analogously, a recent study proposes to use peptides from hydrolyzed hen egg white lysozyme
(HEWL) as a convenient source to prepare an amyloid hydrogel by lyophilization and
resuspension at 37 ◦ C and pH shifting [11]. Antioxidant and antimicrobial peptides derived
from lysozyme enzymatic digestion were also reported [12,13]. Phenylalanine-, tyrosineand tryptophan-containing peptides are easily detected in the 240–310 nm range [14] and
easily quantified and studied by fluorescence [15] or used as platforms for assembly [16]
or chemosensors [17]. Phenylalanine may act as relay amino acid in the electron transfer
process through peptides [18], which imprint antioxidant properties to some peptides [19].
Phenylalanine prefers homologues and heterologous interactions with tyrosine or tryptophane residues [20]. The assembly of uncapped, single- or double-capped phenylalanine
homopeptides was recently revised. The self-assembly thermodynamics and kinetics is
also dictated by N- and C-termini groups [21]. The number of aromatic residues, peptide concentration and incubation conditions are influencing peptides morphology and
polymorphism [22]. The amphiphilic peptide palmitoyl-FFFEEE-COOH self-assembly
of twisted helical ribbons was also reported [23]. The self-assembly kinetics of a model
amyloid peptide YYKLVFF, having a core sequence derived from Aβ peptide, was also
studied in solution by linear dichroism [24].
FTIR spectroscopy and differential scanning calorimetry were previously applied
in order to study the thermal, aggregation and gelation capacity of the lysozyme in selfassembling conditions [25]. Waltz-DB 2.0 is a database of amyloid-like forming peptides that
contains more than 1400 entries. According to this database, FESNFN is a peptide, among
the other 40 phenylalanine based-hexapeptides, derived from hen egg white lysozyme
primary sequence, which stands out by its amyloid propensity [26]. Analogously, by
applying a Tango algorithm [27] prediction a possible amyloidosis conformation of this
peptide was also suggested. According to our knowledge and literature search, experimental studies to elucidate the capacity of FESNFN hexapeptide to from fibril were not
reported. However, the sequence FESNF was identified as (i) an epitope peptide selected
by class MHC molecules [28], (ii) as part of tryptic peptide fragment 34 FESNFNTQATNR45
for relative quantification in size exclusion chromatography coupled with tandem mass
spectrometry [29] and (iii) as interacting moiety for NHS-aryl azido heterobifunctional
cross-linker used to study large-scale protein–protein interactions by chemical cross-linking
mass spectrometry [30]. This is the first time an experimental study indicates the possibility
of long-distance phenyl moieties to be involved in self-assembly.
Inspired by these theoretical data, a pentapeptide NH2 -FESNF-CO-NH2 has been
selected in this study. Initially, we assumed that the presence of aromatic groups at both the
N- and C-termini would cause π-π-stacking interactions and play a significant role in the
self-assembling process. Since serine residue facilitate hydrogen bonding and glutamate
moiety could be involved in electrostatic interaction, these residues are important in the
native self-assembly process and scaffolds [31]. Herein, we present the synthesis, separation
and characterization of C-terminal amidated FESNF peptide by spectroscopy and mass
spectrometry. Atomic force microscopy (AFM), aggregation index and thioflavin (ThT)
test assays and molecular dynamics simulations were used to investigate peptide selfassembling behavior.
2. Materials and Methods
2.1. Reagents
L-Phenylalanine was obtained from Fluka (Buchs, Switzerland). N,N-dimethylformamide
(DMF), sodium acetate trihydrate, trifluoroacetic acid (TFA), dichloromethane (DCM),
2,5-dihydroxy-benzoic acid (DHB) and N-methyl morpholine (NMM) were purchased from
Sigma-Aldrich (Steinheim, Germany). Rink amide resin (50–90 mesh, 0.51 mmol g−1 ) purchased from Sigma-Aldrich was used as a solid support. Acetic acid glacial and the amino
acids protected at N-terminal with Fmoc group (9-fluorenylmethyloxycarbonyl) used in
solid phase synthesis were purchased from Merck (Darmstadt, Germany) and piperazine—
‐
Pharmaceutics 2023, 15, 371
‐
−
‐
‐
‐
‐
‐
‐
3 of 15
‐
′‐
‐
N,N′ -bis (2-ethanesulfonic acid), PIPES buffer was from Carl Roth (Karlsruhe, Germany).
