Article

The Ac-RGD-NH2 peptide as a probe of slow conformational exchange of short linear peptides in DMSO

Department of Chemistry, University of Ioannina, Yannina, Epirus, Greece
Biopolymers (Impact Factor: 2.39). 05/2003; 69(1):72-86. DOI: 10.1002/bip.10335
Source: PubMed

ABSTRACT

According to general belief, the conformational information on short linear peptides in solution derived at ambient temperature from NMR spectrometry represents a population-weighted average over all members of an ensemble of rapidly interconverting conformations. Usually the search for discrete conformations is concentrated at low temperatures especially when sharp NMR resonances are detected at room temperature. Using the peptide Ac-RGD-NH(2) (Ac-Arg-Gly-Asp-NH(2), Ac: acetyl) as a model system and following a new approach, we have been able to demonstrate that short linear peptides can adopt discrete conformational states in DMSO-d(6) (DMSO: dimethylsulfoxide) which vary in a way critically dependent on the reconstitution conditions used before their dissolution in DMSO-d(6). The conformers are stabilized by intramolecular hydrogen bonds, which persist at high temperatures and undergo a very slow exchange with their extended structures in the NMR chemical shift time scale. The reported findings provide clear evidence for the occurrence of solvent-induced conformational exchange and point to DMSO as a valuable medium for folding studies of short linear peptides.

Full-text

Available from: Nikolaos Biris, Mar 31, 2016
The Ac–RGD–NH
2
Peptide
as a Probe of Slow
Conformational Exchange
of Short Linear Peptides
in DMSO
Nikolaos Biris
1
Athanassios Stavrakoudis
1
Anastasia S. Politou
2
Emmanuel Mikros
3
Maria Sakarellos-Daitsiotis
1
Constantinos Sakarellos
1
Vassilios Tsikaris
1
1
Department of Chemistry,
University of Ioannina,
45110 Ioannina, Greece
2
Medical School,
University of Ioannina,
45110 Ioannina, Greece
3
Department of Pharmacy,
University of Athens,
15771 Athens, Greece
Received 27 March 2002;
accepted 21 October 2002
Abstract: According to general belief, the conformational information on short linear peptides in
solution derived at ambient temperature from NMR spectrometry represents a population-weighted
average over all members of an ensemble of rapidly interconverting conformations. Usually the
search for discrete conformations is concentrated at low temperatures especially when sharp NMR
resonances are detected at room temperature. Using the peptide Ac–RGD–NH
2
(Ac–Arg–Gly–Asp–
NH
2
, Ac: acetyl) as a model system and following a new approach, we have been able to
demonstrate that short linear peptides can adopt discrete conformational states in DMSO-d
6
(DMSO: dimethylsulfoxide) which vary in a way critically dependent on the reconstitution condi-
tions used before their dissolution in DMSO-d
6
. The conformers are stabilized by intramolecular
hydrogen bonds, which persist at high temperatures and undergo a very slow exchange with their
extended structures in the NMR chemical shift time scale. The reported findings provide clear
evidence for the occurrence of solvent-induced conformational exchange and point to DMSO as a
valuable medium for folding studies of short linear peptides. © 2003 Wiley Periodicals, Inc.
Biopolymers 69: 72–86, 2003
Keywords: arginine ionic interactions; aspartic acid ionic interactions; fibrinogen inhibitor;
hydrogen bonds; NMR; peptide folding; RGD peptide; slow conformational exchange
Correspondence to: Vassilios Tsikaris; email: btsikari@
cc.uoi.gr
Contract grant sponsor: Greek Secretariat of Research and
Technology
Biopolymers, Vol. 69, 72–86 (2003)
© 2003 Wiley Periodicals, Inc.
72
Page 1
INTRODUCTION
Information on the favored conformational states of
short linear peptides comes mainly from x-ray diffrac-
tion, NMR studies in different solvents, and molecular
modeling calculations.
1–3
For instance, five different
crystal structures for Leu-enkephalin and three for
Met-enkephalin, depending on solvent crystallization
conditions, have been reported.
4,5
By taking advan-
tage of the solubility of peptides in a number of polar
solvents, various solvent-dependent conformational
states can be resolved. It is generally accepted that
these states are found in very fast conformational
exchange. The structures of peptides, especially those
derived from NMR, are believed to represent only
population-weighted averages over all conformers in
a given solvent at ambient temperature. Only slow
transitions in the NMR time scale conformational,
such as the amide bond cistrans interconversion,
have been resolved to date for linear peptides by
NMR spectroscopy. To study very rapid processes,
such as the folding of peptides and proteins, new
methods had to be developed (stopped-flow fluores-
cence, stopped-flow CD, temperature jump, etc.),
which allow the earliest events in folding to be
probed.
6,7
Using temperature jump and photodissocia-
tion techniques, Eaton et al.
8
have shown that some
-helices are formed in a few nanoseconds, whereas
others require microseconds to fold, depending on the
particular amino acid sequence. To date, intermediate
conformational states between folded and unfolded
states in peptides have not been detected.
Some recent studies have indicated that short linear
peptides can adopt different conformational states in
dimethylsulfoxide (DMSO) solutions depending on
the pH value of the aqueous solution they originated
from.
9–12
We have also provided experimental evi-
dence by
17
O-NMR spectroscopy for slow conforma-
tional exchange of Boc–[
17
O]Tyr(2,6-diClBzl)–OH
(Boc: tert-butoxycarbonyl; Bzl: benzyl) in DMSO
solution.
13
Our conclusion was based on the detection
of two, rather than one,
17
O resonances for the car-
boxyl group of the peptide in DMSO, most probably
due to the engagement of the carboxyl group in a
strong hydrogen-bonding interaction. In CDCl
3
solu-
tion, on the other hand, a single
17
O resonance, re-
sulting from the fast exchange between open and
hydrogen bonded states, was observed. The depen-
dence of the peptide conformation on the nature of the
reconstitution medium has also been highlighted by
the recent work of Boden et al., who studied the
three-dimensional (3D) structure of a linear 27-resi-
due peptide in lipid bilayers by Fourier transform
infrared (FTIR) absorption.
14
When that peptide was
reconstituted from methanol, it adopted a
-strand
structure, while in the case of 2,2,2-trifluoroethanol it
formed initially an
-helix, which relaxed very slowly
(within hours) to an equilibrium state between
-helix
and
-sheet.
