Pharmacology & Pharmacy, 2011, 2, 322-331
doi:10.4236/pp.2011.24041 Published Online October 2011 (
Copyright © 2011 SciRes. PP
Anticushing Drug Metyrapone Exhibits Specific
Interactions with Serine Containing Systems. A
Possible Molecular Target?
David Crouzier1, Jean-Claude Debouzy1, Florian Nachon2, Dominique Debouzy3, Guy Lallement4,
Frédéric Canini5
1Effets Biologiques des Rayonnements, IRBA/CRSSA, La Tronche, France; 2Unité d’Enzymologie IRBA/CRSSA, La Tronche,
France; 3Université Pierre Mendes France, St. Martin d’Hères, France; 4Département de Toxicologie, IRBA/CRSSA, La Tronche,
France; 5Unité de Neurophysiologie du Stress, IRBA/CRSSA, La Tronche, France.
Received August 17th, 2011; revised September 13th, 2011; accepted September 28th, 2011.
Metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone) is a drug largely used as inhibitor of glucocorticoid synthesis.
Although its binding to various proteins has been well indentified, its accurate molecular mechanism of action remains
unknown. Therefore, the interactions of metyrapone (MET) with various membrane components such as phospholipids,
cholesterol, their corresponding polar heads and a model serine containing peptide have been investigated by NMR and
ESR methods. It was found that neither cholesterol nor most of the phospholipids tested, nor dimyristin exhibit any in-
teraction with MET, except phosphatidylserine (DMPS). Furthermore, only serine bearing polar head (O-phosphoser-
ine) showed an association with MET (stoechiometry 1:1, Kd = 3200 M-1). As similar observations were also performed
on serine alone and in the presence of the serine containing model peptide, (NASDSDGQDL), a possible implication of
these interactions in the binding recognition of MET on serine-containing active site was finally tested and discussed.
Keywords: Metyrapone, Membranes, Serine, NMR, ESR, Interactions
1. Introduction
In the clinical field, metyrapone (2-methyl-1,2-di-3-pyri-
dyl-1-propanone MET), an inhibitor of glucocorticoid
synthesis [1], is widely used to treat the Cushing disease
[2,3]. Since high blood corticosterone levels have been
linked to a depressive mood [4], metyrapone is also used
to treat Cushing related mental disturbances [5] and ma-
jor depression [6-9]. However, metyrapone exhibits other
properties highlighted by preclinical studies that can not
be explained by its action on the corticotrope axis. At the
physiological level, metyrapone injection is followed by
a slight hypothermia [10] not reversed by glucocorticoid
supplementation [11], a decreased locomotion [12], an
enhanced arousal [13] together with biological signs of
stress [14]. The latter does not mask the protective effect
of metyrapone against psychological stressors [15,16]
and physiological stressors such as brain ischemia [17,18]
and excitotoxicity [19]. At the cell level, metyrapone
modify the energy metabolism [10] with a decreased use
of glucose [20-21] and alters the mitochondrial function-
ing [22]. Lastly, metyrapone also modify the detoxifica-
tion as it modifies the expression of numerous cyto-
chrome P450 such as CYP1A1 and CYP 3A [23].
Besides these integrated levels, the molecular support,
i.e. the identification or the molecular target, still failed.
Metyrapone profoundly modify the steroid metabolism
through its inhibitory activity on the 11β hydroxylase
(CYP11B1), [1], 17β hydroxysteroid deshydrogenase
(17β-HSD) [24] and the 11 β-HSD [25]. Metyrapone also
inhibits P450 [26] in numerous species [27]. Its inhibi-
tory effect is not specific as it targets the CYP11B1 [28]
and the CYP3A [29], but also other enzymes such as
lipooxygenase [30], guanylate cyclase [31] or nitric ox-
ide synthetase as a member of cytochrome P450-like
hemoprotein [32]. Some works have focused on the
mechanisms by which metyrapone interacts with P450.
Its fixation on P450 is independent from the redox state
of the enzyme and th e availability of oxygen [33,34] and
does not modify the conformation of the protein P450A3
[35,36]. Another mechanisms sugg ested was the possible
Anticushing Drug Metyrapone Exhibits Specific Interactions with Serine Containing Systems. 323
A Possible Molecular Target?
implication of membrane components, phospholipids or
cholesterol [37,38]. The investigation of this hypothesis
was the starting point of the present study. NMR and
ESR experiments were recorded on synthetic multilayers
of phospholipids and sphingolipids, their building blocks
(polar head groups and acyl chain backbone) and choles-
terol. This selection led to identify a potential target and
to finally observe the interactions of MET with a model
peptide containin g this molecular target, serine.
