Báo cáo khoa học: Characterization of the bioactive conformation of the C-terminal tripeptide Gly-Leu-Met-NH2 of substance P using [3-prolinoleucine10]SP analogues pdf

Characterization of the bioactive conformation of the C-terminal
tripeptide Gly-Leu-Met-NH
2
of substance P using
[3-prolinoleucine10]SP analogues
Jean Quancard, Philippe Karoyan, Sandrine Sagan, Odile Convert, Solange Lavielle, Ge
´
rard Chassaing
and Olivier Lequin
UMR 7613 Paris 6-CNRS, Universite
´
Pierre et Marie Curie, Paris, France
Residue Leu10 of substance P (SP) is critical for NK-1
receptor recognition and agonist activity. In order to probe
the bioactive conformation of this residue, cis-andtrans-3-
substituted prolinoleucines were introduced in position 10
of SP. The substituted SP analogues were tested for their
affinity to human NK-1 receptor specific binding sites (NK-
1M and NK-1m) and their potency to stimulate adenylate
cyclase and phospholipase C in CHO cells transfected with
the human NK-1 receptor. [trans-3-prolinoleucine10]SP
retained affinity and potency similar to SP whereas [cis-3-
prolinoleucine10]SP shows dramatic loss of affinity and
potency. To analyze the structural implications of these
biological results, the conformational preferences of the
SP analogues were analyzed by NMR spectroscopy and
minimum-energy conformers of Ac-cis-3-prolinoleucine-
NHMe, Ac-trans-3-prolinoleucine-NHMe and model
dipeptides were generated by molecular mechanics calcula-
tions. From NMR and modeling studies it can be proposed
that residue Leu10 of SP adopts a gauche(+) conformation

around the v
1
angle and a trans conformation around the v
2
angle in the bioactive conformation. Together with previ-
ously published results, our data indicate that the C-terminal
SP tripeptide should preferentially adopt an extended con-
formation or a PPII helical structure when bound to the
receptor.
Keywords: substance P; NK-1 receptor; bioactive confor-
mation; prolinoleucine.
The introduction of a cyclic structure into a polypeptide
greatly limits the inherent flexibilities of the peptide
backbone and side chains. Disulfide bridging, lactam
cyclization and substitution by proline residues are the
easiest and therefore the most frequently used strategies.
These constraints are introduced to probe the conformation
of the substituted residue(s) and/or to generate more specific
peptide analogues with high affinities (due to reduction of
entropy). Proline, the only ÔnaturalÕ cyclic amino acid, has a
restricted F-value of around )60° that constrains the
peptide backbone. When inserted into a peptide sequence, a
biologically potent proline-substituted analogue of the
initial peptide gives information on both the F-value of
the substituted residue and the nonimportance of its side
chain. If, in contrast, this side chain is mandatory for full
biological potency, substituted proline analogues on Cb
(position 3), Cc (position 4) or Cd (position 5) may restore
the information carried by the side chain. These proline
analogues may be used to probe both the orientation of

the peptide backbone (/, w) and side chain conformation
[v
1
gauche(+) ¼ ) 60°, trans ¼ 180° and gauche(–) ¼ 60°].
Proline has been widely used as a scaffold and substitu-
tions on the pyrrolidine ring have yielded a large variety of
proline analogues [1–19]. Such constrained templates have
been introduced in peptides to elucidate their bioactive
conformation: for instance, 3-methylthiomethylproline
(3-prolinomethionine) in substance P (SP) [20], 3-(p-hy-
droxyphenyl)proline (3-prolinotyrosine) in opioid peptides
[21] as well as 3-n-propylproline [22], 3- and 4-alkylthio-
prolines [23] in cholecystokinin analogues.
Most of cis-andtrans-3-substituted prolinoamino acids
(P
c
3
aa and P
t
3
aa, Fig. 1) bearing a side chain of a natural
amino acid can be prepared. These prolinoamino acids
chimera may constitute valuable conformational tools
assuming that their preferred three-dimensional structures
overlap some, if not all, canonical (helical, extended)
structures of a-amino acids. Figure 2 shows the dependence
of v
1
torsion angle upon the ring pucker and the cis/trans
diastereoisomerism. In cis-prolinoamino acids, the Cc-endo

ring pucker corresponds to a v
1
around 150° (assimilated to
the trans rotamer) and the Cc-exo ring pucker is associated
with v
1
around 90° [assimilated to the gauche(–) rotamer].
On the contrary, Cc-endo and Cc-exo ring puckers of trans-
prolinoamino acids are related to gauche(+) (v
1
 )90°)
and trans rotamers (v
1
 )150°), respectively. Thus, the
Correspondence to O. Lequin, UMR 7613 Paris 6-CNRS, Structure
et fonction de mole
´
cules bioactives, Case courrier 45, Universite
´
P. et M. Curie, 4, Place Jussieu, 75252 Paris cedex 05, France.
Fax: + 33 1 44 273115, Tel.: + 33 1 44 273843,
E-mail:
Abbreviations:Boc,(tert-butyloxy)carbonyl; CHO, Chinese hamster
ovary; CSD, chemical shift deviation; HBTU, O-benzotriazol-1-yl-
N,N,N¢,N¢-tetramethyluronium hexafluorophosphate; IP, inositol
phosphate; P
3
aa, prolinoamino acid; PLC, phospholipase C; P
c
3

Leu,
cis-3-prolinoleucine; P
t
3
Leu, trans-3-prolinoleucine; P
c
3
Met, cis-3-
prolinomethionine; P
t
3
Met, trans-3-prolinomethionine; PtdIns, phos-
phatidyl inositol; SP, substance P (H-Arg-Pro-Lys-Pro-Gln-Gln-
Phe-Phe-Gly-Leu-Met-NH
2
); SPPS, solid phase peptide synthesis.
(Received 26 February 2003, revised 29 April 2003,
accepted 13 May 2003)
Eur. J. Biochem. 270, 2869–2878 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03665.x
cyclic constraint excludes one v
1
gauche rotamer,
gauche(+) for cis-prolinoamino acids and gauche(–) for
trans-prolinoamino acids, respectively.
The C-terminal conformation of SP (H-Arg-Pro-Lys-
Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH
2
) has been probed
with proline or prolinoamino acids chimera. The biological
activities on the tachykinin NK-1 receptor of [Pro9]SP,

[Pro10]SP, [Pro11]SP and both [P
c
3
Met11]SP and
[P
t
3
Met11]SP have been previously reported [20,24]. In the
case of prolinomethionine incorporation, we have shown
that both [P
c
3
Met11]SP and [P
t
3
Met11]SP are equipotent to
SP, indicating that the v
1
angle of Met11 should be trans.
We now report the incorporation of prolinoleucines at
position 10 of SP. Indeed, the side chain of Leu10 is critical
for NK-1 receptor recognition and agonist activity [25,26].
In contrast to prolinomethionines, only [P
t
3
Leu10]SP reta-
ins affinity and potency towards the NK-1 receptor. The
structural implications of these results on the bioactive
conformation of Leu10 and the C-terminal tripeptide of SP
are analyzed by NMR spectroscopy on SP analogues and

molecular mechanics calculations on model peptides.
Experimental procedures
Materials
[11-
3
H][Pro9]SP (65 CiÆmmol
)1
) was synthesized at Com-
missariat a
`
l’Energie Atomique (Saclay, France) according
to Chassaing et al.[27].[
3
H]propionyl[Met(O
2
)11]SP(7–11)
(95 CiÆmmol
)1
) was synthesized as described previously [28].
Boc-P
t
3
Leu and Boc-P
c
3
Leu were synthesized as described
elsewhere [19].
Peptide synthesis
[P
c