‐ ‐ ‐
‐
‐
‐
Benzotriazol-1-yl-oxy-trispyrrolidinophosphonium-hexafluoro-phosphate (PyBOP) used
as activator was purchased from NovaBiochem (Novabiochem, Merck KGaA, Darmstadt,
Germany). Tris base (ULTROL® Grade) was obtained from the Calbiochem (EMD Chemicals, Inc., San Diego, CA, USA) and Thioflavin T from EMD (Millipore, Bedford, MA, USA).
Acetonitrile (ACN) HPLC grade and piperidine were purchased from Merck (Darmstadt,‐
Germany). All the solutions were prepared using deionized water (18.2 MΩ·cm) produced‐
‐
by a Milli-Q systemΩ∙
(Millipore, Bedford, MA, USA).
All other reagents were used without
further purification.
2.2. Peptide Synthesis
‐
‐ ‐
The amidated peptide, NH2 -FESNF-CO-NH
2 (Figure 1) was synthesized by solid-‐
phase peptide synthesis (SPPS) based on Fmoc/tBu strategy as previously described [32–34].
Rink amide resin (50–90 mesh, 0.51 mmol g−1 )− was used as a solid support. In sum-‐
mary, the protocol used was as follows: (i) Fmoc deprotection for 2, 2, 5 and 10 min
using 20% piperidine in DMF; (ii) washing with DMF; (iii) coupling ‐of Fmoc-amino‐
acid/PyBOP/NMM in DMF for 50 min; (iv) washing with DMF; (v) second coupling‐
of Fmoc-amino acid/PyBOP/NMM in DMF for ‐50 min. A 3-fold excess was used for‐
PyBOP—coupling reagent and double coupling was performed
using 5-fold excess‐ fol‐
lowed by 3-fold excess of each sequential coupled amino acid. Bromophenol blue test has
been used for monitoring the coupling progress [35]. After the synthesis was finished, a‐
chemical solution containing TFA, triisopropylsilane and deionized water (95:2.5:2.5, v/v/v)
was used to cleave the peptide from the resin at room temperature for 2.5 h. The crude
product was afterwards precipitated with cold diethyl ether, solubilized in 5% aqueous
acetic acid prior‐ to freeze-drying using a lyophilizer from Martin Christ Alpha 1–2 LDplus
(Martin Christ, Osterode am Harz, Germany) and stored
at −20 ◦ C until further use.
−
‐
‐
‐ ‐
Figure 1. C-amidated
pentapeptide (NH2 -FESNF-CO-NH
2 ) molecular structure (Balls and sticks
‐
‐
model), where gray is carbon; blue is nitrogen; red is oxygen and white is hydrogen, where F‐
‐
‐
phenylalanine, E-glutamic acid, S-serine and N-asparagine.
‐
‐
2.3. Reversed-Phase-High
Performance Liquid Chromatography
The pentapeptide was purified using an HPLC Dionex UltiMate 3000 UHPLC system
(Thermo Scientific, Waltham, MA, USA) equipped with a Diode Array Detector module.
‐
μ silica, 250 mm × 4.6 mm, 300 Å pore size) from Waters
A Vydac RP-C18
column (5 µm
(Milford, MA, USA) was used as stationary phase. A mixture of two eluents, A (0.1%
TFA in bidistilled water) and B (0.1% TFA in ACN: bidistilled water, 80:20, v/v), was
used as a mobile phase. A linear gradient elution, from 5 to 65% B within 30 min, with a
flow rate of 1 mL/min was employed for HPLC separation. The peptide was detected by
monitoring the typical peptide bond absorption at 215 and 220 nm, and characteristic band
of phenylalanine moieties at 255 nm. Two main elution peaks were observed when crude
peptide was analyzed by chromatography. Thus, beside the peptide elution peak observed
at 11.64 min a byproduct having a retention time of 13.96 min was noticed.
Pharmaceutics 2023, 15, 371
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2.4. MALDI-ToF Mass Spectrometry
Mass spectrometry analysis was carried out using a Bruker Ultraflex MALDI ToF/ToF
mass spectrometer. A 50 mg/mL DHB in 2:1 ACN: 0.1% TFA in MilliQ was used for
peptide mapping. Consequently, the mixture was transferred and allowed to dry at RT.
After samples cocrystallization (sample: matrix 1:1), on a 348-spot target plate, this was
inserted into instrument. The following parameters were used to obtain MALDI-ToF MS
spectra: positive ion mode, an acceleration voltage 20 kV, 140 ns delay, 40% grid voltage,
low mass gate of 500 Da. The mass spectra acquisition was performed in a mass range
of 600–3500 Da. A mixture of five peptides (ACTH, Angiotensin II, Bradykinin, Insulin,
B-chain oxidized P14R and insulin) was used as an external mass calibrator. Each final
mass spectrum was obtained as a result of 300 shots taken per each acquisition. In order to
investigate peptide fragmentation LIFT cell in MALDI–ToF/ToF mass analyzer was used.