2. Materials and Methods
2.1. Chemicals
Dymiristoyl phosphatitdylcholine (DMPC), dymiristoyl
phosphatidylethanolamine (DMPE), dymiristoyl phos-
phatidylserine (DMPS), dimyristin, bovine brain sphin-
gomyelin, ceramids, galactocérébrosides, dipalmitoylphos-
phatidylglycerol DPPG, phosphorylcholin O-phosphos-
erine, serine, O-phosphocolamine, ethanolamine were pu r -
chased from Sigma-Aldrich (La Verpillère, France) and
used without purification.
The model peptide N10L: Asn-Ala-Ser-Asp-Ser-Asp-
Ser-Gly-Gln-Asp-Leu (NASDSDGQDL) was purchased
from NeoMPS, Stasbourg, France. Proton attribution, and
purity control were performed in D2O by using classical
NMR technics (TOCSY, HMBC, HMQC, nOesy [39]) at
298 K. No stable conformation of the peptide could be
identified at this step.
MET was from Sigma. Purity control and proton as-
signment were obtained by classical NMR methods (1H,
COSY, HMBC, HMQC [39]), as indicated on the Figure
2.2. Vesicles Preparation
2.2.1. Multilayer Vesicles (MLV)
The phospholipids or sphingolipids in their chloroformic
solution were evaporated to a film and resuspended in
pure D2O. The liposomes were formed by fast freezing
and thawing cycles. The final lipid concentration was 50
mM and MET/lipid molar ratio were fixed to 1:12, 1:10,
1:7, 1:4 M/M.
2.2.2. Small Unilamelar Vesicle (SUV)
Phospholopids in theirs chloroformic solution (10 mg/ml)
were lyophylized and resuspended in pure D2O for a final
lipid concentration of 10mM. SUV were formed by 1
Hour bath sonication. For MET containing SUV, the
respective chloroformic solutions were mixed before lyo-
2.3. NMR Experiments
High resolution NMR experiments. Standard 1H-NMR
experiments were recorded at 298 K on a Bruker AM-
400 spectrometer using a 4000 Hz spectral width, 32 K
digitization points, a recycling delay o f 2 sec. A presatu-
ration of water resonance was used for all the experi-
ments. Phase sensitive NOESY NMR experiments were
recorded at 298 K to detect MET-serine and MET-pep-
tide vicinities (dipolar correlations) with a presaturation
of the solvent resonance and mixing times of 250 ms.
Solid-state 31P-NMR experiments in multilayers (MLV)
were performed at 162 MHz. Phosphorus spectra were
recorded using a dipolar echo sequence (/2-t--t) [40]
with a t value of 12 sec, a /2 pulse of 3.8 µs and a
broadband two level proton decoupling. Phosphoric acid
(85%) was used as external reference. The sample tem-
perature was regulated within 1˚C by a BVT-1000 unit.
Job plots. For all interaction experiments, in a first step,
a coarse screening was used by preparing equimolar (5
mM) host/MET mixtures and sample containing excess
of MET (9/1, M/M). In the absence of any spectral modi-
fication (mainly chemical shift, peak intensity and
linewidth), the affinity was considered as negligible and
the study was not followed further. Fast exchange kinet-
ics were identified on the spectra by both MET and host
chemical shift variations upon MET addition, and the
classical method described by Job [41,42] was used to
draw the stoechiometry of the complex. Besides, math-
ematic determination method SIMPLEX (EXPREX, or
MURIEL-X) algorithms generously given by Bruno
Perly, CEA Saclay, France, for strong complex and in
order to draw estimations of the apparent association
constant [43].
2.4. ESR Experiments: Spin Label Study
Effect of MET on DMPS SUV fluidity was assessed by
ESR spin label experiments. Two spin labels (Sigma
France) were used: 5-nitroxide stearate (5 NS) and 16-
nitroxide stearate (16 NS). This fatty acids self incorpo-
rate the SUV and the nitroxide groups provide informa-
tion of motional freedom of the label in the system. So
the former probes the superficial part of the membrane
layer, the latter in its hydrophob ic core.
The experiments were performed on SUV made with
DMPS 10 mg/mL. 1 mg of MET was added in 500 µL
SUV solution. 200 µl of the SUV control solution and
SUV MET solution were then labelled with 10 µL of spin
label solution (5 NS 5 × 10–3 M or 16 NS 5 × 10–3 M).