3
Leu10]SP and [P
t
3
Leu10]SP syntheses were carried out
on an ABI Model 431 A peptide synthesizer starting from
an a-p-methylbenzhydrylamine (MBHA resin, substitution:
0.9 mmolÆg
)1
of resin). All Na-Boc-amino acids, in a five- or
10-fold excess, were assembled using N,N¢-dicyclohexylcar-
bodiimide and 1-hydroxybenzotriazole as coupling reagents
and HBTU for P
c
3
Leu and P
t
3
Leu. The residues Na-Boc-
Met, Na-Boc-P
c
3
Leu or Na-Boc-P
t
3
Leu, and Na-Boc-Gly
were coupled manually to the resin. In order to obtain a
lower degree of substitution of the resin, 0.5 mmol of
Na-Boc-Met per gram of resin was used for Na-Boc-Met
coupling, yielding after acetylation (acetic anhydride/

dichloromethane, 1 : 5), a substitution of around
0.25 mmolÆg
)1
of resin for the C-terminal amino acid, as
determined by the Gisin test after Boc-deprotection [29].
The syntheses were then carried out on a 0.06-mmol scale.
After removal of the last Na-Boc-protecting group, the resin
was dried in vacuo. The peptide resin was transferred into
the Teflon vessel of an HF apparatus and the peptide was
cleaved from the resin by treatment with 1.5 mL of anisole,
0.25 mL of dimethyl sulfide, and 10 mL of anhydrous HF
per gram of peptide-resin for 1 h at 0 °C. After lyophiliza-
tion of the extract, the crude peptide was purified by
preparative reverse phase HPLC. The separation was
accomplished using various acetonitrile gradients in aque-
ous 0.1% trifluoroacetic acid at a flow rate of 6 mLÆmin
)1
with UV detection fixed at 220 nm. Before pooling, the
purity of collected fractions was verified by analytical
HPLC in isocratic mode at a flow rate of 1.5 mLÆmin
)1
with
UV detection fixed at 220 nm. Mass spectral analysis was
Fig. 1. Schematic representation of 3-prolinoamino acids and descrip-
tion of torsion angles. The nomenclature used for proteins side chain
rotamers has been adopted.
Fig. 2. Structures showing the two ring puckers and the associated side
chain conformers of prolinoamino acids.
2870 J. Quancard et al. (Eur. J. Biochem. 270) Ó FEBS 2003
performed by MALDI-TOF. The calculated mass for both

[P
c
3
Leu10]SP and [P
t
3
Leu10]SP is (MH
+
) ¼ 1370.70;
found mass: [P
c
3
Leu10]SP (MH
+
) ¼ 1373.76, [P
t
3
Leu10]SP
(MH
+
) ¼ 1373.61.
Cell culture
CHO cells expressing human NK-1 receptors were cultured
in DMEM medium supplemented with 100 IUÆmL
)1
peni-
cillin, 100 IUÆmL
)1
streptomycin, and 10% fetal bovine
serum. Cultures were kept at 37 °C in a humidified

atmosphereof5%CO
2
. Stable transfections were main-
tained by geneticin periodic selection.
Binding assays on CHO cells
Binding assays were carried out at 22 °C on whole cells in
Krebs–Ringer phosphate solution consisting of 120 m
M
NaCl, 4.8 m
M
KCl, 1.2 m
M
CaCl
2
,1.2m
M
MgSO
4
,and
15.6 m
M
NaH
2
PO
4
, pH 7.2, containing 0.04% bovine
serum albumin (w/v), 0.6% glucose (w/v), 1 m
M
phenyl-
methylsulfonyl fluoride, and 1 lgÆmL

)1
soybean trypsin
inhibitor, as described [28].
Measurements of inositol phosphate and cAMP
formation
PI hydrolysis and cAMP accumulation were determined as
described previously [30].
NMR spectroscopy
Lyophilized peptides were dissolved in 550 lL of methanol
(C
2
H
3
OH or C
2
H
3
O
2
H) at concentrations around 4 m
M
.
NMR experiments were recorded on Bruker Avance
spectrometers at a
1
H frequency of 500 MHz and were
processed with
XWIN
-
NMR

software, as described previously
[31]. Spectra were acquired at temperatures from 278 to
298 K. Solvent suppression was achieved by presaturation
during the relaxation delay or with a Watergate sequence
[32]. Proton assignments were obtained from the analysis of
TOCSY (20 and 95 ms isotropic mixing times) [33] and
NOESY experiments (400 ms mixing time) [34].
3
J
HNH
a
and
3
J
H
a
H
b
coupling constants were measured on 1D spectra
acquired with 16K data points and a spectral width of
5000 Hz. The chemical shift deviations of Ha protons were
calculated using random coil values reported in methanol
[35].
1
H assignments of [P
c
3
Leu10]SP and [P
t
3

Leu10]SP are
available as supplementary material.
Molecular mechanics calculations
Ac-P
c
3
Leu-NHMe, Ac-P
t
3
Leu-NHMe, Ac-Leu-NHMe,
Ac-Gly-Leu-NHMe, Ac-Gly-Pro-NHMe, Ac-Gly-P
c
3
Leu-
NHMe,Ac-Gly-P
t
3
Leu-NHMe,Ac-Leu-Met-NH
2
,Ac-Leu-
Pro-NH
2
,Ac-Leu-P
c
3
Met-NH
2
and Ac-Leu-P
t
3

Met-NH
2
were built using InsightII package (Accelrys Inc.). All
peptide bonds were fixed in a trans conformation. Mole-
cular mechanics calculations were performed with the
Discover program and CFF91 forcefield. The electrostatic
potential was calculated in vacuo with a distance-dependent
dielectric screening of 4Ær. One hundred to 1000 structures
were generated by molecular dynamics at 1000 K, saving
snapshots every 2 ps. Each structure was then minimized
using steepest descent, conjugate gradient and Newton–
Raphson algorithms until the gradient was less than
0.001 kcalÆmol
)1
Æ
A
˚
)1
.
NMR structures of SP analogues were calculated in
DISCOVER
by restrained molecular dynamics as described
previously [31].
Results
Peptide synthesis of [P
3
Leu10]SP analogues
The coupling of the bulky amino acids P
c
3

