Exploring the biological consequences of conformational changes in aspartame models containing constrained analogues of phenylalanine

Article abstract of DOI:10.3109/14756366.2015.1076811

The dipeptide aspartame (Asp-Phe-OMe) is a sweetener widely used in replacement of sucrose by food industry. 2′,6′-Dimethyltyrosine (DMT) and 2′,6′-dimethylphenylalanine (DMP) are two synthetic phenylalanine-constrained analogues, with a limited freedom in χ-space due to the presence of methyl groups in position 2′,6′ of the aromatic ring. These residues have shown to increase the activity of opioid peptides, such as endomorphins improving the binding to the opioid receptors. In this work, DMT and DMP have been synthesized following a diketopiperazine-mediated route and the corresponding aspartame derivatives (Asp-DMT-OMe and Asp-DMP-OMe) have been evaluated in vivo and in silico for their activity as synthetic sweeteners.

Full text of DOI:10.3109/14756366.2015.1076811

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–11
2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1076811
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RESEARCH ARTICLE
Exploring the biological consequences of conformational changes in
aspartame models containing constrained analogues of phenylalanine
Adriano Mollica1, Sako Mirzaie2, Roberto Costante1, Simone Carradori1, Giorgia Macedonio1, Azzurra Stefanucci3,
Szabolcs Dvoracsko4, and Ettore Novellino5
1
2
Dipartimento di Farmacia, Universita di Chieti-Pescara ‘‘G. d’Annunzio’’, Chieti, Italy, Department of Biochemistry, Sanandaj Branch, Islamic Azad
`
3
University, Sanandaj, Iran, Dipartimento di Chimica, Sapienza Universita di Roma, Rome, Italy, Institute of Biochemistry, Biological Research
5
Centre, Hungarian Academy of Sciences, Temesvari krt, Hungary, and Dipartimento di Farmacia, Universita di Napoli ‘‘Federico II’’, Naples, Italy
4
`
`
Abstract
Keywords
The dipeptide aspartame (Asp-Phe-OMe) is a sweetener widely used in replacement of sucrose
by food industry. 20,60-Dimethyltyrosine (DMT) and 20,60-dimethylphenylalanine (DMP) are
two synthetic phenylalanine-constrained analogues, with a limited freedom in ꢀ-space due
to the presence of methyl groups in position 20,60 of the aromatic ring. These residues have
shown to increase the activity of opioid peptides, such as endomorphins improving the
binding to the opioid receptors. In this work, DMT and DMP have been synthesized following
a diketopiperazine-mediated route and the corresponding aspartame derivatives (Asp-DMT-
OMe and Asp-DMP-OMe) have been evaluated in vivo and in silico for their activity as synthetic
sweeteners.
Aspartame, computational methods,
20,60-dimethyltyrosine,
20,60-dimethylphenylalanine, sweetener,
T1R2 receptor
History
Received 29 May 2015
Accepted 16 July 2015
Published online 24 August 2015
Introduction
explored the importance of the side chains orientation of the
phenylalanine residue in influencing the sweet potency and the
taste quality. It is well known that the incorporation of unnatural
amino acids into bioactive peptides may lead to unique analogues,
biologically more active and/or more selective than the parent
peptides7. This is the result of strong modifications on the
stereoelectronic properties of the peptide and of the secondary
structure, as the stabilization of a folded conformation. Lately, it
has been proved that the arrangements of the side chains of the
residues are also important for the binding with the target
molecules. For example, the introduction of 20,60-dimethyltyr-
osine (DMT) in place of the native Tyr1 in endomorphin-1 yields
a very potent analogue at the m opioid receptor and a similar effect
has been reported for the incorporation of 20,60-dimethylpheny-
lalanine (DMP) in place of C-terminus Phe8,9. This behaviour can
be explained with the concept of ꢀ space10–12. In fact, side chains
of natural amino acids possess a certain degree of flexibility
around the ꢀ dihedral angles, which allows the peptide to adapt its
overall 3D shape to different receptors. In order to enhance
potency and efficacy, the side chain’s freedom of the native
residue may be reduced in order to obtain a more favourable
topology. Plotting energy versus ꢀ dihedral angle is available in
the Supplemental material. Considering the recent applications
of this approach in peptidomimetics design13,14, in this paper,
we synthesized two new aspartame models in which the
native phenylalanine residue was substituted by two constrained
aromatic surrogates, namely 20,60-dimethyltyrosine and 20,60-
dimethylphenylalanine.