2.5. Spectrophotometric and Spectrofluorimetric Studies
UV-visible absorption spectra of purified pentapeptide were recorded in a 1 cm quartz
cell using a Libra UV-visible spectrophotometer (Biochrom, Cambridge, UK) equipped
with a Peltier thermostat cell holder maintained at 25 ◦ C. Each spectrum was measured
in triplicate using a scan speed of 1856 nm/min (scan step 1 nm), in the spectral range of
200–500 nm. All samples were dissolved and diluted in Tris 30 mM, pH 7.3.
Index aggregation assay was performed at 37 ◦ C for 150–180 min, according to Pignataro et al. [36] and Rajan et al. [37]. The peptide was dissolved in a corresponding buffer
with final concentration of 1.0–2.0 mg/mL. Four different buffer solutions, Acetate 45 mM
pH 6.0, Acetate 50 mM pH 7.0, Tris 40 mM pH 8.0 and Tris 50 mM pH 10.70 were used.
Two solutions (HCl 10 mM and 0.1 mM) were used for acidic conditions. Each spectrum
was recorded with a scan speed of 2649 nm/min (scan step 1 nm), in the spectral range of
200–400 nm at 37 ◦ C. The highest aggregation index (Equation (1)), determined based on
absorbance values recorded at both 258 and 350 nm, was reached in 20–30 min.
AI =
A350
∗ 100
( A X − A350 )
(1)
where A350 is absorbance at 350 nm and AX is the absorbance at 280 or 258 nm
Thioflavin T spectroscopic assay was performed using an λex = 450 nm and an
λem = 482 nm. A stock solution of ThT was prepared in a freshly prepared NaPi 10 mM/
NaCl 150 mM buffer pH 7. The ThT fluorescence was monitored in the presence of aggregate using a diluted dye solution (40 µL of 0.5 mM) at a final concentration of 40 µM using
alpha-synuclein as positive control aggregating protein [38]. The increase of emissions was
observed by adding a volume of 10 µL of peptide solution into the cuvette (final volume
500 µL) and recorded as mentioned above.
All fluorescence 2D and 3D spectra were recorded using a FP-8350 Spectrofluorometer
(JASCO, Tokyo, Japan). The peptide solution was excited at different wavelengths to get
emission spectra. Analogously, excitation spectra were taken at pH 5 in the range from 200
to 272 nm using an λem = 282 nm. The maxima of excitation spectra correspond to 258 nm
from the absorbance.
All measurements were carried out at 27 ◦ C using a transparent quartz SUPRASIL®
cuvette (with 0.5 cm × 0.51 cm light path length, 1.7 mL volume, Hellma, Mulheim,
Germany) and a cell support FMH-802 type. Two-dimensional emission or excitation
spectra were recorded using a scan speed of 1000 nm/min, scan step 0.5 nm, 5 acquisition
per sample; both excitation and emission slit were 5 nm. Emission spectra of peptapeptide
were recorded in the range from 268 to 360 nm using an λex = 258 nm, corresponding to
the maximum wavelength band of the compound. The peptide quenching experiments
were executed in similar conditions following emission decrease at 283 nm.
Three-dimensiona; fluorescence spectra of peptide were performed under the following conditions: excitation 200–350 nm; emission 210–360 nm; data interval 1 nm in
excitation and emission; scanning rate 2000 nm min−1 . Blank subtractions were consid-
Pharmaceutics 2023, 15, 371
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ered for both 2D and 3D measurements and dilution correction were performed for 2D
quenching data.
2.6. Atomic Force Microscopy
The atomic force microscopy measurements were performed using a NTEGRA Scanning Force Microscope device (NT-MDT, Spectrum Instruments, Zelenograd, Moscow,
Russia) equipped with a NSG10 cantilever (resonance frequency νres = 214 kHz) in atmospheric conditions at 23 degrees. Different scan sizes were imaged between 3 × 3 µm2
and 20 × 20 µm2 with a scanning frequency of 0.6 Hz; the scanning velocities were 3.6
and 24 µm/s, respectively. Pentapeptide stock solutions (10 mg/mL) were prepared in
bidistilled water and further diluted 1:10 (v/v) in a 4.5% methanolic or buffer solution
(ammonium acetate 50 mM pH 5.5, Tris 50 mM pH 7.4 or Tris 50 mM pH 8.2). A small
volume (5 µL peptide solution) was transferred on a clean glass slide (10 × 10 mm) and
incubated at room temperature for 48 h.
