Báo cáo khoa học: Structural and biological effects of a b2- or b3-amino acid insertion in a peptide Application to molecular recognition of substance P by the neurokinin-1 receptor ppt

Structural and biological effects of a b
2
-orb
3
-amino acid insertion
in a peptide
Application to molecular recognition of substance P by the neurokinin-1 receptor
Sandrine Sagan, Thierry Milcent, Rachel Ponsinet, Odile Convert, Olivier Tasseau, Ge
´
rard Chassaing,
Solange Lavielle and Olivier Lequin
UMR 7613 CNRS-Paris 6, Universite
´
Pierre et Marie Curie, Paris, France
Molecular mechanics calculations on conformers of
Ac-HGly-NHMe, Ac-b
2
-HAla-NHMe and Ac-b
3
-HAla-
NHMe indicate that low-energy conformations of the
b-amino acids backbone, corresponding to gauche rotamers
around the Ca–Cb bond, may overlap canonical backbone
conformers observed for a-amino acids. Therefore, Sub-
stance P (SP) was used as a model peptide to analyse the
structural and biological consequences of the substitution of
Phe7andPhe8by(R)-b
2
-HPhe and of Gly9 by HGly (R)-b
2
-

HAla or (S)-b
3
-HAla. [(R)-b
2
-HAla9]SP has pharmacolo-
gical potency similar to that of SP while [HGly9]SP and
[(S)-b
3
-HAla9]SP show a 30- to 50-fold decrease in biological
activities. The three analogues modified at position 9 are
more resistant to degradation by angiotensin converting
enzyme than SP and [Ala9]SP. NMR analysis of these SP
analogues suggest that a b-amino acid insertion in position 9
does not affect the overall backbone conformation. Alto-
gether these data suggest that [HGly9]SP, [(S)-b
3
-HAla9]SP
and [(R)-b
2
-HAla9]SP could adopt backbone conforma-
tions similar to that of SP, [Ala9]SP and [Pro9]SP. In con-
trast, incorporation of b
2
-HPhe in position 7 and 8 of SP led
to peptides that are almost devoid of biological activity.
Thus, a b-amino acid could replace an a-amino acid within
the sequence of a bioactive peptide provided that the addi-
tional methylene group does not cause steric hindrance and
does not confine orientations of the side chain to regions of
space different from those permitted in the a-amino acid.

Keywords: b
2
-andb
3
-amino acid; secondary structure;
molecular mechanics calculations; substance P; neurokinin-1
receptor.
Bioactive a-peptides often present conformational equili-
briums in solution but probably adopt one structure (or
family of related structures) when bound to their receptor
[1]. A plethora of studies has been conducted on chemical
modifications of a-amino acids to stabilize this so-called
bioactive conformation that may be present as a minor
conformer in solution, and/or to design peptidomimetics
[2–8]. The corresponding b-amino acids (Fig. 1), should be
better building blocks to design peptidomimetics as
b-peptides are more resistant to degradation by mammalian
enzymes [9]. However, each b-amino acid insertion in a
peptide sequence introduces additional degrees of confor-
mational flexibility with the rotation around the Ca–Cb
bond [10]. Previous quantum mechanics calculations on
protected b-dipeptides (mimics of tetra-b-peptides) have
permitted the identification of many low-energy conforma-
tions, namely six- and eight-membered ring hydrogen-
bonded structures (C
6
,C
8
), extended structures (Ex) and
helical structures (He) [11]. The C

6
structure was found to be
the most stable conformation by ab initio calculation [12].
The Ex and He conformations observed for b-sheet, H
14
and H
12
helices (14- and 12-membered ring hydrogen-
bonded structures) were less stable in the gas phase but gain
stabilization in polar solvents [13]. All these structures have
also been found experimentally in b-peptides either in
solution and/or in the solid state [14–17]. An additional helix
corresponding to the formation of successive 12-, 10-, 12-
hydrogen-bonded structures (C
12
/C
10
/C
12
) has also been
detected [18].
Thewideuseofb-amino acids is prevented by the small
number of enantiomerically pure b
2
-amino acids commer-
cially available and the cost of synthesizing b
3
-amino acids.
Therefore, the design of heterooligomers made of both
a-andb-amino acids could overcome this limitation,

assuming that these chimeric a, b-peptides may keep the
molecular recognition properties of a-peptides and the
biological stability of b-peptides. Only a few data have been
reported in the literature on the structural properties of
these heterooligomers. However, it has been shown that the
c-turn or C
7
structure found with a-amino peptides can be
Correspondence to: O. Lequin, UMR 7613 Paris 6-CNRS,
Laboratoire Structure et Fonction de Mole
´
cules Bioactives,
Universite
´
Pierre et Marie Curie, Paris, France.
Fax: + 33 1 44 27 71 50, Tel.: + 33 1 44 27 26 78,
E-mail:
Abbreviations: NK-1, neurokinin-1; SP, substance P (H-Arg-Pro-Lys-
Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH
2
); NKA, neurokinin A;
HGly, homoglycine; b
2
-HAla, b
2
-homoalanine; b
3
-HAla, b
3
-homo-

alanine; b
2
-HPhe, b
2
-homophenylalanine; Aib (aMeAla), a-amino-
isobutyric acid; Sar, sarcosine (N-MeGly); ACE, angiotensin
converting enzyme (E.C. 3.4.15.1); PtdIns, phosphatidyl inositol;
PLC, phospholipase C; CHO, Chinese hamster ovary; CSD, chemical
shift deviation; Ex, extended structures; He, helical structures.
Enzyme: angiotensin converting enzyme (EC 3.4.15.1).
(Received 3 October 2002, revised 20 December 2002,
accepted 9 January 2003)
Eur. J. Biochem. 270, 939–949 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03456.x
stabilized as a C
8
conformer in cyclic tetrapeptides and
pentapeptides containing one b-amino acid [19], whereas the
introduction of two adjacent b-amino acids induced a
10-membered hydrogen-bonded turn (C
10
) analogous to the
b-turn made of a-amino acids [20]. This propensity of
b-amino acids to mimic canonical structures observed with
a-peptides (oligomers of a-amino acids) has recently been
applied to the syntheses of linear tetrapeptides of somato-
statin [20] and of cyclic tetrapeptide and pentapeptide
analogues of the RGD sequence [19]. With the cyclic
compounds it is uncertain whether the C
8
conformation