Leu and P
t
3
Leu
was inefficient using the standard N,N¢-dicyclohexylcarbo-
diimide/1-hydroxybenzotriazole procedure and required
HBTU as coupling reagent. Consequently, to ascertain the
quality of the coupling, the three C-terminal residues were
coupled manually. Using this strategy, [P
c
3
Leu10]SP and
[P
t
3
Leu10]SP were obtained with purities and yields similar
to SP and [Pro10]SP [36].
Pharmacology of the [P
3
Leu10]SP analogues
The affinities of [P
3
Leu10]SP analogues for the two specific
binding sites associated with the human NK-1 receptor,
NK-1M and NK-1m, have been measured with transfected
CHO cells. The more abundant binding site NK-1M (85%)
is labeled by
3
H[Pro9]SP and is coupled to cAMP produc-
tion, whereas the less abundant binding site NK-1m (15%)

is labeled by
3
H-propionyl[Met(O
2
)11]SP(7–11) and is
associated with IPs production [20,28]. The binding affinit-
ies and agonist potencies of the SP analogues are expressed
as K
i
for NK-1M (major site) and NK-1m (minor site), and
EC
50
values for the cAMP and IPs pathways, and are
reported in Table 1 [20,28]. The affinity of the trans
analogue [P
t
3
Leu10]SP is similar to that of SP and [Pro9]SP
at both binding sites. The EC
50
values of this analogue to
stimulate the cAMP and PLC pathways are in good
agreement with the expected theoretical values, calculated
from previously established correlations between K
i
and
EC
50
[20]. In contrast, the cis isomer [P
c

3
Leu10]SP is a very
weak competitor with high K
i
values for NK-1M and
NK-1m specific binding sites. Its efficacy is lower than that
of SP on both second messenger responses. [P
c
3
Leu10]SP is
a partial agonist (pK
B
5.12) on the cAMP pathway while no
antagonist activity could be detected with this analogue on
IPs production. Surprisingly, the K
i
values of both
[P
c
3
Leu10]SP and [P
t
3
Leu10]SP for NK-1M and NK-1m
specific binding sites are in the same range whereas agonists
generally have K
i
values that differ at least by one order of
magnitude between the two binding sites.
NMR spectroscopy of SP analogues

Due to its inherent flexibility, SP is largely unstructured in
water but folded conformers are stabilized in lower dielectric
constant solvents such as methanol and in lipid environ-
ments [31,37,38]. The stabilized conformations in these
environments are in agreement with the current state of
knowledge on the postulated bioactive conformation of SP
[24]. To analyze the structural effects of a P
3
Leu insertion in
Ó FEBS 2003 Insertion of prolinoleucines in substance P (Eur. J. Biochem. 270) 2871
the sequence of SP, the conformations of [P
c
3
Leu10]SP and
[P
t
3
Leu10]SP have been studied by NMR spectroscopy in
methanol.
Cis/trans isomerism. Weak additional resonances could be
observed in the spectra of SP analogues. These minor forms
involve spin systems in the N-terminus and are likely due to
cis/trans isomerism of peptide bonds preceding Pro2 and/or
Pro4. However the proline isomerism was not further
analyzed, as the proportions of these forms were too weak
to give rise to NOE crosspeaks. In all peptides, the major
form corresponds to a trans conformation of all peptide
bonds, as shown by ad(i)1, i) sequential connectivities for
residues Pro2, Pro4, P
c

3
Leu10 and P
t
3
Leu10. The propor-
tion of minor species remains very low (<5%) except in the
case of [P
c
3
Leu10]SP, for which an additional minor form is
more significantly populated ( 15%). The observation of
an NOE correlation between Ha Gly9 and Ha P
c
3
Leu10 in
this minor form demonstrates that the peptide bond
between Gly9 and P
c
3
Leu10 adopts a cis conformation.
Conformational analysis of prolinoamino acid residues.
The conformation of the pyrrolidine ring was analysed
using the
3
J
H
a
–H
b
coupling constant and intraresidual NOEs.

In P
t
3
Leu, the two ring puckers are characterized
by divergent
3
J
H
a
–H
b
coupling constants (10 Hz for the
Cc-exo form,  1.5HzfortheCc-endo form, using a
Karplus relationship [39]). The
3
J
H
a
–H
b
coupling constant of
P
t
3
Leu10 is 7.0 Hz, indicating that there is a conformational
equilibrium between the two puckerings. Based on
3
J
H
a

–Hb
coupling constant, the proportion of Cc-exo (v
1
trans)
conformers can be estimated to be 70% for P
t
3
Leu. NMR
analysis of [P
t
3
Met11]SP gave a
3
J
H
a
–H
b
coupling constant of
5.2 Hz (unpublished data) which corresponds to 50% of
Cc-exo conformers. These data, together with that reported
for Ac-trans-3-MePro-NHMe (
3
J
H
a
–H
b
3.2 Hz in chloro-
form) [40], suggest that the equilibrium is shifted toward

the Cc-exo form (v
1
trans) on increasing the bulkiness of
the b-substituent on the pyrrolidine ring (3-MePro
<P
t
3
Met < P
t
3
Leu). In cis-prolinoleucine, the two ring
puckers are characterized by close
3
J
H
a
–H
b
coupling con-
stants ( 6–8 Hz), which therefore cannot be used for the
conformational analysis. However the two ring puckers
differ by their intraresidual Ha–Hc and Hb–Hd NOEs.
In [P
c
3
Leu10]SP, the prolinoamino acid exhibits a strong
Hb-Hd NOE and no Ha–Hc NOE, showing that the
Cc-endo form (v
1
trans) predominates.

Secondary structure. The chemical shift deviations of Ha
protons, which depend on the secondary structure of the
peptide backbone [41], are reported in Fig. 3. SP,
[P
c
3
Leu10]SP and [P
t
3
Leu10]SP are characterized by upfield
shifts of Ha protons in the 4–7 region, indicative of helical
conformations. This helical structure is further supported by
the presence of characteristic aN(i, i +3) and aN(i,i+4)
medium-range NOEs and small coupling constants for
residues Gln5 and Gln6 (<6.3 Hz). The incorporation of
a prolinoamino acid in position 10 has no major effect on
the Ha CSD of preceding residues.
Three-dimensional structure. The structures of the two SP
analogues have been calculated by restrained molecular
dynamics. They are similar to that of SP in segment 1–7 and
are only slightly different in the C-terminus (Fig. 4).
Segment 1–3 is found to adopt an extended conformation
whereas residues 4–8 form a helical structure. The confor-
mations of residues 8–9 are ill defined in the peptides. Thus,
the introduction of a prolinoamino acid in the C-terminal
tail of SP does not affect the overall structure of the peptide
backbone and in particular of the core helical region.
Molecular modeling
Conformations of prolinoleucine. Minimum-energy con-
formations of Ac-P

c
3
Leu-NHMe, Ac-P
t
3
Leu-NHMe and
Ac-Leu-NHMe were generated by molecular mechanics
using CFF91 forcefield and results are reported in Tables 2
and 3. The same calculations were performed using
AMBER
forcefield and yielded similar results, which are not shown
here.
Table 1. Affinities and activities of cis-andtrans-3-prolinoamino acid-substituted analogues of SP. The affinities of SP and SP analogues were
measured for the NK-1M (labeled with
3
H[Pro9]SP) and the NK-1m (labeled with
3
H-propionyl[Met(O
2
)11]SP(7–11) binding sites in CHO cells
expressing the human NK-1 receptor, as well as their related potencies to stimulate adenylate cyclase and phospholipase C. The antagonist potency
value, pK
B
, is obtained from pK
B
¼ log(DR–1)–log[B].DRisthedose-ratio[A]¢/[A] where [A]¢ is the concentration of agonist A, in the presence
of the antagonist B, which is equiactive to [A], the concentration of agonist in the absence of antagonist. Values presented are the mean ± SE of
at least three independent experiments run in triplicate.
Peptide
K

i
, NK-1M
(n
M
)
EC
50
, adenylate cyclase
(n
M
)
K
i
, NK-1m
(n
M
)
EC
50
, phospholipase C
(n
M
)
SP
a
1.6 ± 0.4 8 ± 2 0.13 ± 0.02 1.0 ± 0.2
[Pro9]SP
a
1.1 ± 0.1 10 ± 2 0.13 ± 0.02 0.7 ± 0.1
Propionyl[Met(O