The low-calorie sweetener aspartame, namely aspartyl-phenyl-
alanine methyl ester, is a common synthetic dipeptide ester which
possesses a sweetening power around 200 times sweeter than
sucrose. Considering its commercial importance in food industry,
its safety has been widely investigated by studies in several human
subpopulations, including healthy people, obeses, diabetics, etc.
and its massive use has been recently criticized basing on a
theoretical toxicity of its metabolic components, namely aspar-
tate, phenylalanine and methanol. Nonetheless, additional
research has been carried out to understand the possible
association between aspartame and headaches, seizures, behav-
iour, cognition, and mood as well as allergic-type reactions.
The question about its safety remains still unresolved1.
The detection of sweet-tasting compounds (carbohydrates and
non-caloric sweeteners) is mediated by a heterodimeric receptor
composed of two subunits, namely T1R2 and T1R3. Moreover,
this receptor modulates glucose transporters and glucose homeo-
stasis in other ‘‘non-taste’’ organs (gastrointestinal tract, pancreas,
bladder, adipose tissues, and brain) demonstrating to show a
potential as new therapeutic target2,3. Small molecule inhibitors of
sweet receptors have been demonstrated to reduce glucose
absorption and calorie uptake providing an alternative strategy
for the treatment of obesity and its related diseases.
Several structure–activity relationship studies have been
conducted on aspartame derivatives4–6, but none of them have
In 2010, an effort was done to elucidate the molecular
mechanism of the sweet taste enhancers. Umami and sweet taste
receptors share a T1R3 subunit as a common subunit. It has been
Address for correspondence: Adriano Mollica, Dipartimento di Farmacia,
`
Universita di Chieti-Pescara ‘‘G. d’Annunzio’’, Via dei Vestini 31, 66100
Chieti, Italy. E-mail: a.mollica@unich.it

2
A. Mollica et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
shown that the sweet enhancers have no effect on umami taste r.t. and stirred for 3 h, then the solvent was removed in vacuo to
receptor. So, they probably bind to the N-terminal extracellular give HClꢁH-DMP-OMe (2a) which was used for the next reaction
domain of T1R2 subunit. This subunit is composed of two distinct without further purification. Coupling with Boc-Asp(tBu)-OH
domains, Venus flytrap (VFT) and transmembrane domain, as was performed following the general coupling procedure. After
present in T1R family receptors. Generation of hybrid receptors silica gel chromatography using CH2Cl2/EtOAc (from 95:5 to
showed that the interaction mode of some enhancers is mediated 85:15) as eluent, the pure dipeptide was obtained as a colorless oil
by VFT domain of T1R2 subunit15. As a consequence, bioactivity in 65% yield. HRMS calcd.: 478.2679, found: 478.2687. 1H NMR
and in silico evaluation of the binding mode of both derivatives (CDCl3) ꢁ: 7.11 (1H, d, DMP NH), 7.05–6.97 (3H, m, Ar), 5.54
have been explored using an established receptor model.
(1H, d, BocNH), 4.77–4.69 (1H, m, Asp aCH), 4.42–4.35 (1H, m,
DMP aCH), 3.59 (3H, s, OMe), 3.08–2.87 (2H, m, DMP bCH2),
2.84–2.51 (2H, m, Asp bCH2), 2.34 (6H, s, DMP CH3), 1.43, and
1.46 (18H, s, Boc, OtBu).
Materials and methods
Chemistry
The structure of the intermediates and the final compounds was TFAꢁH-Asp-DMP-OMe (4a)
1
confirmed by H NMR spectra recorded on a 300 MHz Varian
Nꢂ-Boc and OtBu deprotections of compound (3a) were per-
Inova spectrometer (Varian Inc., Palo Alto, CA). Chemical shifts
are reported in parts per million (ꢁ) downfield from the internal
standard tetramethylsilane (Me4Si). Homogeneity was confirmed
by TLC on silica gel Merck 60 F254 (Merck, Darmstadt,
Germany). The HR ESI-MS experiments were performed on a
Thermo Scientific Q Exactive (Thermo Fisher Scientific, San
Jose, CA). The MS was operated in the positive mode. The
parameters used were the following: capillary temperature 220 ꢀC,
spray voltage 2.3 kV, and sheath gas 5 units.
formed as reported for TFAꢁH-Val-Gly-OMe. The crude product
was purified by RP-HPLC semi-preparative C18 column (eluent:
ACN/H2O gradient, 5–80% over 20 min) and the TFA salt was
obtained, after freeze drying, in quantitative yield as a white
powder. HRMS calcd.: 322.1529, found: 322.1535. 1H NMR
(DMSO-d6) ꢁ: 8.98 (1H, d, DMP NH), 7.00–6.98 (3H, m, Ar),
4.86–4.60 (1H, m, Asp aCH), 4.09–4.05 (1H, m, DMP aCH),
3.50 (3H, s, OMe), 3.15–2.70 (4H, m, DMP bCH2, Asp bCH2),
2.52 (6H, s, DMP CH3).