It appears, therefore, that the nature of the solvent
and the conditions employed in the conformational
reconstitution might influence the prevalence of a
certain peptide conformation.
The work reported here aims to develop a NMR-
based strategy that would allow us to identify directly
discrete conformational states of short linear peptides,
differentiate them from the average one, and gain new
insight into their folding process. The tripeptide Ac–
RGD–NH
2
(Ac–Arg–Gly–Asp–NH
2
, Ac: acetyl) was
used as a model compound for our NMR and molec-
ular modeling studies.
15–18
Solutions of this peptide in
DMSO-d
6
, reconstituted from aqueous solutions at
different pH values, were studied by
1
H-NMR spec-
troscopy at several temperatures (in the range 300
355 K). We managed to detect discrete conforma-
tional states of the peptide in DMSO-d
6
, which vary
with the initial pH of the solution, and to show that
these states can be in slow exchange depending on the
reconstitution conditions. We present here our find-
ings and discuss their implications for peptide folding.
MATERIALS AND METHODS
Reagents and solvents were used without further purifica-
tion. 2-(1H-benzotriazol-l-yl)-1,1,3,3-tetramethyluronium
(TBTU), 1-hydroxybenzotriazole (HOBt), and Boc amino
acids were purchased from Neosystem (France); solvents
from Labscan Ltd. (Ireland); trifluoroacetic acid (TFA) and
diisopropylethylamine (DIEA) from Merk Schuchardt (Ger-
many); and 4-methyl-benzhydrylamine resin (MBHA) resin
from Saxon Biochemicals (Hannover, Germany). DMSO-d
6
and tetramethylsilane (TMS) were purchased from Euriso-
top (France).
Synthesis of Ac–RGD–NH
2
This was carried out by the stepwise solid-phase procedure
on a MBHA resin following the Boc chemistry.
19
Arg was
introduced as Boc–Arg(Tos)–OH (Tos: toluene-4-sulfonyl)
and Asp as Boc–Asp(OBzl)–OH. Coupling reactions were
performed using the molar ratio of amino acid/TBTU/
HOBt/DIEA/resin 3/2.9/3/3/1. The
NH
2
group, after
cleavage of the Boc protecting group with TFA, was acety-
lated using an excess of Ac
2
O in pyridine (the ratio
Ac
2
O/—NH
2
group was 30:1). Ac–RGD–NH
2
was cleaved
from the resin with anhydrous HF in the presence of phenol
and anisole as scavengers. The crude material (yield 80%)
was subjected to high performance liquid chromatography
The Ac–RGD–NH
2
Peptide 73
Page 2
(HPLC) purification (semipreparative reverse-phase C
18
col-
umn) using gradient elution with the following solvents: A,
H
2
O/0.1% TFA; B, CH
3
CN/H
2
O/TFA (10/90/0.1). A pro-
grammed gradient elution (4 mL/min) was applied (A/B: 90/
10–A/B: 75/25), elution time 20 min (yield 60%). The purity
of the peptide was checked by analytical HPLC and the correct
molecular mass was confirmed by electrospray ionization mass
spectroscopy (ESI-MS) (MW calc.: 387.40; found: 387.62).
Synthesis of Ac–RGd–NH
2
This was carried out on a MBHA resin according to Boc
chemistry as described above. The purity of the peptide was
checked by analytical HPLC and the correct molecular mass
was confirmed by ESI-MS.
1
H-NMR Experiments
The NMR samples were prepared by dissolving the solid
material in H
2
O, and adjusting the pH to the desired value
with NaOH or HCl. The aqueous solutions were lyophi-
lized, and weighted amounts were dissolved in DMSO-d
6
at
concentrations 5mM. The NMR experiments were per-
formed at 295–355 K on Bruker AMX 400 and Avance 500
spectrometers. The standard correlation spectroscopy
(COSY), total COSY (TOCSY), and rotating frame nuclear
Overhauser effect spectroscopy (ROESY) Bruker micropro-
grams were used. The TOCSY spectra were recorded using
a mixing time of 100 ms. The spectral width in F1 was 5600
Hz. Various ROESY experiments were performed using
mixing times of 250 and 350 ms at 300, 305, and 310 K.
The rate constants of rotation about the guanidinium N
–C
(k
), C
–N
1
(k
1
), and C
–N
2
(k
2
) bonds were obtained by
calculating the NMR line shape for a four-site exchange pro-
cess using the general multiple site exchange matrix algo-
rithm.
20
The best fitted simulated spectra were obtained by
using the spectral parameters (chemical shifts, line widths, and
intensities) for guanidinium protons and varying rate constants
k
, k
1
, and k
2
. The natural line widths of guanidinium NH
signals required in these determinations were estimated by
measuring the line widths of nonexchanging Arg amide proton
signals at appropriate temperatures. All calculations were per-
formed using the program Muses (MUltiple Site Exchange
Simulations).
21
The activation parameters were evaluated from
Eyring equations:
lnk/T 23.76 共⌬H*/RT 共⌬S*/R
and
G* RT23.76 ln(k/T]
where R is the universal gas constant.
Structure Calculation
Structure calculation was carried out using the software DY-
ANA (DYnamics Algorithm NMR Applications).
22
The dis-
tance restraints used as inputs in DYANA were derived from
a
1
H-
1
H ROESY spectrum of the Ac–RGD–NH
2
peptide
reconstituted in DMSO-d
6
after lyophilization from an aque-
ous solution at pH 4.9. The ROESY spectrum was recorded at
310 K with a mixing time of 250 ms. The ROE intensities were
converted into distances using the
1-
2 cross peak of Asp as
reference. Thirty-six upper-limit cross-peak intensities, classi-
fied as strong (up to 2.8 Å), medium (up to 3.5 Å), and weak
(up to 5 Å), were used as input restraints for the calculation.
Appropriate corrections for center averaging were added to
DYANA restraints for degenerate proton resonances.
23
No
lower limits were used. Constraints for
,
, and
1
angles
were calculated using the HABAS program of DYANA pack-
age. Six
3
J
␣␤
2
and
3
J
coupling constants were included with
a tolerance of 2.5 Hz. All distance and angle constraints were
assigned the default relative weight of 1. The default tolerance
of 0.05 at target function units was applied. The calculations
were performed using the standard minimization protocol and
the REDAC (REdundant Dihedral Angle Constraints) strategy
implemented in DYANA.