2
)
11
]SP(7–11)
a
1900 ± 450 >5000 10 ± 2 >5000
[P
c
3
Leu10]SP 465 ± 105 >10 000 220 ± 30 260 ± 30
12 ± 3% at 10
)5
M
(49 ± 3%)
pK
B
¼ 5.12 ± 0.06 No antagonism
[P
t
3
Leu10]SP 0.86 ± 0.07 39 ± 9 0.34 ± 0.09 2.1 ± 0.3
[P
c
3
Met11]SP
b
2.1 ± 0.1 35 ± 1 0.08 ± 0.005 0.8 ± 0.1
[P
t
3

Met11]SP
b
3.0 ± 0.5 25 ± 2 0.10 ± 0.007 1.4 ± 0.3
a,b
Results already published in [26] and [20], respectively.
2872 J. Quancard et al. (Eur. J. Biochem. 270) Ó FEBS 2003
The relative energies of ring puckers of P
c
3
Leu and
P
t
3
Leu indicate that the isopropyl substituent destabilizes
the Cc-exo conformer [v
1
gauche(–)] in Ac-P
c
3
Leu-NHMe
and the Cc-endo conformer [v
1
gauche(+)] in Ac-P
t
3
Leu-
NHMe(Tables2and3).Theincreaseoftheenergy
difference between the v
1
gauche(–) and trans rotamers is

particularly important with Ac-P
c
3
Leu-NHMe. Because
of the constraint imposed by the pyrrolidine cycle, the /
torsion angle of these prolinoamino acids is restrained to
values between )95° and )60°,theCc-endo pucker being
associated with more negative values of / torsion angle
(Tables 2 and 3), as previously reported for proline and
proline derivatives [42]. The lower limit values of / ( )90°)
are related to minimum-energy conformers corresponding
to reverse c-turn, for which the puckering is always Cc-endo.
Although structures corresponding to w  )40° (helical)
and w  90° (reverse c-turn) are found, the most stable
conformations correspond to extended structures with w
torsion angle between 110° and 165°.Thew angles of
Ac-P
t
3
Leu-NHMe are smaller for Cc-exo puckering than
for Cc-endo puckering, as observed for proline [42]. The
opposite effect is observed for Ac-P
c
3
Leu-NHMe, for which
the cis isopropyl substituent constrains more the w torsion
angle. As previously reported [40], reverse c-turn is desta-
bilized for cis-prolinoamino acids due to steric interactions
between the b-substituent and the carbonyl oxygen.
Low-energy conformations of all model amino acids are

shown in (v
1
, v
2
) maps (Fig. 5). We fixed a limit of
3kcalÆmol
)1
to the energy difference (relative to the lowest-
energy conformer) for an acceptable structure. Relative
energy values of side chain conformations are in agreement
with classical (v
1
, v
2
) maps for Leu in proteins [43]. The
conformational space of the prolinoleucines side chain
appears to be more limited than that of the parent leucine
amino acid. Among the nine (v
1
, v
2
) conformers of Leu, the
most stable conformers are (t, g
–
)and(g
+
, t). The three
(v
1
, v

2
) conformers corresponding to a trans v
1
, i.e. (t, t)
(t, g
–
)(t,g
+
), are found in both Ac-P
c
3
Leu-NHMe and
Ac-P
t
3
Leu-NHMe. But only one v
1
gauche(+) rotamer
is acceptable for Ac-P
t
3
Leu-NHMe corresponding to a
Fig. 3. Ha chemical shift deviations of SP,
[P
t
3
Leu10]SP and [P
c
3
Leu10]SP analogues.

The chemical shift deviations (CSDs) of Ha
protons are calculated as the differences
between observed chemical shifts and random
coil values determined in methanol [35]. The
random coil value of Pro was used for the
different prolinoamino acids. Therefore, the
CSDs of P
3
Leu10 depend primarily on the
chemical modification of the pyrrolidine ring
and cannot be used for the conformational
analysis.
Fig. 4. NMR structures of SP [31], [P
t
3
Leu10]SP and [P
c
3
Leu10]SP analogues in methanol. Residues 4–8 have been used for backbone superposition
of the selected conformers.
Ó FEBS 2003 Insertion of prolinoleucines in substance P (Eur. J. Biochem. 270) 2873
trans v
2
,andallv
1
gauche(–) rotamers are energetically
excluded in Ac-P
c
3
Leu-NHMe.

Superimposition of prolinoleucine and leucine. In order to
compare the conformational spaces of cis-andtrans-
prolinoleucine to that of leucine, minimum-energy struc-
tures were systematically superimposed. As expected, only
leucine conformations with / torsion angle around )80°
(proline-like) fit with prolinoleucines. Although the
geometrical constraint due to the cyclization induces a 30°
deviation of v
1
from ideal values, all conformers of
prolinoleucine acids fit well with the corresponding struc-
tures of natural amino acids (rmsd values between 0.024 and
0.06 nm for all heavy atoms).
Conformational effects of prolinoamino acids on the
preceding residue. Insertion of P
t
3
Leu in position 10 and
both P
c
3
Met and P
t
3
Met in position 11 [20] yields potent
agonists of NK-1 receptor. In order to analyze the effects
of a prolinoamino acid insertion on the backbone
Table 2. Relative energies (kcalÆmol
-1
)ofminimum-energyconformersofAc-P

t
3
Leu-NHMe and corresponding conformers of Ac-Leu-NH
2
that best
fit the prolinoamino acid. Conformers highlighted in bold characters have energies beyond 3 kcalÆmol
)1
of the global minimum. v
1
¢, torsion angle is
defined by Na,Ca,Cb,Cc¢ atoms of the pyrrolidine cycle. v
1
and v
2
torsion angles correspond to the side chain of the prolinoamino acid and are
defined by Na,Ca,Cb,Cc and Ca,Cb,Cc,Cd(proR) atoms.
Ac-P
t
3
Leu-NHMe
Ac-Leu-NHMe DE
a
w, v
1
, v
2
/wv
1
¢ v
1

v
2
DE
a,t,t -65 -57 -30 -157 177 0.98 2.61
a,t,g
–
-65 -41 -34 -164 60 0.78 1.29
a,t,g
+
-64 -54 -32 -161 -70 1.37 2.72
a,g
+
,t )76 )22 29 )95 173 3.79 1.71
a,g
+
,g
–
)71 )41 21 )106 61 4.48 2.62
a,g
+
,g
+
)70 )42 18 )107 )68 4.39 2.92
b,t,t -71 112 -29 -157 177 0.14 1.01
b,t,g
–
-66 147 -33 -163 59 0.00 0.22
b,t,g
+
-68 118 -31 -161 -68 0.63 1.34

b,g
+
,t - 75 162 30 -93 172 2.53 0.69
b,g
+
,g
–
)75 158 25 )102 61 3.41 1.47
b,g
+
,g
+
)74 158 24 )102 )68 3.38 1.76
c,g
+
,t - 92 78 32 -91 173 2.80 0.57
c,g
+
,g
–
)91 86 28 )98 60 3.36 1.47
c,g
+
,g
+
)92 86 28 )97 )69 3.38 1.76
a
The selected conformers of the amino acid are those that best fit the prolinoamino acid and are not necessarily described by the same
torsion angle values.
Table 3. Relative energies (kcalÆmol