Solutions were routinely dried over anhydrous Na2SO4 prior to
evaporation. Chromatographic purifications were performed for
the intermediate products by Merck 60, 70–230 mesh silica gel
Boc-Asp(tBu)-DMT-OMe (3b)
column and for the final products 4a and 4b by RP-HPLC. All HClꢁDMT-OMe (2b) and Boc-Asp(tBu)-DMT-OMe (3b) were
chemicals used were of the highest purity commercially available. synthesized as reported for compound (3a) and, after column
All dipeptides were synthesized by solution phase peptide chromatographic purification, the dipeptide was obtained as a
synthesis (SPPS) using Boc strategy following well-established colourless oil in 85% yield. HRMS calcd.: 494.2628, found:
procedures16. Na-Boc deprotection was carried out by TFA 494.2640. 1H NMR (CDCl3) ꢁ: 7.88 (1H, d, DMT NH), 6.50 (2H,
treatment. DMP (1a) and DMT (1b) were synthesized following s, Ar), 5.46 (1H, d, BocNH), 4.68–4.55 (1H, m, Asp aCH), 4.32–
the method developed by Mollica and Costante (Patent 4.25 (1H, m, DMT aCH), 3.64 (3H, s, OMe), 3.40–2.21 (2H, m,
Application ‘‘Sintesi enantioselettiva di amminoacidi aromatici DMT bCH2), 2.92–2.56 (2H, m, Asp bCH2), 2.28 (6H, s, DMT
non-naturali’’, Application Number: RM2015A000091, date: CH3), 1.44, and 1.42 (18H, s, Boc, OtBu).
27/02/2015) and transformed in methyl ester hydrochlorides 2a
and 2b by treatment with SOCl2 in MeOH, and used without TFA H-Asp-DMT-OMe (4b)
further purification for the next step16b (Scheme 1).
Nꢂ-Boc and OtBu deprotections of compound (3b) were
performed as reported for compound 3a. The crude product was
Boc-Asp(tBu)-DMP-OMe (3a)
purified by RP-HPLC semi-preparative C18 column (eluent:
Boc-DMP-OH (1a) was dissolved in MeOH, then SOCl2 (2 eq.) ACN/H2O gradient, 5–80% over 20 min) and the TFA salt was
was added dropwise at 0 ꢀC. The mixture was allowed to warm to obtained in quantitative yield as a white powder after freeze
drying. HRMS Calcd.: 338.1478, found: 338.1489. 1H NMR
(DMSO-d6) ꢁ: 9.14 (1H, s, DMT OH), 8.84 (1H, d, DMT NH),
6.40 (2H, s, Ar), 4.56–4.41 (1H, m, Asp aCH), 4.10–3.96 (1H, m,
DMT aCH), 3.53 (3H, s, OMe), 3.15–2.56 (4H, m, DMT bCH2,
Asp bCH2), 2.12 (6H, s, DMT CH3).
R
R
a
b
O
Boc
OH
H N
HCl·
2
N
H
O
O
NMR conformational studies
2a: R = H
2b: R = OH
1a: R = H
1b: R = OH
To gain further information on the preferred conformation of the
1
models, we examined the 2D ROESY H NMR of aspartame and
[DMP2]aspartame. As shown in Table 1 and Figure 1, aspartame
and [DMP2]aspartame derivatives show strong sequential NOEs
Phe NH–Phe CHa. This effect can be observed for both products.
Some differences can be appreciated in the NOEs between the
aromatic ring protons and the backbone of the peptides of
aspartame, and the NOEs between the Ar–CH3 protons and the
backbone of the [DMP2]aspartame. These data strongly suggest
that the aromatic ring of the DMP model is restricted into a well-
defined ꢀ angles, according to the computational energy calcu-
R
R
O
O
Boc
HN
TFA·H2N
HO
OMe
c
OMe
HN
HN
O
O
O
O
3a R: H
4a
R: H
O
3b R: OH
4b R: OH
1
lation. All 1D and 2D H NMR experiments were performed at
300 MHz on a Varian Inova NMR spectrometer (Varian, Palo
Alto, CA) with a constant temperature at 298 K. The ROESY
spectra were obtained using standard pulse programs, with a
Scheme 1. (a) SOCl2/MeOH, r.t., 3 h; (b) Boc-Asp(tBu)-OH, EDCꢁHCl,
HOBt, NMM, DMF, r.t., overnight; (c) TFA/CH2Cl2 1:1, r.t., 1 h,
under N2.

DOI: 10.3109/14756366.2015.1076811
Biological consequences of conformational changes in aspartame models
3
Table 1. Observed NOE Cross Peaks and intensities of aspartame and [DMP2]aspartame in DMSO-d6 and J
coupling constants (Hz)a.