observedwithasingleb-amino acid is enforced by the cyclic
constraints. The C
10
conformation, initially identified in
cyclic b-peptides containing adjacent b
2
-, b
3
-amino acids,
has also been identified in solution with a linear b-tetra-
peptide analogue of somatostatin [20]. These few examples
suggested that there might be spatial overlaps between some
three-dimensional structures of a-amino acids and those of
b-amino acids and posed the following questions. Which
structures of b-amino acids overlap the canonical structures
of a-amino acids? Subsequently, is it possible to substitute
one a-amino acid in the sequence of a linear peptide for one
b-amino acid without drastically affecting the recognition
properties of the resulting peptide? To try to answer these
questions we chose SP (RPKPQQFFGLM-NH
2
)andthe
glycine residue in position 9 of SP that has been extensively
analysed in structure–activity relationship studies. In terms
of peptide affinity for the NK-1 receptor and biological
activity, this achiral amino acid can be favourably replaced
by sarcosine or proline, whereas a b-II¢ turn constraint
around residues 9 and 10 confers antagonist properties
[22,23]. Therefore, this position associated with a plausible
conformational flexibility is considered as a switch point

between agonist and antagonist structures [21–23].
By molecular mechanics calculations the conformers of
Ac-HGly-NHMe, Ac-b
2
-HAla-NHMe and Ac-b
3
-HAla-
NHMe have been generated and compared to the
canonical structures of the corresponding a-amino acid
Ac-Gly-NHMe. The corresponding SP analogues substi-
tuted in position 9 by these b-amino acids have been
synthesized and their conformational preferences analysed
by NMR spectroscopy. These substituted SP analogues
were tested for their resistance to enzymatic degradation,
their affinity for the human NK-1 receptor and their
potency to stimulate adenylate cyclase and phospho-
lipase C (PLC) in CHO cells transfected with the human
NK-1 receptor [24,25]. Modelling and superimposition of
the conformers of the different b-anda-amino acids
inserted in position 9 of SP was performed to analyze
structure–activity relationships.
Experimental procedures
Molecular mechanics calculations
N-acetyl N¢-methyl amide derivatives of HGly, b
2
-HAla and
b
3
-HAla and of a-amino acids were built using
INSIGHTII

(Accelrys Inc.). Molecular mechanics calculations were
performed with the
DISCOVER
program and AMBER force
field [26]. The electrostatic potential energy was calculated
with a distance-dependent dielectric screening of 4Ær and no
cut-off was used.
Minimum-energy conformers of b-anda-amino acids
were generated by molecular dynamics at high tempera-
ture followed by energy minimization. Two thousand
structures were generated by molecular dynamics at
1000 K, saving snapshots every 2 ps. The time step used
was 1 fs and the temperature was controlled by direct
velocity scaling. Each structure was then submitted to
2 ps of dynamics at 300 K and minimized using steepest
descent, conjugate gradient and Newton–Raphson algo-
rithms until the gradient was less than 0.001 kcalÆ
mol
)1
ÆA
˚
)1
.
Conformational grid searches of b-amino acids were
initially carried out at intervals of 30° for each torsion angle
/, h,andw and were subsequently refined by varying /, w
angles in intervals of 10° and setting h angle to ) 60 ± 10°
and 60 ± 10°.Each/, h,andw-value was fixed by applying
a harmonic potential and the structures were minimized
(adiabatic relaxation).

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 1–2 m
M
concentration. NMR
experiments were recorded at 298 K and 278 K on Bruker
Avance spectrometers at a
1
H frequency of 500 MHz and
were processed with the
XWIN
-
NMR
software. 1D spectra
were acquired over 16 K data points using a spectral width
of 5000 Hz. Solvent suppression was achieved by presatu-
ration during the relaxation delay or with a WATERGATE
sequence [27]. Proton assignments were obtained from the
analysis of TOCSY (20 and 80 ms isotropic mixing times)
[28] and NOESY (400 ms mixing time) experiments [29].

Typically 512 t
1
increments were acquired over a spectral
width of 5000 Hz. Prior to Fourier transformation in t
2
and
t
1
, the time domain data were multiplied by a 60–90° shifted
square sinebell function and zero-filled. Baseline distortions
were corrected with a fifth-order polynomial.
1
H-
13
CHSQC
experiments were recorded using pulsed field gradients for
coherence selection [30].
Fig. 1. Schematic representation of a-, b
2
-and
b
3
-amino acids with the torsion angles /, h
and w. For the b
2
and b
3
-amino acid substi-
tuted-SP analogues, the methyl side chain of
b-HAla exhibits the same orientation as the

one in [Ala9]SP (CIP’s rule a-amino acid:
S configuration, thus (R)forb
2
-HAla and
(S)forb
3
-HAla). (CIP, Cahn–Ingold–Prelog.)
940 S. Sagan et al. (Eur. J. Biochem. 270) Ó FEBS 2003
The chemical shift deviations of Ha and Ca were
calculated using random coil values determined in methanol
[31] and water [32], respectively. No sequence correction
(proline effect, for example) was applied for this calculation
as only position 9 in the sequence of these analogues is
modified, the RPKPQQFF and LM-NH
2
domains being
constant.
Enzymatic degradation
Enzymatic cleavage of SP, [Ala9]SP, [HGly9]SP, [(R)-b
2
-
HAla9]SP and [(S)-b
3
-HAla9]SP by ACE was performed
as described previously, with slight modifications [33].
Briefly, peptides (10 nmol) were incubated in 100 lL Tris/
HCl 50 m
M
pH 8.3, NaCl 300 m
M

, with 0.01 U rat lung
ACE(Fluka)at37°C for 60 min. Degradation was
stopped by addition of 1 lL trifluoroacetic acid and
followed by HPLC using a RP8 Lichrospher100 column in
isocratic mode (72% H
2
O, 28% acetonitrile, 0.072%
trifluoroacetic acid) at a flow rate of 1.5 mLÆmin
)1
.
Retention times were 7.3 min for SP, 8.6 min for [Ala9]SP,
7.5 min for [HGly9]SP, 7.1 min for [(R)-b
2
-HAla9]SP and
7.7 min for [(S)-b
3
-HAla9]SP. Enzymatic assays were
performed twice and yielded similar results. All assays
were done in parallel experiments with control at t ¼ 0for
each peptide. The percentage of degradation was calculated
by comparing the area of the peaks of the intact peptide at
t ¼ 0andt ¼ 60 min.
Binding assays
Binding assays were carried out at 22 °C with either
[
3
H][Pro9]SP (0.2–0.5 n
M
,65CiÆmmol
)1