-1
) of minimum-energy conformers of Ac-P
c
3
Leu-NHMe and corresponding conformers of Ac-Leu-NH
2
that best
fit the prolinoamino acid. Conformers highlighted in bold characters have energies beyond 3 kcalÆmol
)1
of the global minimum. v
1
¢, torsion angle is
defined by Na,Ca,Cb,Cc¢ atoms of the pyrrolidine cycle. v
1
and v
2
torsion angles correspond to the side chain of the prolinoamino acid and are
defined by Na,Ca,Cb,Cc and Ca,Cb,Cc,Cd(proR) atoms.
Ac-P
c
3
Leu-NHMe
w, v
1
, v
2
/wv
1
¢ v
1

v
2
DE Ac-Leu-NHMe DE
a
a,t,t )73 )37 30 164 161 3.39 2.61
a,t,g
–
-71 -47 28 159 62 1.29 1.29
a,t,g
+
-72 -44 30 165 -70 2.92 2.72
a,g
–
,t )67 )15 )33 93 170 4.90 3.47
a,g
–
,g
–
)64 )29 )36 87 82 5.72 3.49
a,g
–
,g
+
)64 )21 )34 91 )58 6.79 4.75
b,t,t -74 130 31 166 165 2.27 1.01
b,t,g
–
-80 111 30 161 57 0.00 0.22
b,t,g
+

-76 125 31 166 -70 1.71 1.34
b,g
–
,t )65 164 )34 92 171 3.41 1.91
b,g
–
,g
–
)62 151 )38 84 82 4.75 2.46
b,g
–
,g
+
)62 157 )36 88 )57 5.49 3.45
a
The selected conformers of the amino acid are those that best fit the prolinoamino acid and are not necessarily described by the same
torsion angle values.
2874 J. Quancard et al. (Eur. J. Biochem. 270) Ó FEBS 2003
conformation of the C-terminal tripeptide of SP, the
minimum-energy conformations of model peptides
Ac-Gly-Leu-NHMe, Ac-Gly-Pro-NHMe, Ac-Gly-P
c
3
Leu-
NHMe, Ac-Gly-P
t
3
Leu-NHMe, Ac-Leu-Met-NH
2
,Ac-

Leu-Pro-NH
2
,Ac-Leu-P
c
3
Met-NH
2
and Ac-Leu-P
t
3
Met-
NH
2
were generated (NHMe and NH
2
C-terminal cappings
were chosen to mimic the sequence of SP). Energy
differences between minimum structures corresponding to
helical (negative w) and extended (positive w) conformations
of the first residue are reported in Table 4. The helical
conformation of Leu is destabilized when this residue is
followed in the sequence by a proline or prolinomethionine
and there are no energy minima corresponding to c-turn
conformations. These effects can be ascribed to the steric
hindrance of the pyrrolidine cycle, as already reported
[44,45]. A b-substituent on the pyrrolidine cycle (Met side
chain) does not further affect the conformation of leucine.
In the case of Gly-Xaa dipeptides, the conformation effect
of the pyrrolidine cycle is much smaller on Gly residue and
does not significantly restrict its conformational space

beside the absence of c-turn minima.
Discussion
The nonsubstituted proline analogue [Pro11]SP is only a
weak competitor of SP binding sites. Reintroducing the
methionine side chain in the pyrrolidine ring led to
analogues [P
c
3
Met11]SP and [P
t
3
Met11]SP which are both
400- to 800-times more potent than [Pro11]SP, and therefore
equipotent to SP [20]. [Pro10]SP is only slightly less potent
than SP (10- to 30-times) [26]. The reintroduction of the
leucine side chain restores full potency to [P
t
3
Leu10]SP,
whereas [P
c
3
Leu10]SP is even less potent than [Pro10]SP,
being a very weak competitor of SP binding sites. To our
knowledge, this is the first time that such a selective
recognition is observed between trans and cis-3-substituted
prolinoamino acids. Thus, these analogues incorporating
the restricted prolinoleucine, with its isopropyl substituent,
representedanidealsituationtoascertainthevaluesforthe
(/, w, v

1
, v
2
) torsion angles of leucine in SP, assuming that
variations in the structure of SP were mainly restricted to the
substituted residue. NMR analysis and molecular modeling
studies were performed to reach these torsion angles.
Cis-andtrans-3-substituted prolines induce a local
constraint restricting the / and v
1
angles of the substituted
residue. The v
1
angle is restricted around two values, ± 150°
and ± 90° (+ for cis-andfortrans-prolinoamino acids).
Our NMR and modeling studies show that the v
1
trans
conformers are stabilized on increasing the bulkiness of the
b-substituent. The w angle of the prolinoamino acid residue
is also restrained. In particular, the introduction of a
b-substituent with a cis stereochemistry induces a destabil-
ization of reverse c-turn structures in cis-prolinoamino acids
[46], due to interactions with the following carboxamide
function. In solution, the global three-dimensional struc-
tures of SP incorporating either a cis-ortrans-3-substituted
proline are quite similar to that of SP except locally around
the substituted position. cis/trans isomerism of the Xaa-P
3
aa

amide bond is marginally affected by a C3 substitution of
the pyrrolidine ring. Indeed, only [P
c
3
Leu10]SP in metha-
nolic solution showed a slight increase in the concentration
of cis amide bond around Gly9-P
c
3
Leu10. This is in
agreement with previous studies on 3-alkylprolines [46].
This result justifies the trans orientation fixed to the amide
bond of the model compounds for molecular mechanics
calculations. Minimum-energy conformers of model dipep-
tides, including either a proline or a 3-substituted proline,
revealed slight conformational disturbances on the pre-
ceding (i)1) amino acid, this effect being related to the
proline constraint and not to the presence of a b-substituent
on the pyrrolidine ring. Thus, cis-andtrans-3-substituted
prolinoamino acids chimera constitute valuable tools to
probe relationships between the affinity/activity and the
conformation of the substituted residue, i.e. all its torsion
angles (peptidic backbone: /, w, and side chain conformer
v
1
, v
2
). Differences in receptor affinity of the modified
peptide vs. the initial one can be directly related to variations
in the structure of the substituted residue.