Aspartame
[DMP2]aspartame
J coupling constants (Hz)
Aspartame
Asp NHþ3 —Asp CHa
w
–
w
m
s
w
w
–
m
s
b
Asp NHþ3 —Phe CH2
Phe NH—aromatics
Phe NH—Phe CHa
Phe NH—Asp CHa
Phe NHJ ¼ 7ꢁ8
AB part of ABX system Phe:
Jab ¼ 14; Jax ¼ 6; Jbx ¼ 9.3
AB part of ABX system Asp:
Jab ¼ 15; Jax ¼ 3.3; Jbx ¼ 8.1
DMP[aspartame]
b
Phe NH—Phe CH2
–
–
m
m
–
m
w
m
–
s
m
m
Phe NH—Ar CH3
DMP NHJ ¼ 7ꢁ5
b
Phe CHa—Asp CH2
Phe CH2b—aromatics
Ar CH3—aromatics
Ar CH3—Phe CHa
AB part of ABX system DMP:
Jab ¼ 14.1; Jax ¼ 8.1; Jbx ¼ 6.9
AB part of ABX system Asp:
Jab ¼ 17.4; Jax ¼ 3.3; Jbx ¼ 8.1
–
–
b
Ar CH3—Phe CH2
aNOE intensities are classified as weak (1.6–5.0 B), medium (1.6–3.6 B), and strong (1.6–2.9 B).
O
compliance with the principles of the Declaration of Helsinki.
TFA.Aspartame, commercial aspartame, [DMT2]aspartame and
[DMP2]aspartame were tasted by three volunteers in blind tests
using diluted aqueous solutions of each test compound. Different
concentrations (between 0.1 and 0.001% (w/v)) of each dipeptide
were evaluated at 3–5 min intervals. About 1.5 mL of each
solution was applied via pipettes and allowed to flow over the
volunteer’s tongue for 3 s and then washed out. Sweetness
intensities were determined relative to aqueous sucrose solutions
of 0.5, 2.0, 4.0, and 8.0% (w/v) as the reference. The
[[DMT2]aspartame and [DMP2]aspartame solutions resulted to
be tasteless even at the higher concentration. The absence of taste
of the novel synthesized aspartame analogues was the object of
the in silico investigation.
HO
m
m
H
s
H
H
N
H
m
w
H
H
H
+
H N
3
m
w
O
O
H
MeO
aspartame
O
m
H C
3
s
HO
Computational experiments
s
H
N
m
H
H
Sequence alignment
s
s
w
H
H
H
Due to the lack of experimental data, a three-dimensional
structure of sweet T1R family receptors, the 3D structure of
extracellular VFT domain of T1R2 was constructed by homology
modelling (Figure 2). In the first stage, the amino acid sequence
of human T1R2 was retrieved from Swiss–Prot database with the
accession number Q8TE23. Then, the BLAST search engine from
National Center for Biotechnology Information (NCBI) was
utilized to find the suitable template with pdb format. Among
the listed templates, crystal structure of the extracellular region of
the group II metabotropic glutamate receptor complexed with L-
+
H N
3
CH
w
3
H
O
MeO
m
O
DMP[aspartame]
Figure 1. Relevant interproton correlations as deduced by ROESY
experiments of aspartame and [DMP2]aspartame.
˚
glutamate (pdb code: 2E4U) with resolution 2.35 A, was used as a
template18. Alignment between query sequence and selected
template was done by ClustalW2 from its website19,20 with default
parameters. Produced alignment, which showed 28% identity, was
used for 3D model building.
mixing times of 300 ms, t1 ¼ 1.5 s. The 2D NMR matrixes were
created and analyzed using the Agilent WNMR computer
program (Agilent Technologies Inc., Atlanta, GA). Each two-
dimensional spectrum was acquired in a 1024 ꢂ 1024 data matrix
complex points in F1 and F2. Zero filling in F1 and sine windows
in both dimensions was applied before Fourier transformation.
Chemical shifts (ꢁ) are quoted in parts per million (ppm)
downfield from tetramethylsilane, and values of coupling con-
stants are given in Hz.
T1R2 model building by homology modeling
To construct the T1R2 models, MODELLER 9v14 package
(Andrej Sali Lab, San Francisco, CA) was employed. Using this
package, one hundred models of human T1R2 were constructed
and to select the best model, the one with the lowest value of
Discrete Optimized Protein Energy (DOPE)21 was picked. The
final model was minimized by the steepest descent algorithm
implemented in GROMACS 4.5 package22. The Ramachandran
plots of models also were computed by PROCHECK to assess
the psi and phi angles of model residues and clarify the
model quality23.
Sensory evaluation
Standard [DMT2]aspartame and [DMP2]aspartame were synthe-
17
¨
sized and tested as TFA salts as reported by Dorrich et al.