) for 100 min or
[
3
H]propionyl[Met(O
2
)11]SP(7–11) (2–5 n
M
,95 CiÆ mmol
)1
)
for 80 min on whole CHO cells expressing the human
NK-1 receptor (6 pmolÆmg protein
)1
) as described
previously [24], in 200 lL 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 and containing 0.04% bovine serum albumin
(w/w), 0.6% glucose (w/v), 10 m
M
phenylmethanesulfonyl
fluoride. SP peptide analogues (stored at )20 °Cin
water at a concentration of 1 m
M
)weredilutedinthe
binding buffer to the desired concentration just prior to
the assay.
Phospholipase C and adenylate cyclase assays
PtdIns hydrolysis and cAMP accumulation were deter-
mined as described previously [25]. Briefly, CHO cells
expressing the human NK-1 receptor (6 pmolÆmg pro-
tein
)1
) were labelled with [
3
H]adenine (0.2 lCi per well)
or [
3
H]inositol (0.5 lCi per well) for 15–24 h. PtdIns
hydrolysis assay was performed in 500 lLKrebs-Ringer
phosphate buffer containing 10 m
M

LiCl and the peptide
to be tested for 10 min. The accumulation of cAMP was
performed in 500 lL Krebs-Ringer phosphate buffer
containing 1 m
M
3-isobutyl-1-methylxanthine and the
peptide to be tested for 10 min. SP peptide analogues
(stored at )20 °C in water at a concentration of 1 m
M
)
were diluted in the assay buffer to the desired concen-
tration just prior to the assay.
Results
Molecular modelling
The conformational properties of b-amino acids and b-pep-
tides have already been extensively analysed by ab initio
quantum calculations [11,13] and molecular mechanics [12].
In the present study minimum-energy conformations of
model b-peptides (Ac-HGly-NHMe, Ac-b
2
-HAla-NHMe
and Ac-b
3
-HAla-NHMe) were first generated by molecular
mechanics using AMBER force field. The results were
compared with those obtained previously from ab initio
molecular orbital (MO) calculations and molecular mechan-
ics calculations [11–13]. The potential energy surfaces
accessible to b-amino acids have plenty of local minima.
The minimum-energy conformers (data not shown) are

similar to those obtained with CHARMm 23.1 force field
[12]: six- and eight-hydrogen-bonded conformations with
the methyl group in axial or equatorial orientations
(conformers C
6eq
,C
6ax
,C
8eq
and C
8ax
), extended structure
(Ex) with no hydrogen bonds and helical structures (H
12
and
H
14
). The C
6
and C
8
structures were found to be the most
stable conformations. Most low-energy structures corres-
pond to h torsion angles around 180°,60° and )60°.
Then, in order to compare the conformational spaces of a
and b-amino acids, grid searches were performed and the
generated structures were superimposed with canonical
conformations of a-amino acids. The atoms used in the
rmsd calculation are those involved in the two peptide
bonds and the two N- and C-terminal methyl groups, in

order to compare the backbone orientations on both sides
of the b-amino acid. The systematic fits indicate that
structures with h torsion angle above 90° have large rmsd
values. In particular, the low-energy extended structures,
corresponding to h  180°, cannot overlap any conforma-
tion of a-amino acids. In order to visualize on the potential
energy surfaces the conformers of b-amino acids that fit
with canonical conformations of a-amino acids, two
(/ ) w) Ramachandran maps corresponding to h torsion
angle values of +60°,gauche(+),or)60°, gauche(–), were
calculated (Fig. 2). The calculated conformers are only a
part of the representative statistical ensemble, due to the h
angle restriction. HGly has a wide range of accessible
conformations, whereas b
3
-HAla has a more limited
conformational space (Fig. 2). For both gauche(–) and
gauche(+) conformers, regions of the (/ – w) diagram can
overlap canonical b-sheet, a-helix and reverse c-turn
conformations found in a-amino acids (rmsd lower than
0.05 nm). The gauche(+) conformers which fit to a-helical,
C7 and b-sheet conformations have approximate (/, w)
torsion angles of ()100°, )110°)()130°,20°)and()170°,
80°), respectively, and correspond to favourable regions.
For the gauche(–) conformers, the conformations that fit to
a-helix have high energies and only two low-energy regions
of the diagram can fit to C7 or b-sheet conformations, with
(/, w) torsion angles of ()30°,120°)and()70°, 180°),
respectively.
Secondary structure of the SP analogues

[(R)-b
2
-HAla9]SP, [(S)-b
3
-HAla9]SP as well as [(R)-b
2
-
HPhe7]SP and [(R)-b
2
-HPhe8]SP have been synthesized
Ó FEBS 2003 Effects of b-amino acid insertion in Substance P (Eur. J. Biochem. 270) 941
by solid phase methodology and obtained with purities and
yields comparable to SP and [Pro9]SP [34].
Due to its inherent flexibility, SP is largely unstructured in
water but helical conformation of the 4–8 domain is induced
in lower dielectric constant solvents such as methanol
[35,36]. The
1
H-NMR spectra of all the SP analogues in
methanol are relatively well-dispersed and have been
completely assigned using conventional techniques [37].
The chemical shifts of a carbon (Ca) have been assigned
from HSQC spectra (data not shown). The chemical shift
deviations of Ha protons and Ca carbons (CSD
Ha
and
CSD
Ca
), corresponding to differences between observed
chemical shifts and random coil values, are commonly used

to detect secondary structures in peptides and proteins [38].
TheyarereportedinFig.3.
Stronger upfield shifts of H
a
(Fig. 3A) and downfield
shifts of C
a
resonances (Fig. 3B) in the 4–8 sequence are
observed for [Aib9]SP compared to SP. The positive
variation observed for CSD
Ca
in methanol is weak when
compared with CSD
Ha
. Because the random coil values of
Ca were determined in water, it is possible that Ca chemical
shifts are sensitive to solvent variation, causing an under-
estimation of calculated CSDs. These CSD
Ha
and CSD
Ca
variations demonstrate the formation of more stable and
abundant helical structures for [Aib9]SP than for SP. By
restrained molecular dynamics, [Aib9]SP has been shown to
adopt a stable helix from residues 4–10 while the 4–8
domain of SP adopts a more flexible helix [36]. Taking SP as
a reference, CSD
Ha
and CSD
Ca

indicate that the introduc-
tion of one methyl (Ala) or two methyl groups (Aib) on Ca
carbon of Gly9 increases progressively the 4–8 domain
folding into helical structures. An opposite effect due to the
helix breaker property of Pro is observed for [Pro9]SP, this
decrease in helical structure is limited to the adjacent Phe8
Fig. 2. (/ – w)mapsofb-amino acids with
indication of the conformational space common
with that of a-amino acids. The (/ ) w)maps
corresponding to h-values of )60° and 60° are
indicated for HGly (A), b
2
-HAla (B) and
b
3
-HAla (C). The contours are drawn within
21 kJÆmol
)1
(5 kcalÆmol
)1
)oftheglobal
minimum and are gradually coloured from
blue to red (the energy difference between each
level is 2.1 kJÆmol
)1
). The structures that
exhibit rmsd values smaller than 0.05 nm with
canonical structures of a-amino acids are
indicated with squares of different colours:
blue, a-helix; green, reverse c-turn;red,anti-