Fig. 5. v
1
–v
2
plots of minimum-energy conformations of Leu, P
t
3
Leu
and P
c
3
Leu. Structures with energies beyond 3 kcalÆmol
)1
of the global
minimum were excluded. For the sake of clarity, angle values are
traced between 0° and 360°, i.e. 360° was added to all negative angle
values. Gradual colors were used from blue (most stable conformers)
to red (less stable conformers).
Table 4. Energy differences between helical (w < 0) and extended
(w > 0) conformations of the first residue in model peptides.
Peptide DE (kcalÆmol
)1
)
Ac-Leu-NHMe 0.77
Ac-Leu-Met-NH
2
0.21
Ac-Leu-Pro-NH
2
3.39

Ac-Leu-P
t
3
Met-NH
2
3.31
Ac-Leu-P
c
3
Met-NH
2
2.70
Ac-Gly-NHMe 0.57
Ac-Gly-Leu-NHMe 0.05
Ac-Gly-Pro-NHMe 0.55
Ac-Gly-P
t
3
Leu-NHMe 0.45
Ac-Gly-P
c
3
Leu-NHMe 0.67
Ó FEBS 2003 Insertion of prolinoleucines in substance P (Eur. J. Biochem. 270) 2875
Only the trans isomer ([P
t
3
Leu10]SP) is active, with a
600-fold difference in affinity between [P
c

3
Leu10]SP and
[P
t
3
Leu10]SP. Consequently, only conformations specific to
P
t
3
Leu are liable to mimic the bioactive conformation of
Leu10 in SP. Molecular mechanics calculations and NMR
studies show that v
1
trans conformations are the most stable
in both P
c
3
Leu and P
t
3
Leu. Moreover, for these conforma-
tions, the three v
2
-rotamers have acceptable energies. These
results indicate that all v
1
trans conformations of Leu lie at
the intersection of the conformational spaces of P
c
3

Leu and
P
t
3
Leu. Thus, a v
1
trans canbeexcludedforthebioactive
conformation of Leu10. Furthermore, modeling studies
indicate that the only energetically acceptable (v
1
, v
2
)
conformer corresponds to the (g
+
,t)rotamer.Figure6
shows the superimposition of P
t
3
Leu and Leu in the
postulated bioactive conformation. NMR spectroscopy
shows that this conformer is observed in solution (propor-
tion of 30% in methanol). Therefore, the selective recogni-
tion of [P
t
3
Leu10]SP vs. [P
c
3
Leu10]SP by the NK-1 receptor

allows us to access to both v
1
and v
2
angles of the bioactive
conformation of Leu10. [Pro10]SP is 10 to 30 times less
potent than SP [24]. The reintroduction of the leucine side
chain was spectacular in terms of selectivity of cis-substitu-
tion vs. trans-substitution of the pyrrolidine ring, but the
active analogue [P
t
3
Leu10]SP was almost as potent as SP
and not a superagonist. One likely explanation might be
that the trans-prolinoleucine binds to the NK-1 receptor as a
higher-energy conformer (2.5 kcalÆmol
)1
) compared to the
corresponding conformer of leucine (0.7 kcalÆmol
)1
).
Together, we can conclude that the proline scaffold can
orientate both the peptidic backbone and the side chains of
Leu10 and Met11 residues in appropriate regions of the
space to be recognized by the NK-1 receptor. The torsion
angles of leucine are now more precisely defined, but the
variations still allowed around the w torsion angles of
glycine, leucine and methionine do not allow us to restrict
sufficiently the conformational space. Calculations per-
formed on Ac-Leu-P

c
3
Met-NH
2
and Ac-Leu-P
t
3
Met-NH
2
indicate that helical conformations of Leu are less stable
than b-conformations, with more than 2 kcalÆmol
)1
differ-
ence. Previous studies of SP analogues modified in positions
9 and 10 such as N-methylation or introduction of a
spirolactam, indicate that these residues should preferen-
tially adopt extended conformations [24,31,47]. All these
results suggest that the bioactive conformation of the
C-terminal tripeptide should be more or less extended
instead of helical as stated in some studies from NMR
analysis of SP in micellar medium [37,38,48–57]. Three
canonical structures correspond to extended structures: poly
c-turn, b-strand and PPII helical conformations. Biologic-
ally potent compounds obtained by substitution of the
residues 9, 10 or 11 with a proline or a prolinoamino acid
permit to exclude the poly c-turn structure. In particular,
Met11 should not adopt a c-turn conformation as
[P
c
3

Met11]SP is as potent as SP. Reverse c-turn conforma-
tions for Gly9 and Leu10 can also be excluded on the basis
of the calculations performed with model dipeptides. Other
constrained SP analogues will be necessary to discriminate
between a b-strand and a PPII helical conformation of the
crucial 9–11 tripeptide, which is necessary for the biological
activity of SP.
Conclusion
The comparison of conformational spaces of cis-andtrans-
prolinoamino acid allows one to access to / and v
1
angles.
Energy calculations can further restrict w and v
2
torsion
angles. In our study, the incorporation of both P
c
3
Leu and
P
t
3
Leuallowedustoaccesstoallthetorsionanglesof
Leu10 in the bioactive conformation of SP: /  )60°,
w  150°, v
1
 )60° and v
2
 180°. The use of both cis-
and trans-3-substituted prolines should become a general

tool to probe the bioactive conformation of peptides, and
applications of prolinoamino acids could be enlarged to
other biologically active peptides.
References
1. Chung, J.Y.L., Wasicak, J.T., Arnold, W.A., May, C.S., Nadzan,
A.M. & Holladay, M.W. (1990) Conformationally constrained
amino acids. Synthesis and optical resolution of 3-substituted
proline derivatives. J. Org. Chem. 55, 270–275.
2. Williams, R.M., Zhai, W., Aldous, D.J. & Aldous, S.C. (1992)
Asymmetric [1,3]-dipolar cycloaddition reactions: synthesis of
highly substituted proline derivatives. J. Org. Chem. 57, 6527–
6532.
3. Beak, P., Wu, S., Yum, E.K. & Jun, Y.M. (1994) Intramolecular
cyclizations of a-lithioamine synthetic equivalents: convenient
syntheses of 3-, 5-, and 6-membered-ring heterocyclic nitrogen
compounds and elaborations of 3-membered ring systems. J. Org.
Chem. 59, 276–277.
4. Herdeis, C., Hubmann, H.P. & Lotter, H. (1994) Synthesis of
homochiral 3-substituted glutamic acids and prolines from pyro-
glutamic acid. Tetrahedron Asymm. 5, 351–354.
5. Lavielle, S., Brunissen, A., Carruette, A., Garret, C. & Chassaing,
G. (1994) Highly potent substance P antagonists substituted with
b-phenyl- or b-benzyl-proline at position 10. Eur. J. Pharmacol.
258, 273–276.
Fig. 6. Superimposition of P
t
3
Leu and Leu in the postulated bioactive
conformation. Theselectedconformerscorrespondto(w, v
1