Qualitative taste assays were carried out following the protocol
reported by the same author17. We conduct our studies in

4
A. Mollica et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
Figure 2. Sequence alignment of VFT domain of T1R2 with extracellular region of glutamate receptor (pdb code: 2E4U). Same color of residues
shows the same properties. (Colors in this figure are referred to the web version of this article.)
Docking study of T1R2 model and ligands
temperature coupling (NVT)32 and pressure coupling (NPT)
ensembles33. For each system, two coupling groups including
protein (or protein ligand), solvent and ions were assigned. These
groups were equilibrated separately at 300 K with a coupling
constant of 0.1 ps and time duration of 100 ps. For NPT
equilibration, a pressure of 1 bar, a time duration of 100 ps, and
a time constant of 2 ps were allotted. In both ensembles
mentioned above, PME method, with an interpolation order of
4, was used to compute the long-range electrostatic interactions34.
LINCS algorithm was used to constrain covalent bond length35.
The short-range neighbor list, short-range electrostatic, and short-
range van der Waals cut-offs were set to 1.2 nm. For all MD runs,
a 30 000 ps molecular dynamics was accomplished on the entire
system. The trajectories were analyzed using the standard tools
implemented in the GROMACS package.
To dock the aspartame, DMP and DMT into the binding site of
T1R2 model, AutoDock Vina was employed24. This molecular
docking software profits of knowledge-based potentials and
empirical scoring functions24,25. To generate the 3D structures
of ligands, PRODRG server was utilized26. After generation of 3D
structures of aspartame, DMP and DMT, their non-polar hydrogen
atoms were deleted and subsequently, Gasteiger–Marsili charges
were assigned27. For protein preparation before docking study,
Gasteiger–Marsili charges implemented in Autodock Tools 1.5.4
were allocated for T1R2 model. To assess the binding affinity of
ligands to the T1R2 binding site, a box with a grid map with
˚
30 ꢂ 30 ꢂ 30 points and grid-point spacing of 1 A was set up at the
geometrical center of the residue Asp 278. For all docking runs, a
default value of exhaustiveness parameter was assigned. All poses
were re-clustered with x-score v1.1 and one with the highest
average score was saved for further analysis and studies. The three
Calculation of binding free energy
scoring functions of X-score have been calibrated with a large Modified Molecular Mechanics–Poisson Boltzmann Surface Area
training set of 800 protein–ligand complexes. Thus, it can be (MM-PBSA) method35 was used to compute the interaction free
applied to a wider range of biomolecular systems giving more energy between DMP and DMT with T1R2, separately. In this
robust results28.
method, based on its theory, the free energy of binding can be
expressed as follows:
À
Á
Molecular dynamic simulations
DGbinding ¼ Gcomplex ꢃ Gprotein þ Gligand
Following the molecular docking, molecular dynamic (MD)
simulations were done for T1R2, T1R2:aspartame, and
T1R2:DMP complexes. All MD simulations were executed in
periodic boundary conditions (PBC), using GROMOS96 43A1, a
united atom force field, by GROMACS 4.5 package. To check the
ionization state of ionizable residues, PROPKA 2.0 was used and
the correct states were assigned for them29. GROMACS param-
eters of aspartame, DMP and DMT, were generated by PRODRG
server26. To increase the precision in charge assignment, the atom
charges of aspartame, DMP and DMT, were determined from the
equivalent functional group in a GROMACS-building block
file30,31. Eleven Na+ counter ions were added to all systems to
neutralize the negative charges. During the minimization, the
protein non-hydrogen atoms were kept fixed in their initial
configuration. After neutralization, all systems were introduced to
the steepest descent minimization algorithm with an energy step
size of 0.01. The minimization was converged when the maximum
force was smaller than 10.0 kJ/mol. Successively, position
restraint procedure was carried out in association with
where Gcomplex is the total free energy of the protein–ligand
complex and Gprotein and Gligand are total free energies of the
separated form of protein and ligand in solvent, respectively. In
our study, van der Waals, Electrostatic, Polar solvation, the
solvent accessible surface area, solvent accessible volume y and
Weeks–Chandler–Andersen energies were computed by
g_mmpbsa36.
Results and discussion
Chemistry
DMP and DMT have been synthesized following the method
licensed by Mollica and Costante (patent titled ‘‘Sintesi
enantioselettiva di amminoacidi aromatici non-naturali’’,
Application Number: RM2015A000091, date: 27/02/2015) and
their chemical–physics parameters were equal to those of the
commercial products. Boc-DMP-OH and Boc-DMT-OH were
converted into the corresponding methyl esters hydrochloride by

DOI: 10.3109/14756366.2015.1076811
Biological consequences of conformational changes in aspartame models
Table 2. Stereo-chemical quality of template and model checked by PROCHECK.