RESULTS AND DISCUSSION
Conformational State of Ac–Arg–Gly–
Asp–NH
2
, Reconstituted in DMSO-d
6
from an Aqueous Solution at pH 2.0
The complete assignment of all proton resonances of
Ac–Arg–Gly–Asp–NH
2
was based on the combined
use of COSY, TOCSY, and ROESY experiments. The
1
H-NMR spectrum in DMSO-d
6
solution of the pep-
tide reconstituted after lyophilization from an aqueous
solution at pH 2 is shown in Figure 1 (bottom). The
resonance at 12.3 ppm (not shown) confirms the pro-
tonated state of the Asp
-carboxylic group. The high
absolute temperature coefficient values of all of the
NH protons, including the C-terminal amide protons
(8 ppb/K), suggest that they are exposed to the
solvent. The equal
3
J
N
and
3
J
N
values (5.32 Hz) of
Gly indicate that there is a free rotation about the
NOC
bond of this residue,
24
while the Asp
3
J
␣␤
and
3
J
␣␤
coupling constant values (5.27 and 8.46 Hz)
correspond to a high percentage (80%, Table I) of
the two energetically favored C
–C
rotamers
25–28
(I
and II,
1
60°, 180°), suggesting the absence of
rotational restrictions about the C
OC
bond. On the
other hand, the Arg–N
H and Arg–N
2
H
4
(this latter
symbology indicates both N
atoms and all four pro-
tons linked to them) proton resonances, at 7.63 and
7.13 ppm, respectively, are attributed to their nonhy-
drogen-bonded states.
10,11,29
It must be noted that the
Arg–N
2
H
4
protons under free rotational conditions
can be detected as two broad peaks due to chemical
exchange in the guanidinium group.
10,11,30,31
In this
conformational state two separate, broad peaks at 7.29
74 Biris et al.
Page 3
and 6.91 ppm, respectively, were detected at 295 K
(data not shown), which collapse to a broad peak at
300 K (Figure 1) at 7.13 ppm. Strong sharpening of
this peak is observed as the temperature increases to
355 K (data not shown). The NMR data thus suggest
that Ac–Arg–Gly–Asp–NH
2
is found in the extended
conformational state when lyophilized from aqueous
solution at pH 2 and redissolved in DMSO-d
6
.
NMR spectra recorded back at 300 K after heating
the sample to 355 K and keeping it at 300 K for varying
time periods reveal the presence of a second set of
resonances indicative of a slow aggregation process tak-
ing place under these conditions (data not shown). This
conclusion is supported by the appearance in the ES-MS
spectrum of a low intensity peak originating from a
small amount of the dimeric form of the peptide. Inter-
estingly, this peak was not detected under neutral or
basic conditions (data not shown). Sanderson et al.
32
reported a similar observation for the (SS) Mba–Arg–
Gly–Asp–Man peptide (Mba: 2-mercaptobenzoate;
Man: 2-mercaptoanilide) in DMSO-d
6
solution. These
authors, based on the fact that the Arg–N
H was not
detected in the second set of resonances, concluded that
such a behavior could be the result of a slow hydrolytic
process. This phenomenon can be excluded in our case
by the full set of resonances for the Ac–Arg–Gly–Asp–
NH
2
peptide present in the one-dimensional (1D) NMR
spectrum and in the TOCSY and ROESY NMR spectra
as well.
Conformational State of Ac–Arg–Gly–
Asp–NH
2
, in DMSO-d
6
Reconstituted
from an Aqueous Solution at pH 3.3
The ionization state of the peptide remains the same
as that at pH 2.0, as indicated by the presence of a
broad peak at 12.25 ppm corresponding to the pro-
FIGURE 1 NH (A), C
H (B), and C
H (C) regions of the 400 MHz
1
H-NMR spectra of
Ac–RGD–NH
2
in DMSO-d
6
solution at various temperatures. Conformational reconstitution of the
peptide was performed from an aqueous solution at pH 3.3. For comparison the spectrum of the
peptide reconstituted from an aqueous solution at pH 2.0 is given (bottom). Arg–N
2
H
4
notation
indicates four protons.
The Ac–RGD–NH
2
Peptide 75
Page 4
tonated form of the Asp
-COOH group. Neverthe-
less, the dramatic changes in the resonance frequen-
cies and the line shapes over the entire spectrum are
indicative of a new conformational state of the pep-
tide. Thus, the Asp–NH proton broadens and is down-
field shifted from 8.19 to 8.46 ppm, the Arg–N
H
becomes very broad and vanishes into the baseline at
300 K (Figure 1A), the Arg–N
2
n
H
4
protons appear as
a broad peak at 7.10 ppm, and the two carboxamide
protons are seen as a sharp resonance at 7.10 ppm and
a broad one at 6.92 ppm. The line shape of the
resonances can provide information about the inter-
acting parts of the molecule in the new conforma-
tional state. The observed broadening of many reso-
nances is not due to a change in the rotational corre-
lation time of the entire peptide since not all of the
resonances are broadened to the same extent and the
narrowing of each resonance occurs at different tem-
perature values (Figure 1). Thus, the difference in the
line width broadening of the Gly–NH, Asp–NH, Asp–
C
H resonances, the upfield shifted Gly–C
H, Asp–
C
H, and the C-terminal amide proton resonances, as
well as the downfield shifted Arg–C
H, indicate the
occurrence of a slower exchange process, which af-
fects mainly these groups, compared to the other parts
of the molecule. It is also interesting to note that from
the geminal Gly–C
H
2
and Asp–C
H
2
protons, only
the resonances of the upfield shifted protons appear
broadened with that of the Asp–C
H proton almost
vanishing into the baseline (Figure 1C). Since the
Asp–C
H, Gly–C
H, Asp–C
H, and the Arg–C
H
protons cannot be involved in any other exchange
process, we assume that the origin of their broadening
resides in a conformational exchange process. It is
also clear that the Gly–C
H
2
and Asp–C
H
2
groups
are not involved directly in the exchange process.