, v
2
) ¼
(b,g
+
, t). The rms deviation for heavy atoms is 0.041 nm.
2876 J. Quancard et al. (Eur. J. Biochem. 270) Ó FEBS 2003
6. Udding, J.H., Tuijp, J.M., Hiemstra, H. & Speckamp, W.N.
(1994) Transition metal-catalyzed chlorine transfer cyclizations of
carbon-centered glycine radicals; a novel synthetic route to cyclic
a-amino acids. Tetrahedron 50, 907–918.
7. Sharma,R.&Lubell,W.D.(1996)Regioselectiveenolizationand
alkylation of 4-oxo-N-(9-phenylfluoren-9-yl) proline: synthesis of
enantiopure proline-valine and hydroxyproline-valine chimeras.
J. Org. Chem. 61, 202–209.
8. Sasaki, N.A., Dockner, M., Chiaroni, A., Riche, C. & Potier, P.
(1997) A novel stereodivergent synthesis of optically pure cis-and
trans-3-substituted proline derivatives. J. Org. Chem. 62, 765–770.
9. Karoyan, P. & Chassaing, G. (1997) Asymmetric synthesis
of (2S,3S)- and (2S,3R)-3-prolinomethionines: 3-methylsulfa-
nylmethyl-pyrrolidine-2-carboxylic acids. Tet. Asymm. 8, 2025–
2032.
10. Karoyan, P., Triolo, A., Nannicini, R., Giannotti, D., Altamura,
M., Chassaing, G. & Perrotta, E. (1999) Solid-phase amino-zinc-
enolate cyclization for the synthesis of 3-substituted prolines.
Tetrahedron Lett. 40, 71–74.
11. Evans, M.C. & Johnson, R.L. (2000) Triproline analogues of Pro-
Leu-Gly-NH
2
with Pro/Leu and Pro/Phe chimeric amino acids in

position 2. Tetrahedron 56, 9801–9808.
12. Turner, S.C., Zhai, H. & Rapoport, H. (2000) Enantiospecific
synthesis of annulated nicotine analogues from
D
-and
L
-glutamic
acid pyridotropanes. J. Org. Chem. 65, 861–870.
13. Duan, S. & Moeller, K.D. (2001) Building constrained peptido-
mimetics: a convenient approach to 3-phenyl-5-vinyl substituted
proline derivatives. Tetrahedron 57, 6407–6415.
14.Kamenecka,T.M.,Park,Y J.,Lin,L.S.,Lanza,T.J.&
Hagmann, K. (2001) Enantioselective approach to 3-substituted
prolines. Tetrahedron Lett. 42, 8571–8573.
15. Basak, A., Bag, S.S., Rudra, K.R., Barman, J. & Dutta, S. (2002)
Diastereoselective synthesis of 4-substituted
L
-prolines by
intramolecular radical cyclization of N-aryl sulphonyl-N-allyl
3-bromoalanines. Chem. Lett. 7, 710–711.
16. Karoyan, P. & Chassaing, G. (2002) Short asymmetric synthesis of
(2S,3S)- and (2S,3R)-3-prolinoglutamic acids: 2-carboxy-3-pyrro-
lidine-acetic acids (CPAA). Tetrahedron Lett. 43, 253–255.
17. Karoyan, P. & Chassaing, G. (2002) Asymmetric synthesis of
3-alkyl substituted prolines by alkylation of a chiral sulfone.
Tetrahedron Lett. 43, 1221–1223.
18. Pellegrini, N., Schmitt, M., Guery, S. & Bourguignon, J.J. (2002)
New strategies towards proline derivatives as conformationally
constrained arginine analogues. Tetrahedron Lett. 43, 3243–3246.
19. Karoyan, P., Quancard, J., Vaissermann, J. & Chassaing, G.

(2003) Amino-zinc-enolate carbometalation reactions: application
to ring closure of terminally substituted olefin for the asymmetric
synthesis of cis-andtrans-3-prolinoleucine. J. Org. Chem. 68,
2256–2265.
20. Sagan, S., Karoyan, P., Chassaing, G. & Lavielle, S. (1999) Fur-
ther delineation of the two binding sites (R.*
n
) associated with
tachykinin neurokinin-1 receptors using [3-prolinomethio-
nine
11
]SP analogues. J. Biol. Chem. 274, 23770–23776.
21. Mosberg, H.I., Lomize, A.L., Wang, C., Kroona, H., Heyl, D.L.,
Sobczyk-Kojiro, K., Ma, W., Mousigian, C. & Porreca, F. (1994)
Developmentofamodelforthed opioid receptor pharmaco-
phore. 1. Conformationally restricted Tyr l replacements in the
cyclic delta receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-
Pen]OH (JOM-13). J. Med. Chem. 37, 4371–4383.
22. Holladay, M.W., Lin, C.W., May, C.S., Garvey, D.S., Witte,
D.G., Miller, T.R., Wolfram, C.A.W. & Nadzan, A.M. (1991)
Dual antagonists of platelet activating factor and histamine:
identification of structural requirements for dual activity of
N-acyl-4-(5,6-dihydro-11H-benzo[5,6]cyclohepta-[1,2-b]pyri-
din-11-yliden) piperidins. J. Med. Chem. 34, 457–461.
23. Kolodziej, S.A., Nikiforovich, G.V., Skeean, R., Lignon, M F.,
Martinez, J. & Marshall, G.R. (1995) Ac-[3- and 4-alkylthiopro-
line31]-CCK4 analogs: synthesis and implications for the CCK-B
receptor-bound conformation. J. Med. Chem. 38, 137–149.
24. Lavielle, S., Chassaing, G., Ploux, O., Loeuillet, D., Besseyre, J.,
Julien, S., Marquet, A., Convert, O., Beaujouan, J C., Torrens,

Y., Bergstro
¨
m, L., Saffroy, M. & Glowinski, J. (1988) Analysis of
tachykinin binding site interactions using constrained analogues of
tachykinins. Biochem. Pharmacol. 37, 41–49.
25. Lavielle, S., Chassaing, G., Brunissen, A., Rodriguez, M., Marti-
nez, J., Convert, O., Carruette, A., Garret, C., Petitet, F., Saffroy,
M., Torrens, Y., Beaujouan, J C. & Glowinski, J. (1993)
Importance of the leucine side-chain to the spasmogenic activity
and binding of substance P analogues. Int. J. Peptide Protein Res.
42, 270–277.
26. Sagan, S., Milcent, T., Ponsinet, R., Convert, O., Tasseau, O.,
Chassaing, G., Lavielle, S. & Lequin, O. (2003) Structural and
biological effects of a b
2
-orb
3
-amino acid insertion in a pep-
tide: application to molecular recognition of substance P by the
neurokinin-1 receptor. Eur. J. Biochem. 270, 939–949.
27. Chassaing, G., Lavielle, S., Julien, S. & Marquet, A. (1985)
Homocysteine thiolactone as precursor of methionine amide:
application to the modification of peptides of the tachykinin
family. Tetrahedron Lett. 26, 623–626.
28. Sagan, S., Beaujouan, J C., Torrens, Y., Saffroy, M., Chassaing,
G., Glowinski, J. & Lavielle, S. (1997) High affinity binding of
[
3
H]propionyl-[Met(O
2