Ramachandran plot quality (%)
5
Goodness factor
Covalent
PROCHECK
Core
Allowed
General
Disallowed
Dihedral
Overall
Template (2E4U)
T1R2 model
86.1
87.1
12.6
10.4
1.3
1.6
0
0.9
0.08
ꢃ0.09
0.49
ꢃ0.28
0.25
ꢃ0.16
Figure 3. Ramachandran plot of template (2E4U) (A), TIR2 model (B), and cartoon view of superposition of template and TIR2 model (C). In the
superimposed image, template and TIR2 model are shown in green and brown, respectively. (Colors in this figure are referred to the web version of this
article.)

6
A. Mollica et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
treatment with SOCl2 in MeOH16b,37. The so obtained building also the N-terminal ligand-binding domain, which is often called
blocks were coupled in the next step to Boc-Asp(Otbu)-OH. The the VFT module38,39. An agonist is included in the template pdb
protected aspartame derivatives were then purified by silica gel file, which can help to model the orientation of T1R2 active site.
chromatography and deprotected by TFA in CH2Cl2. TFA salt The best template selected by BLAST had just 28% identity.
were purified by RP-HPLC, C18 (ACN/H2O, gradient, 1% TFA). Because of low identity between the template and query
sequence, validation of the generated 3D model is necessary to
elucidate the model validity. Thus, the quality of the model was
evaluated by PROCHECK.
Computational modelling of T1R2
As shown in Table 1, 86.1% of template residues are located in
favored core regions with no one in disallowed region. For our
model, in comparison with the template, a bit more residues are
located in core regions (87.1) with a relatively low percentage
of residues 1.3% in generously allowed regions and 0.9% in
disallowed regions. For acceptable structures, it is expected that
more than 85% of residues fall into the core regions. The second
The VFT domain of T1R2 subunit was modeled by the homology
modeling method to investigate the interaction mode of aspar-
tame, DMP and DMT. Sequence alignment between VFT domain
of T1R2 and the template (2E4U) is shown in Figure 1. The
residues with same physicochemical properties are shown in same
color. The selected template is an extracellular region of
metabotropic glutamate receptor (mGluR)18. All mGluRs have
Figure 4. (A) Schematic two-dimensional representations of the binding interactions between aspartame and binding site of T1R2 model. Black dashed
lines are hydrogen bonds. Hydrophobic interactions are shown by green solid line; (B) schematic two-dimensional representations of the binding
interactions between DMP and binding site of T1R2 model. Black dashed lines are hydrogen bonds; (C) schematic two-dimensional representations of
the binding interactions between DMT and binding site of T1R2 model. Black dashed lines are hydrogen bonds. (Colors in this figure are referred to the
web version of this article.)

DOI: 10.3109/14756366.2015.1076811
Biological consequences of conformational changes in aspartame models
7
set of parameters, Goodness factor, was evaluated and summar- comparison with aspartame, is slightly different (Figure 4B). The
ized in Table 2. Reasonable values of G-factor in PROCHECK are bulkier aromatic ring of DMP prevents to form a hydrophobic
between 0 and ꢃ0.5 with the best models exhibiting values close interaction. Based on this behaviour, Ser303 interacts with DMP
to zero40. The overall G-factor for TIR2 model (ꢃ0.16) shows a through hydrogen bonding. In contrast to aspartame and DMP,
good and acceptable quality.
DMT binds to T1R2 with a completely different pattern. With
Ramachandran plots of template and T1R2 model are shown in regard to Figure 4(C), NH of Ser144 and Ile167 formed a
Figure 3(A) and (B), respectively. As seen in Figure 3(B), 97.5% hydrogen bond with the carboxyl moiety of DMT. An additional
of T1R2 model residues were either in the most-favored regions hydrogen bonding is formed between carboxyl moiety of Glu 61
or the additionally allowed regions. Moreover, Figure 3(C) shows and hydroxyl group of DMT. Based on our docking study, the only
the superposition of the template and T1R2 model. The most hydrophobic interaction of the binding site is present in
conformational differences between the template and T1R2 are T1R2:aspartame complex. For further analysis, aspartame and
related to loops.
DMP were selected to perform MD simulation.
Molecular dynamic simulation
Molecular docking analysis
Protein folding, enzymatic reaction, molecular recognition,
and substrate–receptor bonding take place as a result of inter
and intra-molecular interactions between biomacromolecules and
ligands. MD simulation of the biological process is a powerful
tool to generate such information at the microscopic level. In this
stage, MD simulations of free T1R2, T1R2:aspartame and
T1R2:DMP complexes were done to demonstrate and elucidate
the mode of interaction between peptides and T1R2 during 30 ps.