parallel b-sheet. The corresponding (/, w)
values of the canonical structures are ()57°,
)47°)()90°,68°), and ()139°,135°).
942 S. Sagan et al. (Eur. J. Biochem. 270) Ó FEBS 2003
residue. Homologation of Gly9 in [HGly9]SP does not
decrease the helix proportion. Methylation of HGly in
position two, in [b
2
-HAla9]SP, has no effect on the helical
structure when compared to SP, whereas methylation of
HGly in position three, in [b
3
-HAla9]SP, induces a helical
folding of the 4–8 domain similar in amplitude to that
observed for [Ala9]SP. Whatever the chemical modifications
carried out in position 9, the
3
J
NH-Ha
coupling constant of
Gln5 remains close to 5 Hz, indicating that Gln5 always
adopts a helical conformation. This invariable folding of
Gln5 is related to the helix initiator propensity of Pro4 and
decreases from Gln5 to Gly9. In methanol, the helical
structures in the 4–8 domain increase with [Pro9]SP < SP
 [HGly9]SP  [b
2
-HAla9] < [b
3
-HAla9]SP < [Ala9]SP

<[Aib
9
]SP. The presence of helical structures was con-
firmed by the observation of NN (i, i +1),aN(i, i +2),aN
(i, i +3),aN(i, i +4)andab (i, i + 3) NOEs along the 5–8
domain of SP and [Aib
9
]SP. The aN(i, i +4) and ab (i, i
+ 3) NOEs were not detected in [b
2
-HAla9]SP, [b
3
-
HAla9]SP and [HGly9]SP, indicating more flexible helical
structures.
The C-terminal part of SP undergoes a complex con-
formational equilibrium between more or less extended
structures. On the basis of CSD
Ha
, mono- or di-methylation
of Gly9 (Ala, Aib) appears to induce helical structure for
Leu10. This effect is also observed for [b
3
-HAla9]SP. The
analysis of coupling constants and NOEs indicate that the
local conformation of residue 9 is not well-defined in the
b-amino acid-substituted SP analogues.
The lack of NOEs prevented three-dimensional structure
calculations by restrained molecular dynamics. Neverthe-
less, the similar patterns of Ha and Ca CSDs suggest that

the different SP analogues may adopt conformations of
their peptide backbone close to that of SP, which has been
previously described [36]. The decreased number of observed
NOEs in [HGly9]SP, [b
2
-HAla9] and [b
3
-HAla9]SP
is consistent with a higher flexibility around the b-amino
acid-substituted position.
Potency of the SP analogues
The binding potencies of these analogues for the two specific
binding sites, NK-1M and NK-1m, associated with the
human NK-1 receptor have been measured with transfected
CHO cells [24,25]. The more abundant binding site NK-1M
(85%) is labelled by [
3
H][Pro9]SP and is coupled to cAMP
production, whereas the less abundant binding site NK-1m
(15%) is labelled by [
3
H]propionyl[Met(O
2
)11]SP(7–11) and
associated with the production of inositol phosphates. The
binding and agonist potencies of the SP analogues are
Fig. 3. Chemical shift deviations of Ha (A) and Ca (B) for the SP analogues modified in position 9. The CSD
Ca
are not shown for [Pro9]SP. Residue X
is either Gly (SP), Ala, Pro, Aib, HGly, b

2
-HAla or b
3
-HAla.
Ó FEBS 2003 Effects of b-amino acid insertion in Substance P (Eur. J. Biochem. 270) 943
expressed as K
i
for NK-1M (major site) and NK-1m (minor
site), and EC
50
values for the cAMP pathway and for the
inositol phosphates pathway.
SP, [Ala9]SP and [Pro9]SP are almost equipotent at the
major binding site NK-1M (K
i
between 0.64 n
M
and 1.6 n
M
and EC
50
values between 4.8 and 8 n
M
). [HGly9]SP and
[(S)-b
3
-HAla9]SP are 30 to 45 times less potent than SP (K
i
and EC
50

values). [(R)-b
2
-HAla9]SP is 10 times more potent
than the corresponding b
3
-analogue and is only three to five
times less potent than SP (Table 1).
Regarding the minor binding site NK-1m, SP and
[Pro9]SP show a 10-fold increase in affinity compared to
their affinity for the NK-1M binding site, with K
i
in the
subnanomolar range (0.13 n
M
)andEC
50
values in the
nanomolar range (1.0 and 0.7 n
M
, respectively). Surprisingly,
[Ala9]SP exhibits one of the highest affinity ever found for
the NK-1m specific binding site (K
i
¼ 7p
M
), as it is almost
20 times more potent than SP and [Pro9]SP and even 10
times more potent than [Gly9(wCH
2
CH

2
)(S)Leu10]SP.
[HGly9]SP and [(S)-b
3
-HAla9]SP present similar K
i
values
for the NK-1m specific binding sites (3.2 and 2.3 n
M
,
respectively), being 20–30 times less potent than SP. [(R)-b
2
-
HAla9]SP is only 4.5 times less potent than SP and
[Pro9]SP, as observed for the NK-1M binding site. The
EC
50
values for inositol phosphates production of these
three b-amino acids-substituted SP analogues [HGly9]SP,
[(S)-b
3
-HAla9]SP and [(R)-b
2
-HAla9]SP are almost identi-
cal (EC
50
 2n
M
). For comparison, the affinities and
potencies of different analogues of SP substituted at

position(s) 9 or/and 10 are also reported in Tables 1, i.e.
[Aib9]SP [36], [Pro10]SP, [Gly9w(CH
2
CH
2
)Gly10]SP,
[Gly9w(CH
2
CH
2
)(S)Leu10]SP and [Gly9w(CH
2
CH
2
)
(R)Leu10]SP.
In contrast, [(R)-b
2
-HPhe7]SP and [(R)-b
2
-HPhe8]SP
are very weak competitors for NK-1M and NK-1m
specific binding sites, being 2000 times less potent than
SP.
Table 1. Affinities of b-amino acid-containing peptide analogues of SP for the NK-1M (labelled with [
3
H][Pro9]SP) and the NK-1m (labelled with
[
3
H]propionyl[Met(O

2
)11]SP(7–11)) binding sites and their related potency to stimulate adenylate cyclase and phospholipase C in CHO cells expressing
the human NK-1 receptor. All experiments have been performed in triplicate in at least three independent experiments. Numbers in parentheses refer
to structures in Fig. 5.
Peptide K
i
, NK-1M (n
M
)EC
50
, adenylate cyclase K
i
, NK-1m (n
M
)EC
50
, phospholipase C
SP (1)
a
1.6 ± 0.4 8 ± 2 0.13 ± 0.02 1.0 ± 0.2
Propionyl[Met(O
2
)
11
]SP(7–11)
a
1900 ± 450 >5000 10 ± 2 37 ± 4
[Pro9]SP (2)
a
1.1 ± 0.1 10 ± 2 0.13 ± 0.02 0.7 ± 0.1