2.7. Replica Exchange and Coarse-Grain Molecular Dynamics Simulations
The structural models of the FENSF peptide were built in an extended conformation
using the Schrödinger BioLuminate graphical environment (BioLuminate, Schrödinger,
LLC, New York, NY, USA, 2021) [39]. The protonation states of the N-terminus and
the aspartate residue in position 2 were chosen according to the pH of the simulation
environment (acidic, neutral and slightly basic). The peptides were solvated in cubic
simulation boxes so that there was a minimum distance of 9 Å between the peptides
and the box boundaries. The peptides and ions were modelled based on the OPLS_2005
forcefield [40], while the TIP3P model was selected for water representation [41]. Replica
exchange with solute scaling (REST2) [42] was used to efficiently sample the peptide
conformational space using 6 replicas for the single peptide and 8 replicas for the dipeptide simulations respectively. All simulations, each 30 ns long, have been performed
using the Desmond [43] molecular dynamics package at constant temperature (300 K)
and pressure (1 atm) using the Nosé–Hoover chains thermostat [44] and Martyna–Tobias–
Klein barostat [45]. Conformations resulted from the replica exchange simulations were
clustered based on their root mean square deviation of atom position using the VMD
software [46]. The dominant cluster was considered the one containing the largest number
of structures. The coarse-grain (CG) simulations (0.8 µs long) were performed using the
MARTINI [47] force field with the GROMACS 2019 software suite [48]. Visualization and
coloring of molecular structures was done with the Schrödinger BioLuminate and VMD
graphical solutions.
2.8. Data Analysis
The spectral 2D data were analyzed using the KaleidaGraph v.4.0 (Synergy Software,
Reading, PA, USA) and 3D-surface emission plot was created in Excel version 2207 (Microsoft 365 package). Mass spectrometry data were analyzed using a Bruker’s Flex Analysis
v.3.4 software (Bruker Daltonics, Bremen, Germany). The AFM data acquisition and analysis were performed using Nova 1.1.1.19891 and Image Analysis 3.5.0.20102 software
developed by NT-DMT Spectrum Instruments.
3. Results and Discussion
3.1. Peptide Synthesis, Purification and MS Characterization
The pentapeptide inspired from the hen egg white lysozyme primary structure was
successfully synthesized by SPPS and purified by HPLC. Initially, the raw peptide was
characterized by two signals in the separation profile (Figure S1, left). The initial peptide
purity was estimated to be 79% and 77% based on the peak areas monitored at 215 and
220 nm. The by-product was eluted with a 2 min delay form the column. A peptide purity
of 96% was attained after two purification steps (Figure S1, right). Both the peptide and byproduct molecular weights were investigated by mass spectrometry using a MALDI-TOF
Pharmaceutics 2023, 15, 371
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MS and MALDI-ToF/ToF (Figure 2). The MALDI-ToF spectrum of FES peptide indicated an
expected molecular weight of m/z 642.48 corresponding to [M+H]+ . Moreover, the sodium,
[M+Na]+ and potassium, [M+K]+ adducts were observed at m/z 664.46 and m/z 680.44,
respectively (Figure 2A). The experimental values were compared with predicted values
calculated using GPMAW software. MS/MS peptide sequence analysis was also used to
confirm the primary structure of the FES peptide. As expected, the m/z 642.48 peptide
parent ion fragmentation in LIFT mode led to several mono-charged b- and y-type ions
and some dehydrated species (Figure 2B). Single positive charged peptide fragments were
identified, such as y2+ (m/z 278.82), y3+ (m/z 365.86), b4+ (m/z 477.85) and y4+ (m/z 495.86).
The peak intensity and cleavage preference is also influenced by amino acids situated near
to the breakage site, which allow a different malleability for fragmentation depending on
the structure of their side chain [49]. Additionally, the tandem mass spectrometric spectrum
had several fragments, such as those resulted from the removal of a molecule of water,
[M+H]+ -H2 O at m/z 623.71 fragment ion, ammonia, y2+ -NH3 at m/z 261.75, y3+ -NH3 at
m/z 348.81, b4+ -NH3 at m/z 460.82 fragment ion or carbon dioxide, [M+H]+ -CO2 at m/z
460.82 fragment ion. The water loss can be attributed to glutamate and serine moiety
and a 17 Da deviation from ammonia release assigned to the pentapeptide N-terminus
or water release along with an isotopic shift [50]. On the other hand, the loss of carbon
dioxide observed for the molecular ion and its dehydrated species originated from the
decarboxylation of the glutamate moiety.