In addition, binding free energy and receptor conformational
changes in the absence and presence of peptides were
investigated.
Molecular docking of aspartame, DMP and DMT, was performed
to evaluate the potential binding modes of the three peptide
derivatives. The docking results were clustered and screened
based on the algorithm of binding energy calculation imple-
mented in AutoDock Vina. Figure 4(A) shows the interaction
mode between aspartame and T1R2 model. Within a hydrophobic
pocket of T1R2, Ile67 forms a hydrophobic interaction with the
phenyl moiety of aspartame. Also, hydroxyl and carbonyl
moieties of Ser303 interact with carbonyl and amino groups of
aspartame via hydrogen bonding, respectively. Carboxyl moiety
of Asp307 can form a salt bridge with NHþ3 of aspartame. In 2006,
Cui et al., based on a homology modeling study, demonstrated
that Asp307 and Ser303 are the main residues involved in
interaction with aspartame, due to the formation of hydrogen
bonding and salt bridging41. Zhang et al. showed that Lys65,
Leu279, Asp278, and Asp307 of T1R2 interact with the enhancer
SE-315. It seems that the interaction mode of DMP with T1R2, in
Root mean square deviation (RMSD)
To assess the equilibration of MD trajectories, RMSD is
considered a crucial parameter. RMSD values of the backbone
atoms of T1R2 are plotted as a function of time to verify the
stability of each system during 20 ps MD simulation. The RMSD
Figure 5. (A) RMSD values of T1R2:aspartame (red line), T1R2:MDP (green line), and free T1R2 (blue line) during MD simulation; (B) RMSD
values of aspartame and DMP in binding site of T1R2 model. (Colors in this figure are referred to the web version of this article.)

8
A. Mollica et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
values of free T1R2, T1R2:aspartame and T1R2:DMP complexes differences among residues. Figure 6 shows the backbone RMSF
are shown in Figure 5(A). RMSD values of free T1R2 are versus residue number. In Figure 6(A), RMSF of free T1R2
increased up to 0.6 nm at 9100 ps. After a short decrease of against T1R2:aspartame complex is depicted. In addition,
RMSD to 0.53 nm, the system is equilibrated at the last 10 000 ps. RMSF of free T1R2 against T1R2:DMP complex is shown in
In the case of T1R2:aspartame complex, RMSD is variable Figure 6(B). VFT domain of T1R2 consists of two globular sub-
between 0.3 and 0.7 nm. Steady increase of RMSD is occurred till domains joined by a three-stranded flexible hinge14. The first
21 700 ps. RMSD value for T1R2:DMP complex is between 0.2 connection flexible strand is composed of Thr183, Thr184,
and 0.48 nm. After increasing up to 0.44 nm at 7900 ps, the Pro185, and Ser186 with RMSF values of 0.972, 1.093, 1.208, and
˚
system approximately is equilibrated till 30 000 ps. It seems that 1.208 A, respectively. These values approximately remained
binding of the DMP to T1R2 decreases the backbone flexibility of unchanged in T1R2:DMP complex (Figure 6B). In the case of
T1R2 and preserves it in a ‘‘rigid state’’. Conversely, binding of T1R2:aspartame complex, these values are increased up to 1.156,
˚
aspartame to T1R2 can promote some backbone flexibility, which 1.238, 1.405, and 1.412 A (Figure 6A). The second strand is made
may be necessary to form the active T1R2:T1R3 heterodimer. of Ala445, Leu446, His447, and Leu448. These residues connect
The RMSD values of aspartame and DMP to T1R2 model were the two b-sheets belonging to sub-domains. Gly324, Ile325,
acquired on the MD simulation of two systems to get information Thr326, Ile327, and Gln328 form the third strand. Based on our
on position fluctuations. As shown in Figure 5(B), the RMSD of data, RMSF of all three strand residues of T1R2:aspartame
aspartame atoms rose to 0.26 nm after 300 ps and then leveled off complex, in comparison with free T1R2 and T1R2:DMP complex,
to 0.25 nm. In the case of DMP, RMSD increased up to 0.3 nm, are increased. So, the mentioned ‘‘hinge’’ may have a critical role
then a short decreasing to 0.15 nm is occurred and finally, the in T1R2 conformational changes and causes emerging the sweat
atoms were equilibrated approximately at the second half time of taste response. Moreover, some researchers claimed that brazzein,
MD simulation. In comparison with DMP, aspartame has been a well-known sweet-tasting protein, binds to the ‘‘hinge’’ region
reached to equilibrium more quickly.
of T1R242. Previously, it has been shown that the ‘‘hinge’’ region
of metabotropic glutamate receptors allows T1R2 conformational
changes between open and closed conformations43. Sweet taste
receptors have the highest similarity with metabotropic glutamate
receptors in ‘‘hinge’’ region15,42. Thus, these ‘‘open’’ and
‘‘close’’ conformational changes can be true for our T1R2
model. Based on our MD simulations, binding of aspartame to
Root mean square fluctuation (RMSF) and induced
conformational changes
RMSF computation with respect to the average MD simulation
conformation can be used to evaluate and assess the flexibility
Figure 6. (A) RSM fluctuation of residues during MD simulation. RMSF comparison between free T1R2 (blue line) and T1R2:aspartame complex (red
line); (B) RMSF comparison between free T1R2 (blue line) and T1R2:DMP complex (red line). (Colors in this figure are referred to the web version of
this article.)