[Aib9]SP (3)
b
44 ± 4 125 ± 30
c
3.8 ± 0.4 5.5 ± 1.5
[Pro10]SP 24 ± 2 375 ± 50 3.7 ± 0.5 3.0 ± 1.0
[Gly9(YCH
2
CH
2
)Gly10]SP (4) 190 ± 30 1250 ± 50 3.0 ± 0.7 1.2 ± 0.5
[Gly9(YCH
2
CH
2
)(S)Leu10]SP (5) 2.6 ± 0.5 24 ± 6 0.07 ± 0.01 0.6 ± 0.1
[Gly9(YCH
2
CH
2
)(R)Leu10]SP (6) 73 ± 8.5 1130 ± 370 4.6 ± 0.8 2.5 ± 0.7
[Ala9]SP (7) 0.64 ± 0.070 4.8 ± 1.2 0.0070 ± 0.00065 0.69 ± 0.03
[HGly9]SP (8) 64 ± 3.5 290 ± 74 3.2 ± 0.4 2.0 ± 0.2
[(R)-b
2
-HAla9]SP (9) 5.2 ± 0.70 40 ± 5 0.54 ± 0.12 2.0 ± 0.4
[(S)-b
3
-HAla9]SP (10) 53 ± 6 360 ± 55 2.3 ± 0.3 2.9 ± 0.7
[(R)-b

2
-HPhe8]SP 2400 ± 300 >10 000 240 ± 25 215 ± 25
[(R)-b
2
-HPhe7]SP 2500 ± 400 >10 000 230 ± 30 210 ± 30
a, b
results already published in [24] and [36], respectively.
c
Efficacy 73% that of [Pro9]SP taken as the peptide of reference.
Fig. 4. Relation between (A) affinity for the NK-1M binding site and
potency to activate adenylate cyclase and (B) affinity for the NK-1m
binding site and potency to activate phospholipase C of b-amino acid-
containing peptide analogues. Symbols are the experimental results
obtained with data in Table 1. Dotted lines represent theoretical values
obtained from equations previously determined with 53 (A) and 22 (B)
SP analogues, respectively [39].
944 S. Sagan et al. (Eur. J. Biochem. 270) Ó FEBS 2003
We have previously established that a good correlation
exists between EC
50
values and the corresponding K
i
values,
i.e. EC
50
for cAMP production and K
i
for the NK-1M
binding site (log(EC
50

) ¼ 0.8 log(K
i
)–0.6),andEC
50
for
inositol phosphates production and K
i
for the NK-1m site
(log (EC
50
) ¼ 0.9 log (K
i
) – 1.0), respectively [39]. The data
obtained in this study (Fig. 4) square with the equations
determined previously with the exception of SP, [Pro9]SP,
[Ala9]SP, [Gly9w(CH
2
CH
2
)(S)Leu10]SP and [(R)-b
2
-
HAla9]SP, which show apparently abnormal behaviour in
their affinity for the NK-1m binding and their potency to
stimulate PLC (Fig. 4B). Whatever the affinity measured
for the NK-1m binding site for these agonists (from 7 p
M
to
0.54 n
M

), the corresponding potency to stimulate PLC is
always close to 1 n
M
(from 0.6 n
M
to 2 n
M
), the highest
EC
50
/K
i
ratio being close to 100 for [Ala9]SP (Table 1). This
apparent discrepancy between the varying affinities and the
nonvarying response of these agonists could be explained by
the number of receptors to be occupied by these agonists to
get activation of the second messenger cascade as reported
[40].
Enzymatic degradation of SP analogues
ACE is a dipeptidyl carboxypeptidase known to hydrolyze
the peptide bonds between residues 8–9 and 9–10 of SP [33].
The peptide cleavage after one hour incubation with ACE
was monitored by HPLC. Percentages of degradation were
found to be 54 ± 1% for SP, 55 ± 3% for [Ala9]SP,
33 ± 5% for [HGly9]SP, 26 ± 3% for [b
2
-HAla9] and
14.5 ± 2.5% for [b
3
-HAla9], thus showing that these

b-amino acid-containing peptides have increased stability
towards cleavage by ACE compared to the corresponding
a-peptides.
Discussion
Computational studies confirm that the conformational
space of b-aminoacidsislargerthanthatofa-amino
acids, but low-energy conformations of the b-amino acids
backbone, corresponding to gauche rotamers around the
Ca–Cb bond, can overlap canonical backbone conformers
observed for a-amino acids. Therefore the addition of a
backbone methylene group could have minor effects on
the overall conformation and biological activity of the
peptide.
Thus, SP was used as a model peptide to analyse the
structural and biological consequences of a single b-amino
acid incorporation. When Phe7 or Phe8 are replaced by
b
2
-HPhe, the corresponding analogues are weak competi-
tors of specific NK-1 binding sites. These amino acids are in
the helical domain of SP which extends from residues 4–8
[35]. Any modification of Phe7 causes a dramatic loss in
receptor affinity for the corresponding peptide [21,41].
Indeed, the backbone conformation, the aromatic ring and
the orientation (v
1
and v
2
) of this phenylalanine have to be
conserved for full biological potencies of the peptide [41]. It

is possible that the side chain of b
2
-HPhe may not fit in the
binding subsite devoted to the aromatic ring of Phe7. Yet,
molecular calculations indicate that low-energy structures of
Fig. 5. Schematic representation of the amino acid sequence 9–10 of the SP analogues 1–10 (pharmacological data in Table 1). Rectangles under
analogues indicate compounds that have affinity for the two (NK-1M/NK-1m) binding sites similar to those of SP. Ovals under analogues point out
compounds compared to SP that lose at least a factor 20 in affinity for the two (NK-1M/NK-1m) binding sites. (1) SP; (2) [Pro9]SP; (3) [Aib9]SP; (4)
[Gly9(YCH
2
CH
2
)Gly10]SP; (5) [Gly9(YCH
2
CH
2
)(S)Leu10]SP; (6) [Gly9(YCH
2
CH
2
)(R)Leu10]SP; (7) [Ala9]SP; (8) [HGly9]SP; (9) [(R)-b
2
-
HAla9]SP; (10) [(S)-b
3
-HAla9]SP.
Ó FEBS 2003 Effects of b-amino acid insertion in Substance P (Eur. J. Biochem. 270) 945
b
2
-HPhe could fit the bioactive conformation of Phe7