Therefore, the differential broadening of their upfield
shifted protons must originate from conformational
interconversion of adjacent groups, which affects
their local magnetic environment. Bogusky et al.
33
have reported a similar broadening for the Gly–C
H
2
protons brought about by freezing to 80°C a meth-
anolic solution of a cyclic (S,S) CRGDC peptide with
a viscosity similar to that of DMSO at room temper-
ature. In this case, molecular dynamics simulations
have shown the presence of conformers differing by
rotational inversions of the peptide-bond planes be-
tween the Arg–Gly and Gly–Asp residues.
The very fast interconverting rate between the con-
formers builds up gradually by raising the temperature
from 300 to 350 K (Figure 1). The first groups of the
molecule to achieve the very fast interconverting rate
are located around Gly (at 310 K, Figure 1B). Most
notable is the fact that at 330 K the upfield shifted
Asp–C
H and especially the Arg–N
H protons are
still undergoing considerably slower exchange as re-
vealed by their resonance broadening (Figures 1A,
1C, and 2). This finding probably indicates that there
is a contact between the Arg–N
H proton and the Asp
-COOH group responsible for this conformational
exchange.
Besides the chemical shift changes observed be-
tween the conformational states reconstituted from pH
2.0 and 3.3, respectively, the temperature coefficient
Table I Chemical Shift Differences of the Geminal Gly–C
H
2
, Asp–C
H
2
, Arg–C
H
2
, Arg–C
H
2
, and Arg–C
H
2
Protons of Ac–Arg–Gly–Asp–NH
2
in DMSO-d
6
Solution at 300 K, Reconstituted from Aqueous Solutions
at Various pH Values
Conformational
Reconstitution
Solvent
(ppm)
Arg–C
H
2
Arg–C
H
2
Arg–C
H
2
Gly–C
H
2
Asp–C
H
2
Rotamers
a
of the
Asp C
–C
Bond (%)
I II III
H
2
O, pH 2.0 0.15 0.00 0.00 0.08 0.16 26 54 20
H
2
O, pH 3.3 0.25 0.03 0.06 0.08 0.16
b
25 31 44
b
H
2
O, pH 4.0 0.36 0.04 0.11 0.20 0.35
c
14 22 64
c
H
2
O, pH 4.4 0.41 0.08 0.16 0.28 0.45 5 25 70
d
H
2
O, pH 4.9 0.47 0.08 0.17 0.30 0.51 5 24 71
a
Rotamer I (
1
⫽⫺60°), II (
1
180°), III (
1
60°). The values of J
g
and J
t
used for estimation of the rotamer populations were 2.32
and 13.70 Hz, respectively.
b
Measured at 340 K.
c
Measured at 330 K.
d
Measured at 310 K.
76 Biris et al.
Page 5
values have been greatly modified in the latter case.
Figure 3 shows the chemical shift variations with
temperature of both C-terminal amide protons of Ac–
RGD–NH
2
in DMSO reconstituted from solutions at
several pH values. For comparison the same plot is
also shown for the Ac–RGd–NH
2
peptide (which con-
tains a D-Asp instead of L-Asp) under similar experi-
mental conditions. The upfield shifted C-terminal
amide proton is not accessible to the solvent, as
judged by its low temperature coefficient value (1.8
ppb/K) in the 310–330 K range. This is not true for
the downfield shifted C-terminal amide proton (7.5
ppb/K). It is evident from Figure 3 that temperature
increase results in coalescence of the two resonances
due to the increase of the rotational rate around the
C-terminal primary amide bond, the double character
of which is expected to be reduced in comparison to
that of a secondary amide bond. The considerably
higher temperature of coalescence (Figure 3) in Ac–
RGD–NH
2
compared to Ac–RGd–NH
2
(more than
20°C), indicating a slower rotation around the CON
bond in the former case, can originate from confor-
mational restrictions. Comparing the temperature co-
efficients in the temperature range 310–320 K and the
temperatures of coalescence of the conformational
states presented in Figure 3 we can safely conclude
that the upfield shifted C-terminal amide proton is
protected from the solvent for the states originating
from aqueous solutions of the Ac–RGD–NH
2
peptide
at pH 3.3 and 4.9. It is worth noting that similar low
temperature coefficients for the upfield shifted amide
proton were found previously for an Ac–Arg–Pro–
Asp–NH
2
peptide.
12
A combination of NMR and mo-
lecular modeling data supported in that case the par-
ticipation of this proton in hydrogen bonding with the
Arg–CO group stabilizing a type I
-turn.
The broad peak of the Asp–NH of Ac–RGD–NH
2
in the temperature range of 300–330K and its over-
lapping with the resonance of the Gly–NH proton
(Figure 4) does not allow an accurate determination of
the temperature coefficient in this case.
As already mentioned, the Arg–N
H proton under-
goes a conformational exchange at an intermediate
rate up to 330 K. This is evidenced by the absence of
a detectable peak. Raising the temperature from 330
to 355 K, a broad peak appears at 8.92 ppm, which
progressively sharpens (Figures 1A and 2). This be-
havior is indicative of a chemical exchange process,
most probably between the hydrogen bonded and the
open state. Taking into account that the Arg–N
H, the
downfield shifted Arg–C
H, and the upfield shifted
Asp–C
H proton resonances show a similar temper-
ature dependent broadening, it is reasonable to as-
sume that the Asp
-COOH would be the interacting
group with the Arg–N
H proton. This hypothesis is
further supported by the Asp
3
J
␣␤
and
3
J
␣␤
coupling
constant values (5.32 and 4.88 Hz, respectively) mea-
sured from the 1D high-resolution spectrum at 340 K.
The reduced values indicate the presence of a high
percentage (44%, Table I) of the g
(x
1
60°)
FIGURE 2 Plot of the Arg–N
H resonance line width (
1/2
) vs temperature of Ac–RGD–NH
2
in DMSO-d
6
solution reconstituted from aqueous solutions at different pH values.
The Ac–RGD–NH
2
Peptide 77
Page 6
rotational state about the C
OC
bond, which is the
least energetically favored rotamer
25,28
under free rota-
tional conditions. Comparing these data with those
found for the Asp C
OC
rotational state at pH 2, we
can conclude that conformational restrictions induce this
unusual rotamer distribution. This is in agreement with
the differential broadening of the two Asp–C
H reso-
nances.