After 30 min incubation at room temperature, sample
was transferred by capillarity in 20 µL Pyrex capillary
tube. This tube was placed in a 3 mm diameter quartz
holder, and insert into the cavity of t he ESR spectrometer.
The ESR spectra were recorded at different controlled
temperature (303, 308, 310, 313, 315, 318 and 323 K)
with the following conditions: microwave power 20.00
Copyright © 2011 SciRes. PP
Anticushing Drug Metyrapone Exhibits Specific Interactions with Serine Containing Systems.
324 A Possible Molecular Target?
mW, modulation frequency 100 kHz, modulation ampli-
tude 2.05 G, receiver gain 6.3 × 105 conversion time
81.92 ms, time constant 81.92 ms. Sweep range was 100
G with a central field value of 3435 G for 5 NS probe,
and in the same condition except, modulation amplitude
1.03 G, receiver gain 105 conversion time 40.96 ms, time
constant 81.9 2 ms for 16 NS probe.
The complete membrane incorporation of the spin la-
bels was ascertained by the absence on the spectra of the
extremely resolved ESR lines corresponding to free ro-
tating markers.
5 NS experimentations: The values of outer and inner
hyperfine splitting were measured (2T// and 2T respect-
tively), on ESR spectra (Figure 3(b)), and order parame-
ter S was calculated following the equation [44]:
S1.723 T2TC
C1.4 0.053TT
 
The increase in the order parameter v alue means a de-
crease of local membrane fluidity.
16 NS experimentations: The changes in freedom mo-
tion of 16 NS were analysed with the calculation of c
the rotational correlation time. c
was calculated fol-
lowing the formula [45]:
TcKWh h1
10 1
 
In this formula, W0 is the peak-to-peak line width of
the central line; h0 an d h–1 are the peak height of the cen-
tral and high-field lines respectively (Figure 3(d)).
The increase in the rotational correlation time means a
decrease of local membrane fluidity.
3. Results
3.1. 31P-NMR Experiments in MLV
A first attempt for MET affinity and interaction screen-
ing was performed by using phospholipid dispersions as
membrane model. This model allows to observe the
structural and dynamics consequences of the presence of
MET at the polar head (31P-NMR) by recording NMR
spectra under different temperature conditions. Thus the
dependence of the interactions with MET following the
nature of the polar head group was tested by using dif-
ferent phospholipids, i.e. DMPC, sphingomyeline, DMPE,
DMPS, DPPG, and also galactocerebrosides and cera-
mides. At this point, it is noteworthy to recall several
basic principles about 31P-NMR in membranes.
31P-NMR chemical shift (the resonance frequency)
depends on the orientation of phosphorus nuclei in the
field (shielding). The chemical shift difference between
the low field and the hi ghfield edges of a 31P-NMR spec-
trum is called Chemical Shift Anisotropy (CSA, ppm)
and is directly related to the fluidity—reorientation—at
the polar head level where the phosphorus nuclei are lo-
cated. On such spectra a mobile phosphorus group gives
a single narrow resonance (several Hz) as detected in true
solutions or for small structures (micelles), while phos-
phorus groups in solid state gives extremely broad con-
tributions (CSA values exceeding 100 ppm). Note that
membrane fluidity increases (and CSA decreases) with
temperature, with a special jump at the transition tem-
perature between gel phase and liquid crystal structure (e.g.
around 297 K for DMPC [46]). Thus the plot of CSA as a
function of temperature provides a good overview of mem-
brane dynamics at the polar head level where phosphorus
nuclei are located, while the lineshape allows identifying
the overall membrane organization (bilayer, hexagonal,
isotropic phases). Such plots are presented on the traces of
the Figure 1 (top traces).
The bottom spectrum (Figure 1, column A) shows the
spectrum of pure DMPC dispersion (MLV), typical of an
axia11y symmetric powd er pattern, with a chemical shift
anisotropy of 65 ppm, classical of a phospholipid (here
DMPC) bilayers in their liquid crystalline phase around
phase transition (297 K) [47].
As expected for pure DMPC dispersions a CSA de-
crease (around 18 - 20 ppm) was me asured on pure DM PC
systems by increasing the temperature (and the mem-
brane fluidity) with the transition-related jump around
297 K [48]. This was also the case for MET containing
MLV (MET/DMPC ratios of 1/10 and up to 1/4 M/M,
middle and top traces, column A). Also temperature de-
pendence of CSA values was found almost surperim-
posed on the entire temperature range Figure 1(c)). Be-
sides, no significant isotropic contribution typical of de-
tergent effect was observed, thus finally indicating the
absence of any interaction with the membrane. Similarly,
no interaction with MET was found for cholesterol (up to
20%) containing DMPC dispersions and also for galac-
tocerebrosides, ceramides, and DPPG MLV (not shown).