DOI: 10.3109/14756366.2015.1076811
Biological consequences of conformational changes in aspartame models
9
VFT domain triggers some conformational changes. As shown in between 11 and 15 ns, the distance increased up to approximately
˚
Figure 7, side chain NH3 of Lys60 from upper lobe interacts with 29 A. Due to binding of aspartame to the T1R2 model, a hydrogen
backbone carbonyl moiety of Ile306 belonging to lower lobe via bonding is formed between backbone carbonyl moiety of Ser165
hydrogen bonding. This interaction can be inspected during 30 ns and hydroxyl moiety of Thr184 (Figure 9A). In the presence of
MD simulation. Figure 8 exhibits the distance between a nitrogen DMP in binding site of T1R2 model, these residues are too far to
atom of Lys60 and oxygen of Ile306. For free T1R2, the distance be able to interact to gather (Figure 9B).
˚
˚
is about 13 A till 7.4 ns. Then, increased up to 24 A and followed
˚
by a reduction to 14 A. In the case of T1R2:aspartame complex, High-throughput MM-PBSA for binding free energy
˚
˚
after a steady variation about 5.4 A, it fell down to 2.9 A. In an calculations
opposite manner, for T1R2:DMP complex, after a short fall
To estimate the interaction free energy between aspartame/DMP
and VFT domain of the T1R2 model, MM-PBSA method was
used36. Recently, a combination of MM-PBSA and molecular
dynamic method has been used to re-score the docked poses. By
applying this new method, the scoring function has been increased
significantly44–46. The snapshots were extracted from the last
10 ns of MD trajectories for the analysis of the binding free
energy. In Table 2, the binding free energy of aspartame and DMP
to T1R2 model is ꢃ86.539 and ꢃ39.61, respectively. Based on our
results, van der Waals energy is the most effective energy, which
favors the binding of aspartame and DMP to binding site of T1R2
model. As shown in Table 3, binding of DMP, in comparison with
aspartame, increases the van der Waals and electrostatic energies.
Thus, increasing the hydrophobicity of aspartame opposes to the
binding. This result suggests that the optimizations of van der
Waals interactions between the agonists and T1R2 model may
lead to the potent sweeteners.
Conclusion
Conformational constricted amino acids are important tools to
explore the topographical preferences of bioactive peptides. DMP
and DMT, in particular, have been recently used in the field of
opioid peptides together with DzPhe and other constrained
residues with remarkable results11. Using our recent developed
method to gain access to DMT and DMP and their N- or C-
terminal protected derivatives, we have incorporated these two
Figure 7. The computed binding mode of Lys60 with Ile306 in the
binding site of T1R2 model. The hydrogen bonds are shown by the yellow
dashed lines. (Colors in this figure are referred to the web version of this amino acids as phenylalanine surrogates in aspartame. The effects
article.)
of this approach on the sweetener power have been evaluated. Our
Figure 8. Distance changes between nitrogen atom of Lys60 and oxygen of Ile306 in free T1R2 (blue line), T1R2:aspartame (red line) and T1R2:DMP
complexes (green line) during MD simulation. (Colors in this figure are referred to the web version of this article.)

10 A. Mollica et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
Figure 9. The computed binding mode of Ser165 with Thr184 in the binding site of T1R2 model in T1R2:aspartame (A) and T1R2:DMP (B)
complexes. The hydrogen bonds are shown by the yellow dashed lines. (Colors in this figure are referred to the web version of this article.)
Table 3. Molecular energy terms for the T1R2:aspartame and
conformational changes do not consent to the proposed molecules
T1R2:DMP complexes.
to correctly positioning in the binding pocket of the receptor, with
a final lack of the signal transduction.
Energy
(kcal/mol)
T1R2:aspartame
complex
T1R2:DMP
complex
Declaration of interest
DEvdw
DEelect
DEsolv
DESASA
DGbinding
ꢃ151.380
ꢃ136.582
215.904
ꢃ109.635
ꢃ82.218
164.286
ꢃ12.043
ꢃ39.610
The authors report that they have no conflicts of interest.
ꢃ14.482
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Supplementary material available online
Supplemental material.