Although this intermediate conformational state
appears stabilized by at least one hydrogen bond,
the aliphatic parts of the Arg and Asp side chains
still appear flexible. This is evident not only from
the broadening of the Arg–N
H, the downfield
shifted Arg–C
H, and the upfield shifted of Asp–
C
H protons, but also from the observed chemical
shift differences for almost all the geminal Arg and
Asp side-chain protons (Table I). These differences
are small compared either to that observed in the
case of the intermediate conformational state recon-
stituted from aqueous solution at pH 4.9 (see be-
low, Table I) or to the reported data for the same
side chains when they are involved in very strong
interactions.
12
Contrary to the behavior of the peptide at pH 2.0,
the spectrum at pH 3.3 is completely recovered after
heating at 355 K and annealing back to 300 K, indi-
cating that there is no (or negligible) peptide aggre-
gation in this case.
In short, at pH 3.3 the peptide seems to adopt a
conformation distinctively different from that at pH
2.0, which appeared fully extended.
The observed changes in the NMR spectra of
Ac–RGD–NH
2
for the conformational states recon-
stituted in DMSO from aqueous solutions at pH 4.0
and 4.4 further support the conformational analysis
described above (Figure 4). More precisely, the
guanidinium–
-carboxylate interaction seems en-
hanced, as judged by the following: (a) the further
Arg–N
H downfield shifting, (b) the broadening
and upfield shifting of the Arg–N
2
H
4
proton reso-
nances, and (c) the gradual increase of the chemical
shift difference between all the Arg and Asp side-
chain geminal protons (Table I). The same proton
resonances, as in the preceding conformational
states (pH 3.3), appeared broadened, although their
sharpening occurred at different temperature val-
ues. It must be noted that the Asp
-carboxylic
proton resonance was detected as a very broad peak
at pH 4.0, while it was no longer observed at
pH 4.4.
FIGURE 3 Plot of chemical shifts vs temperature of the C-terminal amide protons of Ac–RGD–
NH
2
in DMSO-d
6
solution, reconstituted from aqueous solutions at pH 3.3 (A), pH 4.9 (B), and of
Ac–RGd–NH
2
at pH 3.3 (C) and pH 4.9 (D).
78 Biris et al.
Page 7
Conformational State of Ac–Arg–Gly–
Asp–NH
2
in DMSO-d
6
Reconstituted
from Aqueous Solution at pH 4.9
The complete 1D spectrum of Ac–RGD–NH
2
in
DMSO-d
6
after reconstitution from an aqueous solu-
tion at pH 4.9 is shown in Figure 5B. Although the
changes in the spectrum tend to confirm the previous
conformational analysis, almost all of the resonances
are very sharp in this case. More specifically, the
strong downfield shifting of the Arg–N
H proton and
the substantial chemical shift differences between the
Asp–C
H
2
, Arg–C
H
2
, and Arg–C
H
2
geminal pro-
tons (Table I) point to a further stabilization of the
conformation that involves the interaction between
the Arg and Asp side chains. Taking into account that
the Asp
-COO
group is now in the deprotonated
form, it is reasonable to expect that ionic forces con-
tribute to this stabilization. On the other hand, the
overall appearance of the spectrum, with only one set
of resonances clearly present, and the sharpness of all
FIGURE 4 The 400 MHz
1
H-NMR spectra of the NH region of Ac–RGD–NH
2
in DMSO-d
6
solution, reconstituted from aqueous solutions at different pH values, at 300 K (A) and 350 K (B).
The expanded spectrum of the Arg–N
2
H
4
region in the y direction is also shown. Arg–N
2
H
4
notation indicates four protons while Arg–N
H
2
two protons.
The Ac–RGD–NH
2
Peptide 79
Page 8
peaks, hint to either a very fast exchange process
between folded and unfolded states or to a very slow
conformational exchange with the equilibrium quan-
titatively shifted toward the folded state. From these
observations the following questions may be ad-
dressed: Is there any conformational exchange pro-
cess taking place under these experimental condi-
tions? If so, what is the rate of this process?
To answer these questions, we relied on the mon-
itoring and the detailed analysis of the behavior of
FIGURE 5 The 400 MHz
1
H-NMR spectra of the NH region of Ac–RGD–NH
2
in DMSO-d
6
solution, reconstituted from an aqueous solution at pH 4.9, at different temperatures (A). The
complete spectrum of Ac–RGD–NH
2
at pH 4.9 and 300 K is shown at the bottom (B). Notation as
in Figure 4.
80 Biris et al.
Page 9
several resonances at different pH and temperature
values. In this respect the resonances originating from
the Arg–N
H
4
protons provided the most valuable
information. Comparing the 1D spectra of Ac–RGD–
NH
2
recorded in DMSO-d
6
after reconstitution from
aqueous solutions at pH 2.0, 3.3, 4.0, 4.4, and 4.9
(Figure 4), it is evident that the Arg–N
H
4
protons
contribute progressively, but differently, to the struc-
ture stabilization.
Two protons of Arg–N
, when reconstitution takes
place at pH 4.9, appear as a sharp resonance at 7.01
ppm at 300 K, whereas the other two protons are
almost not detectable (Figure 5A). However, the spec-
trum recorded at 295 K, when expanded in the y
direction, reveals the presence of a broad peak at 9.78
ppm (Figure 5A). By increasing the temperature, this
peak broadens and cannot be detected in the temper-
ature range of 305–325 K. Up to this temperature the
resonance at 7.01 ppm slightly broadens and shifts
upfield (from 7.01 to 6.94 ppm). At 325 K a new
broad peak appears at 8.05 ppm, which sharpens when
the temperature is increased to 335 K. Obviously, this
new peak is the result of coalescence between two
peaks. The chemical shift of the second broad, unre-
solvable at 295–300 K, peak, calculated from the
chemical shifts of the peaks at 9.78 and 8.05 ppm, was
found centered at 6.28 ppm. The 1D 500 MHz spec-
trum at 295 K confirms the presence of the second
upfield shifted broad peak at around 6.45 ppm, in good
agreement with our calculated value. Upon further heat-
ing to 355 K, the new peak at 8.05 ppm and the sharp
one at 6.94 ppm become progressively equivalent,
broaden, and coalesce at 7.40 ppm (Figure 5A).