around the peptide bond and the aromatic ring. Because
Ca-methylation of Phe7 leads to an inactive compound [36],
it is likely that the additional methylene group of b
2
-HPhe
causes steric hindrance within the receptor as does the
methyl group in Ca-MePhe. Although position 8 of SP can
accept larger aromatic substituents than position 7 [41],
Ca-methylation is also prohibited, suggesting that steric
hindrance can again be evoked to explain the lack of affinity
of [(R)-b
2
-HPhe
8
]SP for the NK-1 receptor.
Gly9 in the sequence of SP constitutes a hinge between
the helical domain and the C-terminal residues which adopt
more or less extended conformations, Phe7, Phe8, Leu10,
and Met11 being key elements of the SP pharmacophore
[21]. N-methylation of Gly9, i.e. [Sar
9
]SP, as well as proline
substitution, i.e. [Pro9]SP, yield potent and selective NK-1
agonists [21]. Therefore Gly9 should be a more favourable
candidate for b-amino acid substitution than Phe7 or Phe8.
The SP analogues modified in position 9 are schematically
represented in Fig. 5. Their binding potencies for the two
binding sites (NK-1M and NK-1m) associated with the
NK-1 receptor in CHO transfected cells [24,25] have been
classified in two groups. In one group, peptides are as potent

as SP, or even more potent, whatever the binding site
considered, i.e. [Pro9]SP, [Gly9(wCH
2
CH
2
)(S)Leu10]
SP, [Ala9]SP and [b
2
-HAla]SP. In the second group, SP
analogues are more than 20 times less potent than SP,
whatever the binding site considered, i.e. [Aib9]SP,
[Gly9(wCH
2
CH
2
)Gly10]SP, [Gly9(wCH
2
CH
2
)(R)Leu10]SP,
[HGly9]SP and [b
3
-HAla9]SP. Previous structure–activity
A
B
C
Fig. 6. Superposition of selected conformers of
b
2
-HAla (A), b

3
-HAla (B) and Aib (C) with
Pro. The (/, w) values of the selected Pro
conformer are ()72°, 153°). The low-energy
conformers of b-HAla generated in the grid
calculationhavebeenselectedonthebasisof
thebestfitwithPro.Theycorrespondto(/, h,
w)anglesof()110°,70°,100°). The energy
differences relative to the global energy mini-
mum are 0.88 and 0.86 kcalÆmol
)1
for b
2
-HAla
and b
3
-HAla, respectively. Nitrogen atoms are
coloured in blue, oxygens in red, hydrogens in
grey. Carbon atoms of Pro are coloured in
cyan, those of other amino acids in green. The
side chain methyl carbon of b-HAla and the
pro R methyl carbon of Aib are coloured in
magenta.
946 S. Sagan et al. (Eur. J. Biochem. 270) Ó FEBS 2003
relationship studies [42] have established that the leucine
side chain orientation is crucial for full binding potency,
[Gly9(wCH
2
CH
2

)(S)Leu10]SP being even more potent than
SP whereas [Gly9(wCH
2
CH
2
)(R)Leu10]SP is a weak com-
petitor at the NK-1 receptor (at that time NK-1M and
NK-1m binding sites were not differentiated). The amide
bond between residues 9 and 10 is not involved per se in any
stabilizing interaction within the NK-1 receptor because
[Gly9(wCH
2
CH
2
)(S)Leu
10
]SPisaspotentasSP.More
important is the length of the spacer between residues 8 and
10.Indeed,[Gly9(wCH
2
CH
2
)Gly10]SP is a weak compet-
itor of specific NK-1 binding sites while homologation
of one carbon, such as [Gly9(wCH
2
CH
2
CH
2

)Gly10]SP
(D. Loeuillet & S. Lavielle, unpublished data), led to
an analogue completely devoid of binding potency for
NK-1 binding sites. Interestingly, the presence of an amide
bond such as in the homologated analogues [HGly9]SP,
[b
2
-HAla9]SP and [b
3
-HAla9]SP restores part of the
potency to recognize the NK-1 receptor. In view of this
observation it can be proposed that the improved affinity
is indicative of an energetically favoured bioactive
conformation stabilized by the amide function. In
[Gly9(wCH
2
CH
2
CH
2
)Gly10]SP, the backbone of the ami-
nohexanoic moiety must be unable to adopt two consecu-
tive gauche(+) rotamers.
The pyrrolidine ring of proline does not hamper the
correct positioning of Leu10 and Met11, because [Pro9]SP is
as potent as SP. Ca monomethylation of Gly9 drastically
increases the affinity for the NK-1m binding site of
[Ala9]SP, supporting the formation of a new stabilizing
interaction between this methyl group and a hydrophobic
subsite within the specific NK-1m binding site. The CH

2
b of
the pyrrolidine ring of [Pro9]SP probably fits within this
hydrophobic subsite and thus compensates destabilizing
interactions due to CH
2
c and CH
2
d of the pyrrolidine ring
or non optimal F-value. However, the introduction of a
second methyl group on the Ca of Gly9 in [Aib9]SP induces
a strong repulsive interaction. Although the three b-amino
acid substitutions in position 9 of SP are tolerated by the
NK-1 receptor, the substitution of the flexible Gly by the
even more flexible HGly causes a 30- to 40-fold decrease in
affinity and biological activity. Substitution by b
3
-HAla has
similar effects. Only the b
2
-HAla substitution yields an
analogue that is nearly as potent as SP. Thus, a peculiar
orientation of the methyl group in b
2
-HAla-substituted SP
might be at the origin of a stabilizing interaction.
Modelling studies and NMR analysis suggest that the
three b-amino acid-substituted SP analogues [HGly9]SP,
[b
2