‐
Figure 2. MALDI-ToF
MS spectrum of FES peptide (A) and the MS/MS spectrum in LIFT mode of
[M+H]+ ion (B).
‐
Pharmaceutics 2023, 15, 371
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Analogously, the second compound observed after synthesis was studied by MS and
MS/MS analysis. A molecular weight shift of 106 Da was noticed for this by-product and
was attributed to a different cleavage of the peptide from the resin. This cleavage pattern
was also earlier reported for other peptides when rink amide was used as solid support
and TFA as cleaving agent [51].
3.2. Spectral Properties Studies
The pentapeptide under the present study has two distinctive fingerprints due to
phenylalanine residues at both the N- and C-terminal sites, which may predispose to a
self-assembly behavior. The presence of these aromatic rings was investigated using UVvisible spectroscopy as well as fluorescence techniques in order to find another potential
application for peptide-metal ion interactions.
The UV-visible spectrum of pentapeptide was recorded in Tris buffer in order to
establish the typical characteristic fingerprint of phenylalanine moieties. Thus, the highest
peak noticed at 258 nm was accompanied by four spectral shoulders at 247, 252, 263 and
267 nm at both pH 7.3 and 8.0. A similar spectroscopic profile was noticed in PIPES buffer
30 mM pH 6.5 at a 200 µM peptide concentration (Figure S2). These findings are in the
frame of previously reported data for phenylalanine containing peptides [52,53]. Another
spectral band, characteristic of an amide group, was displayed at 203 nm (A 0.97) when‐
a diluted peptide solution (41 µM) was analyzed at pH 7.3. Phenylalanine may act as a‐
relay for long-distance electron transfer through the peptide structure [18] and its modest
μ
contribution to peptide intrinsic fluorescence is related
to its low quantum yield [54].
Fluorescence studies also reveal specific features for peptides containing phenylalanine
moieties. The emission spectrum of FES peptide in Tris 30 mM pH = 7.3 at excitation
wavelengths of 258 nm displays a maximum at 282 nm and a shoulder at around 290 nm.
‐
A good linearity
was noticed in the range of 25–250 µg/mL of pentapeptide at the emission
μ
wavelength of 282 nm (Figure S3, inset). Analogously,
the emission peptide spectrum in
sodium acetate 50 mM pH = 5.05 was measured and a similar profile was noticed. As
expected, the excitation spectra display a maximum at 258 nm (Figure 3A).
Figure 3. 2D and 3D spectra of peptapeptide. Emission spectra (A) at four different peptide concen-‐
μ
trations (39, 78, 132 and 176 µM—a,
b, c and d; continuous lines) obtained at λλex 258 nm and the
corresponding excitation spectra (a’, b’, c’ and d’; discontinuous lines) recorded at an λλem 282 nm.
‐
‐
μ
Three-dimensional spectra of FESNF-NH2 peptide (156 µM, (B)) in acetate 50 mM pH 7.0. Contour
map for pentapeptide collected at three different pH values (pH 5.0; 6.0, and 7.0 in (C–E)).
‐
‐
Pharmaceutics 2023, 15, 371
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Three-dimensional fluorescence spectra were also investigated at pH 5, 6 or 7 in 40 mM
acetate buffer at 27 ◦ C. Peptide alone (156 µM) displays 2 peaks centered at 213 and 283 nm
(Figure 3B–E). These bands are characteristic to phenylalanine residues and not to peptide
backbone [55].
3.3. Peptide Aggregation Studies
Peptides’ and proteins’ physical stability is usually influenced by a number of factors.
Amino acid sequence, peptide net charge and its concentration might influence the peptide’s
ability to form aggregates [56]. A recent guide focused on the investigation of protein
aggregation was recently published. Some peptide aggregation propensity prediction tools
and various experimental methods are available to study aggregating species including
their shape and size [57].
The aggregation index is an important parameter that could indicate a peptide’s ability
to form aggregates in solution, usually when the aggregation index (AI) exceeds the value
of three. Phenylalanine alone has the capacity to form fibrils in PBS at 37 ◦ C [58]. Moreover,
the presence of phenylalanine residues in hexapeptides in cross-β aggregates and amyloidlike species was previously reported in the literature [27] and aggregation propensity is
strongly influenced by the number of phenylalanine residues present in the molecule [59].