If the downfield shifting of the Arg–N
H proton,
due to its participation in hydrogen bonding, is also
taken into account, the chemical shift dispersion of
the guanidinium group can be explained according to
the scheme depicted in Figure 6. Thus, the very fast
rotation around the C
–N
2
bond could explain the
sharp resonance observed for the two Arg–N
2
H pro-
tons at 7.01 ppm. The very slow rotation around the
bond C
–N
1
resulting from hydrogen bonding could
give rise to two separate peaks at 9.78 and 6.45 ppm.
The hydrogen bonding of both the Arg–N
1
H
1
and
Arg–N
H protons are possibly responsible for the
slow rotation around the N
–C
bond. As the temper-
ature increases, the rotation around the C
–N
1
be-
comes fast due to hydrogen-bond breaking, resulting
in the appearance of a broad peak at 8.05 ppm for the
two Arg–N
1
H protons at 325 K. Increase of the rate
of rotation around the N
–C
bond leads to the equiv-
alence of the two Arg–N
H
2
groups with a new co-
alescence of the resonances at 7.40 ppm at 355 K.
A three-step resonance coalescence between Arg–
N
1
H
1
/Arg–N
1
H
2
, Arg–N
2
H
1
/Arg–N
2
H
2
, and Arg–
N
1
H
2
/Arg–N
2
H
2
at temperature values of 238, 246,
and 303 K, respectively, has also been reported in the
past for the Arg–N
2
H
4
protons of Ac–Arg(HCl)O
i
Pr
(O
i
Pr: isoproxy) in a 5:95 DMSO/CD
2
Cl
2
(v/v) solution
at 400 MHz.
30
In the case of intermolecular interaction of Arg–
guanidinium with ligand carboxylate groups, four
NMR signals were observed for the four N
H pro-
tons.
34,35
The two lowest
1
H signals (9.33 and 10
ppm) assigned to NH
12
and NH
22
were attributed to
their hydrogen bonding with the ligand carboxylate
oxygens. At 313 K the high field pair of signals
coalesced into a single broad signal, but the coales-
cence temperature for all four guanidinium protons
was not resolved. This pattern was attributed to a fast
rotation about the N
–C
bond, but to a slow rotation
about the C
–N
bond, on the NMR chemical shift
time scale.
In our study the coalescence temperatures at 400
MHz for Arg–N
1
H
1
/Arg–N
1
H
2
, and Arg–N
1
H
2
/
Arg–N
2
H
2
are considerably higher compared either
to the free Arg or the one complexed to a carboxylate
ligand, suggesting slower rotation rates around the
C
–N
1
and N
–C
bonds. The reversed chemical shift
and rotation pattern about the N
–C
and C
–N
FIGURE 6 Proposed rotational pattern of the guanidinium N
–C
,C
–N
1
and C
–N
2
bonds of
Ac–RGD–NH
2
in the intramolecular hydrogen-bonded interaction with Asp
-COO
according to
the experimental 400 MHz
1
H-NMR data.
The Ac–RGD–NH
2
Peptide 81
Page 10
bonds found for Ac–RGD–NH
2
is consistent with the
Arg–N
H and Arg–N
1
H
1
(Figure 6) hydrogen-
bonded interaction. Interestingly, it has been proposed
in the past
36
that Arg–N
H and Arg–N
1
protons
participate in intramolecular interactions, while Arg–
N
1
and Arg–N
2
protons are more often involved in
intermolecular interactions.
The occurrence of a guanidinium–carboxylate in-
teraction, which is responsible for the restricted rota-
tion about the N
–C
and C
–N
1
bonds, is further
supported by the behavior of the Arg and Asp side
chains, as well as by the observed ROE effects. Thus,
the Arg and Asp side-chain geminal protons exhibit
unusually strong chemical shift differences (Table I),
probably as a result of their restricted mobility. In
addition, the Asp
3
J
␣␤
1
and
3
J
␣␤
2
coupling constant
values (2.85 and 5.29 Hz, respectively) indicate a very
high percentage (71%) of the less energetically fa-
vored rotamer g
(x
1
60°) (Table I). Under these
rotational conditions one of the
-COO
oxygens
must be oriented in close proximity to the Asp–NH
proton.
25
This fact can explain the low absolute tem-
perature coefficient observed for the Asp–NH (0
ppb/K). At the same time, the hydrogen bonding of
the Arg–N
H with the Asp
-COO
group would
bring this proton in the proximity of the Asp–NH.
This result is clearly confirmed by the observed ROE
effects between Arg–N
H/Asp–NH and Arg–C
2
H/
Asp–NH protons (Figure 7). These ROE effects were
accurately resolved by recording the ROESY spec-
trum at 310 K in order to overcome the overlapping of
Asp–NH and Gly–NH resonances. Table II indicates
the number of the observed ROE effects for the Ac–
RGD–NH
2
in DMSO-d
6
solution when the reconsti-
tution was performed in aqueous solution at pH 4.9.
The numerous ROEs detected for this small molecule
strongly suggest that its conformation is very com-
pact. In agreement with this finding are also the small
absolute temperature coefficient values measured for
the Asp–NH (0 ppb/K), the upfield shifted
—CONH
2
(1.3 ppb/K in the temperature range
300–325 K), and the behavior of the Arg–N
H proton
resonance. It is interesting to note that, in contrast to
the conformational states reconstituted from aqueous
solution at pH 2.0 and 3.3, in this case the line width
of the Arg–N
H resonance remains unchanged on
heating to 355 K (Figure 2). This finding suggests that
the hydrogen-bonded network and the ionic interac-
tion, which stabilizes the structure, are very strong,
and there is no conformational exchange involving
breaking of the hydrogen bond between Arg–N
H and
Asp
-COO
even at temperatures as high as 355 K.
FIGURE 7 The NH/C
H (A) and NH/NH (B) regions of the 400 MHz ROESY spectrum of
Ac–RGD–NH
2
in DMSO-d
6
, reconstituted from an aqueous solution at pH 4.9, at 310 K and t
m
250 ms.
82 Biris et al.
Page 11
Based on the data just described and their detailed
analysis, we conclude that the Ac–RGD–NH
2
peptide
when reconstituted in DMSO from pH 4.9 quantita-
tively adopts a very stable and folded conformation,
which undergoes an unusually slow exchange with the
extended conformation.