)
11
]substance P (7–11), a tritiated septide-
like peptide, in chinese hamster ovary cells expressing human
neurokinin 1 receptors and in rat submandibular glands. Mol.
Pharmacol. 52, 120–127.
29. Gisin, B.F. (1972) Monitoring of reactions in solid-phase peptide
synthesis with picric acid. Anal. Chem. Acta. 58, 248–249.
30. Sagan, S., Chassaing, G., Pradier, L. & Lavielle, S. (1996) Tack-
ykinin peptides affect differently the second messenger pathways
after binding to CHO-expressed human NK 1 receptors.
J. Pharmacol. Exp. Ther. 276, 1039–1048.
31. Sagan, S., Lequin, O., Frank, F., Convert, O., Ayoub, M., Lavi-
elle, S. & Chassaing, G. (2001) C
a
methylation in molecular
recognition: application to substance P and the two neurokinin-1
receptor binding sites. Eur. J. Biochem. 268, 2997–3005.
32. Piotto, M., Saudek, V. & Sklenar, V. (1992) Gradient-tailored
excitation for single-quantum NMR spectroscopy of aqueous
solutions. J. Biomol. NMR 2, 661–665.
33. Bax, A. & Davis, D.G. (1985) MLEV-17-based two-dimensional
homonuclear magnetization transfer spectroscopy. J. Magn.
Reson. 65, 355–360.
34. Kumar, A., Ernst, R.R. & Wu
¨
thrich, K. (1980) A two-dimen-
sional nuclear Overhauser enhancement (2D NOE) experiment for
the elucidation of complete proton–proton cross-relaxation net-
works in biological macromolecules. Biochem. Biophys. Res.

Commun. 95, 1–6.
35. Berlose, J.P., Convert, O., Brunissen, A., Chassaing, G. &
Lavielle, S. (1994) Three-dimensional structure of the highly
conserved seventh transmembrane domain of G-protein-coupled
receptors. Eur. J. Biochem. 225, 827–843.
36. Sachon, E., Girault-Lagrange, S., Chassaing, G., Lavielle, S. &
Sagan, S. (2002) Analogs of substance P modified at the C-ter-
minus which are both agonist and antagonist of the NK-1 receptor
depending on the second messenger pathway. J. Pept. Res. 59,
232–240.
37. Chassaing, G., Convert, O. & Lavielle, S. (1986) Preferential
conformation of substance P in solution. Eur. J. Biochem. 154,
77–85.
38. Keire, D.A. & Fletcher, T.G. (1996) The conformation of sub-
stance P in lipid environments. Biophys. J. 70, 1716–1727.
Ó FEBS 2003 Insertion of prolinoleucines in substance P (Eur. J. Biochem. 270) 2877
39. Haasnoot, C.A.G., De Leeuw, F.A.A.M. & Altona, C. (1980) The
relationship between proton-proton NMR coupling constants and
substituents electronegativities-I. Tetrahedron 36, 2783–2792.
40. Delaney, N.G. & Madison, V. (1982) Novel conformational
distributions of methylproline peptides. J. Am. Chem. Soc. 104,
6635–6641.
41. Wishart, D.S., Sykes, B.D. & Richards, F.M. (1991) Relationship
between nuclear magnetic resonance chemical shift and protein
secondary structure. J. Mol. Biol. 222, 311–333.
42. Improta, R., Benzi, C. & Barone, V. (2001) Understanding the role
of stereoelectronic effects in determining collagen stability: 1. A
quantum mechanical study of proline, hydroxyproline, and
fluoroproline dipeptide analogues in aqueous solution. J. Am.
Chem. Soc. 123, 12568–12577.

43. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton,
J.M. (1993) Procheck: a program to check the stereochemical
quality of protein structures. J. Appl. Cryst. 26, 283–291.
44. MacArthur,M.W.&Thornton,J.M.(1991)Influenceofproline
residues on protein conformation. J. Mol. Biol. 218, 397–412.
45. Hurley, J.H., Mason, D.A. & Matthews, B.W. (1992) Flexible-
geometry conformational energy maps for the amino acid residue
preceding a proline. Biopolymers 32, 1443–1446.
46. Beausoleil, E., Sharma, R., Michnick, S.W. & Lubell, W.D. (1998)
Alkyl 3-position substituents retard the isomerization of prolyl
and hydroxylprolyl amides in water. J. Org. Chem. 63, 6572–6578.
47. Ward, P., Ewan, G.B., Jordan, C.C., Ireland, S.J., Hagan, R.M. &
Brown, J.R. (1990) Potent and highly selective neurokinin
antagonists. J. Med. Chem. 33, 1848–1851.
48. Sumner, S.C., Gallagher, K.S., Davis, D.G., Covell, D.G., Jerni-
gan, R.L. & Ferretti, J.A. (1990) Conformational analysis of the
tachykinins in solution: substance P and physalaemin. J. Biomol.
Struct. Dyn. 8, 687–707.
49. Grdadolnik, S.G., Mierke, D.F., Byk, G., Zeltser, I., Gilon, C. &
Kessler, H. (1994) Comparison of the conformation of active and
nonactive backbone cyclic analogues of substance P as a tool
to elucidate features of the bioactive conformation: NMR and
molecular dynamics in DMSO and water. J. Med. Chem. 37,
2145–2152.
50. Young, J.K., Anklin, C. & Hicks, R.P. (1994) NMR and
molecular modeling investigations of the neuropeptide substance
P in the presence of 15 m
M
sodium dodecyl sulfate micelles.
Biopolymers 34, 1449–1462.

51. Keire, D.A. & Kobayashi, M. (1998) The orientation and
dynamics of substance P in lipid environments. Protein Sci. 7,
2438–2450.
52. Cowsik, S.M., Lucke, C. & Ruterjans, H. (1997) Lipid-induced
conformation of substance P. J. Biomol. Struct. Dyn. 15, 27–36.
53. Wymore,T.&Wong,T.C.(1999)Moleculardynamicsstudyof
substance P peptides in a biphasic membrane mimic. Biophys.
J. 76, 1199–1212.
54. Wymore,T.&Wong,T.C.(1999)Moleculardynamicsstudyof
substance P peptides partitioned in a sodium dodecylsulfate
micelle. Biophys. J. 76, 1213–1227.
55. Lee, S., Suh, Y.H., Kim, S. & Kim, Y. (1999) Comparison of the
structures of beta amyloid peptide (25–35) and substance P
in trifluoroethanol/water solution. J. Biomol. Struct. Dyn. 17,
381–391.
56. Qi, X.F., Zhorov, B.S. & Ananthanarayanan, V.S. (2000) CD,
1
H
NMR and molecular modeling studies of the interaction of Ca
2+
with substance P and Ala7-substance P in a non-polar solvent.
J. Pept. Sci. 6, 57–83.
57. Auge, S., Bersch, B., Tropis, M. & Milon, A. (2000) Character-
ization of substance P–membrane interaction by transferred
nuclear Overhauser effect. Biopolymers 54, 297–306.
Supplementary material
The following material is available from: http://www.
blackwellpublishing.com/products/journals/suppmat/EJB/
EJB3665/EJB3665sm.htm
Table S1.

1
Hassignmentof[P
t
3
Leu10]SP (in methanol,
278 K).
Table S2.
1
H assignment of [P
c
3
Leu10]SP (in methanol,
278 K).
2878 J. Quancard et al. (Eur. J. Biochem. 270) Ó FEBS 2003