-HAla9]SP and [b
3
-HAla9]SP may adopt conforma-
tions around residue 9 that are analogous to those
adopted by a-amino acids (Gly, Ala, Sar, Pro). To explain
the slightly higher biological potency of [b
2
-HAla9]SP
compared to [HGly9]SP and [b
3
-HAla9]SP, the structures
(backbone and side chain) of the different a and b amino
acids were superimposed. Pro, the most constrained
residue, was used as a template for the superimposition
and an extended conformation was chosen, in accordance
with structure–activity relationship studies [21,24]. Low-
energy structures of the b-aminoacidsthatbestfitare
shown in Fig. 6. They all correspond to gauche(+) values
of the h angle. The methyl group of b
2
-HAla occupies a
position close to that of CH
2
b of Pro or the pro S methyl
in Aib or Ala. Conversely, the methyl group of b
3
-HAla
occupies a position similar to the pro R methyl of Aib, on
the opposite side of the pyrrolidine ring of Pro. A parallel
can be drawn regarding the differences in biological

activities between [Ala9]SP and [Gly9]SP on the one hand,
and [b
2
-HAla9]SP and [HGly9]SP on the other hand. In-
deed, the higher pharmacological potency of [b
2
-HAla9]SP
compared to [HGly9]SP suggests that the methyl group of
b
2
-HAla9 may fit within the hydrophobic subsite devoted
to the methyl group of Ala9. The similar biological
potencies of [b
3
-HAla9]SP and [HGly9]SP indicate that
even though the backbone of [b
3
-HAla9]SP may adopt
the bioactive conformation, the methyl group of b
3
-HAla9
may not be orientated towards this hydrophobic stabi-
lizing subsite.
Finally, as shown herein with the use of ACE that has
been reported to cleave SP between residues 8–9 and 9–10
[33], the peptides containing a b-amino acid substitution in
position 9 have increased stability compared to the corres-
ponding a-amino-acid-containing peptides. Therefore it is
possible to increase peptide stability at the expense of a
minimal decrease in its activity.

HGly (named by the authors b-alanine) has been
previously introduced in the sequence of the C-terminal
heptapeptide of NKA, another peptide of the tachykinin
family that binds the NK-1 and NK-2 receptors. [HGly8]
NKA(4–10) is as potent as NKA and [Ala8]NKA on rabbit
pulmonary artery and rat portal vein, two NK-2 receptor
bioassays [43]. More recently, the same Ala substitution was
reported to cause a significant decrease in biological
activities of [Ala8]NKA measured in human tissues [44].
Indeed, [Ala8]NKA(4–10) was shown to be a weak partial
agonist ([HGly8]NKA(4–10) was not tested in this study).
These discrepancies have been attributed to the differences
in sequences of rabbit, rat and human NK-2 receptors
( 85% homology).
In conclusion, a b-amino acid could replace an a-amino
acid within the sequence of a bioactive peptide provided that
the additional methylene group does not cause steric
hindrance and does not confine orientations of the side
chain to regions of space different from those permitted in
the a-amino acid. Thus, insertion of a single b-amino acid in
a bioactive peptide could be favourably applied to improve
both biological potency and enzymatic stability of the
original peptide, as already shown with the empiric design of
a metalloendopeptidase tripeptide inhibitor [45].
References
1. Marshall, G.R. (2001) Peptide interactions with G-protein
coupled receptors. Biopolymers 60, 246–277.
2. Kemp, D.S. (1990) Peptidomimetics and the template approach to
nucleation of b-sheets and a-helices in peptides. Trends Biotechn 8,
249–255.

3. Rizo, J. & Gierasch, L.M. (1992) Constrained peptides: models of
bioactive peptides and protein substructures. Annu.Rev.Biochem.
61, 387–418.
4. Marshall, G.R. (1993) A hierarchical approach to peptidomimetic
design. Tetrahedron 17, 3547–3558.
5. Hruby, V.J. (1993) Conformational and topographical consid-
eration in the design of biologically active peptides. Biopolymers
33, 1073–l082.
Ó FEBS 2003 Effects of b-amino acid insertion in Substance P (Eur. J. Biochem. 270) 947
6. Hruby, V.J. & Balse, P.M. (2000) Conformational and topo-
graphical considerations in designing agonist peptidomimetics
from peptide leads. Curr. Med. Chem. 7, 945–970.
7. Saviano,M.,Iacovino,R.,Menchise,V.,Benedetti,E.,Bonora,
G.M., Gatos, M., Graci, L., Formaggio, F., Crisma, M. &
Toniolo, C. (2000) Conformational restriction through C
a
i
· C
a
i
cyclization: Ac
12
c, the largest cycloaliphatic C
a,a
-disubstituted
glycine known. Biopolymers 53, 200–212.
8. Halab,L.,Gosselin,F.&Lubell,W.D.(2000)Design,synthesis,
and conformational analysis of azacycloalkane amino acids as
conformationally constrained probes for mimicry of peptide
secondary structures. Biopolymers 55, 101–122.

9. Frackenpohl, J., Arvidsson, P.I., Schreiber, J.V. & Seebach, D.
(2001) The outstanding biological stability of b-andc-peptides
toward proteolytic enzymes: an in vitro investigation with fifteen
peptidases. Chembiochem. 2, 445–455.
10. DeGrado, W.F., Schneider, J.P. & Hamuro, Y. (1999) The twists
and turns of b-peptides. J. Peptide Res. 54, 206–217.
11. Wu, Y.D. & Wang, D.P. (1998) Theoretical studies of b-peptide
models. J. Am. Chem. Soc. 120, 13485–13493.
12. Gu
¨
nther, R., Hofmann, H.J. & Kuczera, K. (2001) Searching
for periodic structures in b-peptides. J. Phys. Chem. B 105, 5559–
5567.
13. Mo
¨
hle, K., Gu
¨
nther, R., Thormann, M., Sewald, N. & Hofmann,
H.J. (1999) Basic conformers in b-peptides. Biopolymers 50,
167–184.
14. Lopez-Carrasquero, F., Aleman, C. & Munoz-Guerra, S. (1995)
Conformational analysis of helical poly (b-
L
-aspartate) s by IR
dichroism. Biopolymers 36, 263–271.
15. Seebach,D.,Schreiber,J.V.,Abele,S.,Daura,X.&vanGun-
steren, W.F. (2000) Structure and conformation of b-oligopeptide
derivatives with simple proteinogenic side chains: circular
dichroism and molecular dynamic investigations. Helv. Chim.
Acta 83, 34–57.

16. Appella, D.H., Barchi, J.J., Durell, S.R. & Gellman, S.H. (1999)
Formation of short, stable helices in aqueous solution by b-amino
acid hexamers. J. Am. Chem. Soc. 121, 2309–2310.
17. Appella, D.H., Christianson, L.A., Klein, D.A., Richards, M.R.,
Powell, D.R. & Gellman, S.H. (1999) Synthesis and structural
characterization of helix-forming b-peptides: trans-2-amino-
cyclopentanecarboxylic acid oligomers. J. Am. Chem. Soc. 121,
7574–7581.
18. Seebach, D., Abele, S., Gademann, K., Guichard, G., Hinter-
mann, T., Jaun, B., Matthews, J.L., Schreiber, J.V., Oberer, L.,
Hommel,U.&Widmer,H.(1998)b
2
-andb
3
-peptides with
proteinaceous side chains. Synthesis and solution structures of
constitutional isomers, a novel helical secondary structure, and the
influence of solvation and hydrophobic interactions on folding.
Helv. Chim. Acta 81, 932–982.
19. Schumann, R., Muller, A., Koksch, M., Mu
¨
ller, G. & Sewald, N.
(2000) Are b-amino acids c-turn mimetics? Exploring a new design
principle for bioactive cyclopeptides. J. Am. Chem. Soc. 122,
12009–12010.
20. Gademann, K., Kimmerlin, K., Hoyer, D. & Seebach, D.J. (2001)
Peptide folding induces high and selective affinity of a linear and
small peptide to the human somatostatin receptor 4. J. Med.
Chem. 44, 2460–2468.
21. Lavielle, S., Chassaing, G., Loeuillet, D., Convert, O., Torrens, Y.,