Rates of Rotation About the N
–C
,
C
–N
1
, and C
–N
2
Bonds
Depending on the environment and the nature of the
interactions involving the guanidinium protons, vari-
ous patterns can be observed for the rotation rates
around the N
–C
bond. Under free rotational condi-
tions the Arg residue shows a single broad, coalesced
resonance for N
1
H
2
and N
2
H
2
protons in the 400
MHz
1
H-NMR spectrum in DMSO-d
6
solution at 300
K. (See Figure 1.) The coalesced signal arises from
exchange between the N
H protons caused by rota-
tions about the N
–C
and C
–N
bonds. For Ac–Arg–
O
i
Pr in 5:95 DMSO/CD
2
Cl
2
(v/v) solution at 298 K,
rates of 130, 7800, and 17500 s
1
for N
–C
,C
–N
1
,
and C
–N
2
bond rotations, respectively, have been
reported.
30
The rates of N
–C
and C
–N
bond rota-
tions are dramatically reduced for an Arg complexed
to a ligand carboxylate group in which both N
1
H
2
and NH
2
H
2
groups are involved in stable hydrogen-
bonding interactions.
35
This is the case of L. casei
dihydrofolate reductase (DHFR) complexed either
with methotrexate (MTX) or with MTX and
-nico-
tinamide adenine dinucleotide phosphate (NADPH).
The rates about the N
C
and C
N
bonds were found
to be 565 and 117 s
1
for the DHFR MTX com-
plex, and 930 and 71 s
1
for the DHFR MTX
NADPH complex, respectively, at 313 K.
Based on the scheme depicted in Figure 6, an
altered pattern for the rotation rates about the N
–C
and C
–N
bonds is expected for our peptide in com-
parison to that of the DHFR complexes. The rates of
rotation about the N
–C
,C
–N
1
, and C
–N
2
bonds
estimated from temperature- dependent line shape
analysis are 10, 642, and 5350 s
1
, respectively, at
295 K. The rates for the N
–C
and C
–N
1
bond
rotations are 13-fold lower than the corresponding
rates reported for Ac–Arg–O
i
Pr
30
in a 5:95 DMSO/
CD
2
Cl
2
(v/v) solution at 298 K. On the contrary, the
rate for the C
–N
2
bond rotation, the group that does
not participate in a hydrogen-bonded interaction, is
only 3-fold lower.
The most surprising finding in this study is that,
even at 355 K, the hydrogen-bonded interactions be-
tween the guanidinium and carboxylate groups are
still strong enough, resulting in a rate of rotation about
the N
–C
bond of 561 s
1
. Especially, the hydrogen-
bonded interaction involving the Arg–N
H proton is
very stable, as evidenced by the slight broadening
(20%) of the resonance on heating to 355 K. The
Eyring diagram yields the activation parameters, for
rotation about the N
–C
bond, H* 56.4 kJ mol
1
,
S* ⫽⫺35.8 J mol
1
, and G*(303) 67.3 kJ
mol
1
. The free energy of activation for the N
–C
bond rotation is significantly higher in our case than
that estimated for the same bond (61 kJ mol
1
)in
Ac–Arg–O
i
Pr in a 5:95 DMSO/CD
2
Cl
2
(v/v) solu-
tion
30
or for Arg (54 kJ mol
1
) in a 50% aqueous
DMSO solution.
31
The increased value of G*is
probably the result of the guanidinium hydrogen-
bonded interactions.
Structure Calculation
The structure of the Ac–RGD–NH
2
peptide in
DMSO-d
6
reconstituted from pH 4.9 was calculated
on the basis of 36 upper distance constraints and 6
Table II Classification of Restraints and Structure
Statistics
Restraint Classification
Total number of NOEs 36
Number of backbone–backbone NOEs 10
Number of backbone–side-chain NOEs 23
Number of side-chain–side-chain NOEs 3
Number of intraresidue NOEs 13
Number of interresidue NOEs 23
Number of strong NOEs (2.8 Å) 4
Number of medium NOEs (3.5 Å) 11
Number of weak NOEs () 21
Total number of J 6
Number of
3
J
N
4
Number of
3
J
␣␤
2
Number of hydrogen bonds 2
Calculation Results
Number of structures calculated 100
Number of final structures 10
Min/max target function of
final structures 1.79/5.56 10
2
Number of distance restraint
violations 0.3 Å 0
Number of distance restraint
violations 0.2 Å 1 (in 3/10 structures)
Number of torsion angle
violations 0
Mean global heavy atom
RMSD 0.68 0.26 Å
Mean global backbone RMSD 0.37 0.13 Å
The Ac–RGD–NH
2
Peptide 83
Page 12
torsion angle constraints using the REDAC strategy of
the DYANA software.
22
Of the 100 structures gener-
ated by DYANA, the best 10 were selected on the
basis of their target functions and the low number of
restraint violations. After a first run of REDAC, two
side-chain–side- chain hydrogen bonds were imposed
from those present in at least 3 out of the 10 best
resulting structures between Arg–N
H and Arg–
N
1
H
2
(donors) and Asp
-COO
(acceptor). When
these hydrogen bonds were added as upper distance
constraints to a second run of DYANA, the number of
violated distance constraints was reduced from 3 to 1,
and a new hydrogen bond present in over half of the
10 best structures was identified between Arg–N
1
H
2
and Asp–CO. No dihedral angle violation was ob-
served. The final 10 structures were characterized by
very low target functions, the absence of any viola-
tions 0.3 Å, and relatively low root mean square
FIGURE 8 Experimental 400 MHz NMR (A) and calculated spectra of the Ac–RGD–NH
2
in
DMSO-d
6
solution reconstituted from an aqueous solution at pH 4.9 (B). For each pair of
experimental and calculated spectra, the experimental temperature is indicated. The rotation rate
about the N
–C
bond (k
) is also shown. Notation as in Figure 4.
84 Biris et al.
Page 13
displacement (RMSD) values. A classification of the
experimental restraints and a statistical analysis of the
best 10 of the calculated structures are given in Table II.