Additionally, the asparagine or glutamine could play an important role in seeding peptide
and protein aggregation [60,61]. As expected, the obtained aggregation index was higher
than 40 at pH 6.00, 8.00 and 10.7 when A280 was considered. These values, obtained
applying Equation (1), could be particularly assigned only for protein aggregation studies.
Conversely, more appropriate results were obtained for our pentapeptide when small
AI values (less than 3) were calculated based on A258 and A350 . A dynamic aggregation
profile is shown in Figure 4. The highest AI (2.8) was obtained at pH 6 after 20 min
incubation at 37 ◦ C. Similarly, pentapeptide aggregation was studied at pH 2. Interestingly,
in these conditions the plateau was reached after 30–40 min when a solution of 2 mg/mL
peptide was used. The aggregation index was also determined as a function of pH after
30 min incubation at 37 ◦ C. The bell-shape profile (Figure S4 inset) clearly suggests that the
peptides need a slightly acidic environment to promote a propensity for aggregation.
Phenylalanine forms aggregates and ThT fluorescence intensity increases due to the
entrapment of the dye in these self-assembled structures [62]. The peptide aggregation
capacity was also investigated using the ThT assay. In this assay, the response could be also
influenced by FESNF total charge. Thus, after an incubation time at 50 degrees for 145 ha,
an 18% fluorescence increase was noticed at pH 8 (Figure S5). This electrostatic interaction
of positively charged ThT with the investigated partially negative peptide could enhance
the response in the dye fluorescence signal. This prominent role of electrostatic interactions
between fibrils and ThT was demonstrated as a major parameter affecting fluorescence
response [63].
Thus, after optimizing the pH and ionic strength the pentapeptide self-assembly potential was experimentally revealed by aggregation index studies providing new possibilities
for developing higher ordered structures using small molecules, such as peptides, peptide
conjugates and peptoids. To avoid misinterpretation of experimental data, a careful analysis
should be performed. The ThT assay shows a slight aggregation tendency; however, this
method is costly, time consuming and difficult to be applied for short peptides.
Pharmaceutics 2023, 15, 371
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‐
Figure 4. Two-dimensional
(2D) AFM images collected on 20 × 20μµm2 and 3 × 3μ µm2 areas and the
‐
‐ ‐ solutions
‐
corresponding cross-section
profiles of the samples obtained from various FESNF-CO-NH
2
(1 mg/mL) in (A,A’,A”) ammonium acetate 45 mM pH 5.5; (B,B’,B”) Tris 45 mM pH 7.4; (C,C’,C”) Tris
45 mM pH 8.2; and (D,D’,D”) 4.5% methanolic solution.
3.4. Atomic Force Microscopy (AFM)
‐ ‐
‐
The morphology of the aggregated species derived from FESNF-CO-NH
2 under
different solution media were analyzed using AFM and presented in Figure 4.
‐
Both ammonium acetate pH 5.5 and methanolic solutions (Figure 4A,A’,D,D’, respectively) appear to induce the clumping of fibrils. Although, this effect is more noticeable in
‐
the case of Figure 4D,D’,D”, which confirms once again that short-chain
alcohols induce
retardation on fibril formation as previously described in another study [64]. Moreover,
‐
methanol has been reported to substantially change the prevailing morphology by significantly decreasing the drying time [65]. The fibrils observed on the sample derived from the
ammonium acetate pH 5.5 appear similar to a real neural network. In this case, the average
width of the fibrils is approximately 130 nm, compared to that of the fibrils obtained for the
‐
sample from methanolic solution, where the average width is higher, around 235 nm, as
can be seen from the measurements based on the cross-section profiles from
‐ Figure 4A”,D”.
The calculated isoelectric point (pI) of FESNF-CO-NH2 peptide was 6.99, according
to BACHEM Peptide Calculator tool [66]. At pH‐7.4 (Figure
4B,B’), under physiological
‐
conditions and near the pH value of pI, peptide fibrillation is completely absent. Dense
clusters were formed, with dimensions ranging between 116 and 128 nm, according to the
Pharmaceutics 2023, 15, 371
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cross-section profile (Figure 4B”). This is a similar behavior exhibited by bovine insulin
when exposed to the corresponding pH value to its pI [64]. In contrast, fibrillation of
Alzheimer amyloid peptides tends to be favored when the net electrical charge is zero due
to pH conditions [67].