Beaujouan, J.C., Saffroy, M., Petitet, F., Bergstrom, L. &
Glowinski, J. (1990) Selective agonists of tachykinin binding sites.
Fund. Clin. Pharmacol. 4, 257–268.
22. Ward, P., Ewan, G.B., Jordan, C.C., Ireland, S.J. & Hagan, R.M.
(1990) Potent and highly selective neurokinin antagonists. J. Med.
Chem. 33, 1848–1851.
23. 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 the position 10. Eur. J.
Pharmacol. 258, 273–276.
24. 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 gland. Mol.
Pharmacol. 52, 120–127.
25. Sagan, S., Chassaing, G., Pradier, L. & Lavielle, S. (1996)
Tachykinin peptides affect differently the second messenger
pathways after binding to CHO-expressed human NK-1
receptors. J. Pharmacol. Exp. Ther. 276, 1039–1048.
26. Weiner, S.J., Kollmann, P.A., Nguyen, D.T. & Case, D.A. (1986)
An all-atom forcefield for simulations of proteins and nucleic
acids. J. Comput. Chem. 7, 230–252.
27. Piotto, M., Saudek, V. & Sklenar, V. (1992) Gradient-tailored

excitation for single-quantum NMR spectroscopy of aqueous
solutions. J. Biomol. NMR 2, 661–665.
28. Bax, A. & Davis, D.G. (1985) MLEV-17-based two-dimensional
homonuclear magnetization transfer spectroscopy. J. Magn.
Reson. 65, 355–360.
29. Kumar, A., Ernst, R.R. & Wu
¨
thrich, K. (1980) A two-
dimensional nuclear Overhauser enhancement (2D NOE)
experiment for the elucidation of complete proton-proton cross-
relaxation networks in biological macromolecules. Biochem.
Biophys. Res. Commun. 95,1–6.
30. Schleucher, J., Schwendinger, M., Sattler, M., Schmidt, P.,
Schedletzky, O., Glaser, S.J., Sorensen, O.W. & Griesinger, C.
(1994) A general enhancement scheme in heteronuclear
multidimensional NMR employing pulsed field gradients.
J. Biomol. NMR 4, 301–306.
31. 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.
32. Wishart,D.S.,Bigam,C.G.,Holm,A.,Hodges,R.S.&Sykes,
B.D. (1995)
1
H,
13
Cand
15
N random coil NMR chemical shifts of
the common amino acids. I. Investigations of nearest-neighbor

effects. J. Biomol. NMR 5, 67–81.
33. Strittmatter,S.M.,Thiele,E.A.,Kapiloff,M.S.&Snyder,S.H.
(1985) A rat brain isozyme of angiotensin-converting enzyme.
Unique specificity for amidated peptide substrates. J. Biol. Chem.
260, 9825–9832.
34. Sachon, E., Girault-Lagrange, S., Chassaing, G., Lavielle, S. &
Sagan, S. (2002) Analogs of Substance P modified at the
C-terminus which are both agonist and antagonist of the NK-1
receptor depending on the second messenger pathway. J. Peptide
Res. 59, 232–240.
35. Chassaing, G., Convert, O. & Lavielle, S. (1986) Preferential
conformation of substance P in solution. Eur. J. Biochem. 154,
77–85.
36. Sagan, S., Lequin, O., Frank, F., Convert, O., Ayoub, M., Lavi-
elle, S. & Chassaing, G. (2001) Ca methylation in molecular
recognition: application to substance P and the two neurokinin-1
receptor binding sites. Eur. J. Biochem. 268, 2997–3005.
37. Wu
¨
thrich, K. (1986) NMR of Proteins and Nucleic Acids. Wiley,
New York.
38. 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.
39. Sagan, S., Karoyan, P., Chassaing, G. & Lavielle, S. (1999)
Further delineation of the two binding sites (Rn*) associated with
tachykinin neurokinin-1 receptors using [3-prolinomethionine 11]
SP analogues. J. Biol. Chem. 274, 3770–23776.
40. Kenakin, T.P. & Morgan, P.H. (1989) Theoretical effects of
single and multiple transducer receptor coupling proteins on

948 S. Sagan et al. (Eur. J. Biochem. 270) Ó FEBS 2003
estimates of the relative potency of agonists. Mol. Pharmacol. 35,
214–222.
41. Sagan, S., Josien, H., Karoyan, P., Brunissen, A., Chassaing, G. &
Lavielle, S. (1996) Tachykinin NK-1 receptor probed with
constrained analogues of Substance P. Bioorg.Med.Chem.4,
2167–2178.
42. 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.
43. Rovero, P., Pestellini, V., Rhaleb, N E., Dion, S., Rouissi, N.,
Tousignant, C., Te
´
le
´
maque,S.,Drapeau,G.&Regoli,D.(1989)
Structure–activity studies of neurokinin A. Neuropeptides 13,263–
270.
44. Warner, F.J., Mack, P., Comis, A., Miller, R.C. & Burcher, E.
(2001) Structure–activity relationships of neurokinin A (4–10) at
the human tachykinin NK
2
receptor: the role of natural residues
and their chirality. Biochem. Pharmacol. 61,55–60.
45. Steer,D.L.,Lew,R.A.,Perlmutter,P.,Smith,A.I.&Aguilar,
M.I. (2000) Design and synthesis of inhibitors incorporating
b-amino acids of metalloendopeptidase EC 3.4.24.15. J. Peptide

Sci. 6, 470–477.
Supplementary material
The following material is available from http://www.
blackwellpublishing.com/products/journals/suppmat/EJB/
EJB3456/EJB3456sm.htm
Table S1. Minimum-energy conformers of Ac-HGly-
NHMe obtained with DISCOVER and AMBER forcefield.
Table S2. Minimum-energy conformers of Ac-b
2
-HAla-
NHMe obtained with DISCOVER and AMBER forcefield.
Table S3. Minimum-energy conformers of Ac-b
3
-HAla-
NHMe obtained with DISCOVER and AMBER forcefield.
Table S4. NMR Parameters of SP and SP analogues.
Ó FEBS 2003 Effects of b-amino acid insertion in Substance P (Eur. J. Biochem. 270) 949