The elements of intramolecular structuring indi-
cated by the conformational analysis presented in
detail above and summarized in the model depicted in
Figure 6 are confirmed by the calculated structure
(Figure 9). The existence of a compact “pseudocy-
clic” structure stabilized by hydrogen bonding in a
head-to-tail fashion, also supported by previous work
with peptides having an Arg residue in the third
position from the C-terminus,
9–12
is consistent with
the pattern of hydrogen bonds resulting from the
calculations. More specifically: (a) there seems to be
a bifurcated hydrogen bond between the Asp
-COO
and the Arg–N
and one of the Arg–N
1
protons; (b) the conformation might be even more
stabilized through an additional interaction of the
guanidinium group with the terminal carbonyl group
of Asp; (c) the trans proton of the C-terminal NH
2
group is pointing to the interior of the pseudocyclic
structure in more than half of the best 10 structures,
being thus shielded from the solvent, as also indicated
by its temperature coefficient values; (d) the compact-
ness of the peptide conformation is confirmed by the
short distances in all calculated structures between the
Asp–NH proton and the Arg–N
(3.2 Å) and Arg–
C
2
H(2.7 Å) protons, due to the restricted mobility
of these side chains.
CONCLUSIONS
In this study, focused on the Ac–RGD–NH
2
peptide
as a model system, we provided experimental evi-
dence for some important general aspects related to
the conformational states of short linear peptides in
solution. The novel information deduced from this
work can be summarized as follows:
The discrete conformational states of the peptides
in DMSO-d
6
can undergo very slow exchange. Thus,
the species normally observed by NMR might repre-
sent only local conformational changes, which are fast
in the NMR time scale, rather than an overall average
structure.
The approach based on the rational selection of the
conditions employed before the conformational re-
constitution of a peptide in DMSO allows a more
accurate characterization of its folded structure and of
the folding process, since the driving interactions and
forces become in this manner more discernible and
can be quite easily evaluated. Moreover, the hypoth-
esis that short linear peptides in solution are found in
fast conformational exchange in the NMR time scale
FIGURE 9 Best structure (the one with the lowest value of target function) calculated with use
of the REDAC strategy of DYANA. Thin lines represent hydrogen bonds.
The Ac–RGD–NH
2
Peptide 85
Page 14
is not always valid, and has to be explored and veri-
fied in each separate case.
Intramolecular hydrogen-bonding interactions can
persist and stabilize the conformation of short linear
peptides even at very high temperatures.
The folded structure in DMSO-d
6
, of Ac–RGD–
NH
2
, is characterized by an ionic bridge between Arg
guanidinium and Asp
-carboxylate groups. Two hy-
drogen bonds involving the Arg–N
H and Arg–N
H
protons stabilize the interaction between the oppo-
sitely charged groups.
This work was supported by a grant from the Greek Secre-
tariat of Research and Technology. The authors thank V. I.
Polshakov for providing the program Muses.
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  • [Show abstract] [Hide abstract] ABSTRACT: Chemical exchange in NMR spectra was discussed. In solids, a richness of the interactions was present that led to many unusual effects. It was concluded that with modern spectrometers, fast computers and good software, the dynamics can be analyzed quite easily.
    No preview · Article · Dec 2003 · Progress in Nuclear Magnetic Resonance Spectroscopy
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    [Show abstract] [Hide abstract] ABSTRACT: The ability of an integrin to distinguish between the RGD-containing extracellular matrix proteins is thought to be due partially to the variety of RGD conformations. Three criteria have been proposed for the evaluation of the structure-activity relationship of RGD-containing peptides. These include: (i) the distance between the charged centres, (ii) the distance between the Arg Cbeta and Asp Cbeta atoms, and (iii) the pseudo-dihedral angle defining the Arg and Asp side-chain orientation formed by the Arg Czeta, Arg Calpha, Asp Calpha and Asp Cgamma atoms. A comparative conformation-activity study was performed between linear RGD peptides and strongly constrained cyclic (S,S) -CDC- bearing compounds, which cover a wide range of inhibition potency of platelet aggregation. It is concluded that the fulfilment of the -45 degrees < or = pseudo-dihedral angle < or = +45 degrees criterion is a prerequisite for an RGD compound to exhibit inhibitory activity. Once this criterion is accomplished, the longer the distance between the charged centres and/or between the Arg and Asp Cbeta atoms, the higher is the biological activity. In addition, the stronger the ionic interaction between Arg and Asp charged side chains, the lower the anti-aggregatory activity.
    Full-text · Article · Aug 2004 · Journal of Peptide Science
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    [Show abstract] [Hide abstract] ABSTRACT: The Arg-Gly-Asp RGD motif of adhesive proteins is recognized by the activated platelet integrin alpha(IIb)beta3. Binding of fibrinogen (Fg) to activated alpha(IIb)beta3 causes platelet aggregation and thrombus formation. Highly constraint cyclic (S,S) -CXaaC- containing peptides incorporating the (S,S) -CDC- and (S,S) -CRC- motifs were tested for their ability to inhibit platelet aggregation and Fg binding. Our results suggest that the above cyclic scaffolds stabilize a favorable structure for the antiaggregatory activity (IC50-values ranged from 1.7 to 570 microm). The peptides inhibited Fg binding with IC50-values up to 30-fold lower than those determined for the inhibition of the adenosine diphosphate (ADP)-induced platelet aggregation. Importantly, peptides (S,S) PSRCDCR-NH2 (peptide 11) and (S,S) PRCDCK-NH2 (peptide 10) did not inhibit PAC-1 binding to the activated platelets at a concentration in which they completely inhibited Fg binding. Moreover, (S,S) PSRCDCR-NH(2) (peptide 11), one of the more active peptides, inhibited ADP-induced P-selectin exposure. By contrast, peptide (S,S) Ac-RWDCRC-NH2, incorporating the inverse (S,S) -DCRC- sequence (peptide 16), failed to inhibit P-selectin exposure whereas at the same concentration, it effectively inhibited PAC-1 and Fg binding. It is concluded that peptides containing the (S,S) -CDC- as well the (S,S) -CRC- sequences, exhibit a broad range of activities toward platelets, and could be helpful tools for elucidating the structural interaction of Fg with the integrin receptor alpha(IIb)beta3, in its activated form. Furthermore, the (S,S) -RCDC- sequence can be used as a scaffold for developing potent non-RGD-like Fg-binding inhibitors.
    Full-text · Article · Nov 2005 · Journal of Thrombosis and Haemostasis
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