However, at pH 8.2 when negative charges outnumber positive charges, the peptide
is negatively charged. The salts could trigger peptide fibrillation [68]. The glutamate
deprotonation could discourage intra and intermolecular H-bond interactions triggered by
glutamate, serine and asparagine sidechains from the peptide main core and additionally
enhances intermolecular interactions via π-π staking. In these conditions, the pentapeptide
self-assembles near to the pKa value of Tris buffer into long homogenous and distinct
nanofibrils (Figure 4C,C’) with an average width of about 165 nm (see the section image
from Figure 4C”). Therefore, we should consider contributions of both partially dissociatedNH2 groups from both peptide N-terminus and buffer. The fibrils seem to be the result
of multiple globular structures stacked together, as observed in the zoomed image placed
in the corner of the height profile (Figure 4C”). These results are framed with our recent
study when a sensor for the detection of verbascoside in various olive oils by immobilizing
FESNF peptide using glutaraldehyde as a crosslinking agent on screen-printed carbon
electrodes that was modified with graphene oxide. A peptide fibrillary network was also
observed on the surface [69]. Thus, Tris could act as a trigger to form more evolved 3D
structures. A regular dense network of fibers having small diameters (4–5 nm) was noticed
for the FEFEFKFK peptide by AFM and TEM [70].
As a result, a clear peptide assembly capacity was demonstrated by AFM, indicating that aggregated species grow in a pH-dependent manner. The aggregation index
studies are in the frame with AFM investigations demonstrating the pH responsiveness
of self-assembling.
3.5. Replica Exchange Molecular Dynamics Simulations
Molecular simulations were used in order to extract information at the atomic scale on
the conformational preference of the ‘FENSF’ peptide, which could be related to experimental results. In general, peptides do not adopt a single conformation in solution but rather
they exist as an ensemble of conformations. However, if the peptides contain residues
that participate in strong salt bridge interactions or are highly hydrophobic in nature, this
may bias the conformational landscape towards local basins with reduced flexibility [71].
Constructing the ensemble of conformations of even small peptides is a difficult task to be
achieved by conventional molecular dynamics simulations [72] due to the trapping of the
system in local free energy minimum conformations for long periods of time. Thus, we
used a replica exchange with solute tempering (REST2) [42] simulations to improve the
sampling of the peptide conformational space.
We were mainly interested in following whether the Phe residues present at the peptide
ends may participate in intramolecular π-π interactions. We postulate that the preference
for such a conformation would impair hydrophobic-driven growth of large aggregates
due to steric clashes between many adjacent molecules. The results of the isolated peptide
replica exchange simulations revealed that regardless of the pH value the peptides adopt
a preferred extended (“open”) conformation with the Phe aromatic side chains placed
distantly from each other (Figure 5A). Conformations involving intramolecular π-π stacking
(“closed” conformations) are very rarely visited as it can be seen from the histograms of
the end-to-end distance between Cγ –Cγ’ carbon atoms of Phe1 and Phe5, respectively
(Figure 5B).
When two peptides were simulated together, intermolecular interactions involving
terminal Phe side chains dominated the conformation of the binary ensemble (Figure 5D).
These interactions were possible as the terminal Phe side chains were free to participate in
π-π stacking interactions between two adjacent molecules. Interestingly, the intramolecular
conformational preference of each peptide was modified by the presence of the interaction
partner. The histogram of the end-to-end Cγ –Cγ distance now shows a second peak at ~6 Å
Pharmaceutics 2023, 15, 371
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(Figure 5C) compared with only one dominant peak centered at ~12 Å for single peptide
simulations. This increased tendency towards the ‘closed’ conformation is stabilized by
simultaneous intra and intermolecular π-π interactions (Figure 5D). These results show
that the ‘open’ conformation presents an advantage in terms of aggregation, leaving free
endings available for the hydrophobic-driven aggregate growth.
‐
Figure 5. Conformational analysis from single (A,B) and di-peptide
(C,D) peptide REST2 simulations. ‐
(A)—dominant conformation extracted fromγ cluster
analysis at acidic, neutral and slightly basic pH;
γ
‐ ‐
(B)—histogram of end-to-end Cγ –Cγ carbon atom distance distributions ofγ terminal
Phe residues in
γ
‐ ‐
single ‐peptide simualtions; (C)—histogram of end-to-end Cγ –Cγ distance distributions in di-peptide
‐
simulations; and (D)—conformations of peptide dimers extracted from di-peptide simulations.
To further evaluate the tendency of the ‘FENSF’ peptide to aggregate we conducted
coarse grain (CG) molecular simulations. In this type of calculation, chemical groups
composed of several atoms are represented as a single interaction center. This allows the
use of a much larger number of molecules and reaching time scales of at least an order
π‐π
‐
of magnitude higher. Figure 6A shows the results of a 0.8 µs CG simulation including
400 FENSF peptides at pH 5.5. In the initial configuration,
the peptide molecules were
γ
γ
‐ ‐
randomly placed in the simulation box. During the simulation, they self-organize into
increasingly larger aggregates and finally form a fibrillar structure detailed in Figure 6B.
Concisely, coarse grain (CG) simulation of the ‘FENSF’ peptide self-assembly in slightly
π‐π
basic conditions reveals the peptide tendency to form fibril-like structures. The theoretical
simulation data are consistent with AFM data and π-π stacking interactions and the open‐
state conformation might play an important role in the final 3D architecture.
‐
μ
‐
‐
‐
Pharmaceutics 2023, 15, 371
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Figure 6. Coarse grain (CG) simulation of ‘FENSF’ peptide self-assembly
at slightly basic pH. (A)—time
‐
series of peptide aggregates growth; (B)—detail of a large fibrillar aggregate formed after ~0.8 µs.
μ
4. Conclusions
‐
The self-assembly of short new peptides with phenylalanine residues separated
by
‐
three to five amino acids could be tailored to design new biomaterials with
diverse applicaπ‐πselected, synthesized,
tions. In this work, a FESNF peptide from a hen egg lysozyme was
‐
and characterized,
and its propensity for fibrillation was investigated. AFM data clearly
show that FESNF pentapeptide displays the ability to self-assembly into ordered and high
dense fibrils in slightly alkaline condition, while their length decreased in methanolic
solution. This‐ fibrillation is most likely caused by interaction between the two aromatic
groups that flank the peptide ends as confirmed by simulated data.
‐
In terms of potential applications, the results presented in this study are promising and
could be expanded by exploiting the fibrillation capacity of short new aromatic peptides
and their coresponding mutants in the development of ‐a scaffold for cell cultures and
designing new drug delivery systems or peptide-based biosensors. When compared to‐
native protein, the FESNF peptide self-assembly process was more challenging to evaluate
using conventional spectroscopic approaches or all-atom strategies.
These data imply that self-assembly properties of short phenylalanine peptides containing few cleavage sites in biogenic conditions should be multidisciplinary approached,
beginning with a rational design, novel synthesis strategies, molecular simulation and
spectral and modern imaging methods.
‐
‐
‐
Supplementary Materials: The following supporting information can be downloaded at: https:
‐
//www.mdpi.com/article/10.3390/pharmaceutics15020371/s1. Figure S1: Chromatograms of crude
‐ S2: UV-visible spectra of pentapeptide in Tris 30 mM pH 7.3‐
and purified FESNF peptide. Figure
(a and b, 826 µM and 41 µM) and acetate 40 mM pH 7.0 (600 µM, spectrum c, dotted). Figure S3:
Emission spectra of pentapeptide (1, 2,3 and 4 at 26; 80; 132 and 238 µg/mL) in Tris 30 mM pH 7.3. The
inset: fluorescence intensity as function of peptide concentration. Figure S4: Normalized aggregation
index (AI) calculated for both 258 nm and 280 nm values. The maximum values for AI were 2.8
(full dots) and 44 (empty dots), respectively. Inset: aggregation index (AI) variation as function of
pH values. Figure S5: Fluorescence emission (λex 440 nm and λem 482 nm) of pentapeptide with
‐ at pH 8.0 (black squares), pH 7.0 (black triangles) and pH 2.0 (open μ
μ
40 µM ThT
circles) as a function
of incubation time at 50 ◦ C. μ
μ
Pharmaceutics 2023, 15, 371
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Author Contributions: Conceptualization, V.-R.G.; methodology, V.-R.G., G.D.; validation, S.-C.J.,
L.D.-I. and B.-A.P.; formal analysis, A.N.; molecular dynamics simulations, S.-C.J.; investigation, V.R.G., L.D.-I., I.S. and S.-C.J.; data curation, V.-R.G., L.D.-I., B.-A.P. and S.-C.J.; writing—original draft
preparation, V.-R.G., G.D. and S.-C.J.; writing—review and editing, V.-R.G. and B.-A.P.; supervision,
V.-R.G.; project administration, V.-R.G.; funding acquisition, V.-R.G. All authors have read and agreed
to the published version of the manuscript.
Funding: This work was supported by a grant from the Romanian Ministry of Education and
Research, CCCDI-UEFISCDI, project number PN-III-P2-2.1-PED-2019-2484, within PNCDI III.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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