Besides, when DMPE systems were used, only a limited
increase in the fluidity (i.e. a limited CSA reduction) was
observed all over the temperature range, never exceeding
2ppm, even for high MET/DMPE ratios of 1/4.
Such was not the case for the spectra recorded under
the same conditions on DMPS (Figures 1(b)-(d)) disper-
sions in the presence of MET. As expected, pure DMPS
dispersions gave typical spectra of bilayer structures (see
Copyright © 2011 SciRes. PP
Anticushing Drug Metyrapone Exhibits Specific Interactions with Serine Containing Systems. 325
A Possible Molecular Target?
Figure 1(b) bottom), with a decrease of the CSA with
temperature. The same spectra recorded on MET con-
taining DMPS systems resulted in a reduction of the CSA
for MET/DMPS molar ratios exceeding R = 1/10 M/M
(trace of the Figure 1(d)), while an isotropic contribution
appeared for higher R values, with a relative contribution
(a) (b)
(c) (d)
Figure 1. 31P-NMR (a) spectra of DMPC dispersion (50 mM,
D2O) at 297 K, pure (bottom trace) and in the presence of
MET, molar ratios of R = 1/10 and 1/4, M/M, (middle and
top traces, respectively); horizontal bracket represent the
CSA; (b) spectra of DMPS dispersion (50 mM, D2O) at 313
K, pure (bottom trace) and in the presence of MET, molar
ratios of 1/10 and 1/4, M/M, (middle and top traces, respect-
tively; (c) plot of temperature dependence CSA measured
on DMPC dispersion spectra of DMPC (), and in the
presence of MET, R = 1/4 M/M (); (d) plot of temperature
dependence CSA measured on DMPS dispersion spectra of
DMPS (), and in the presence of MET, R = 1/10 M/M (),
and R = 1/7 M/M (); bottom trace () represents the line
width of the isotropic contribution observed at R = 1/4
being developed at the expense of the main structure
(Figure 1(b) middle and top traces). Finally, a single line
of less than 50 Hz with was exclusively detected for R =
1/4, indicating that the native structure had been com-
pletely replaced by very mobile systems of high fluidity,
consistent with a detergent effect or micelle formation.
At this step, the existence of MET/DMPS bilayers ap-
pears as highly probable. In order to obtain more preci-
sions, the different building blocks of the phospho- or
sphingolipids were considered separately, i.e. the polar
headgroups and the diacylglycerol backbone. These ex-
periments are the topic of the next section.
3.2. 1H-NMR and ESR of Polar Headgroups and
Headgroups. As both polar groups and MET are soluble
in D2O, the first step was achieved by recording spectra
on equimolar mixtures and compared to those of pure
species of O-phosphocholine (OPC), O-phosphoserin
(OPS), O-phosphocolamine(OPCO), and sphingosine. A
special care was paid to the aromatic resonances of MET,
clearly isolated in the 7 - 9 ppm region (see Figure 2(a)
for MET structure and nomenclature, bottom trace for the
corresponding s pe ctrum of MET).
In the case of OPC, and sphingosine, as no detectable
spectrum variation was observed both in chemical shift
and linewidth even MET was p resent in excess (9 /1 M/M
ratios MET/headgroup) it was assumed that no interact-
tion occurred and the investigation was not studied fur-
OPCO-MET systems only provided minor chemical
shift variations that did not allow to propose a precise
mode of interaction, stoechiometry or affinity constant
(attempts made using Benesi-Hidebrandt method only
showed it should not excess 30 M–1). The affinity of
MET for OPCO was thus considered as very weak.
As significant chemical shift variations were observed
on both OPS and MET resonances upon addition of OPS,
the continuous variations method was used by recording
spectra of different molar ratios MET/OPS, keeping total
concentration constant (5 mM, see examples of such
spectra on Figure 2). This allowed us to use the method
to characterize these interactions, as classically described
by Job [41].
In such plots, the curves built using concentration
weighted chemical shift variations as a function of the
molar fraction show a maximum for the molar fraction
corresponding to the stoechiometry, here 0.5, which
means a 1/1 association (2B). This also allowed the use
of numerical simulations (EXPREX2 [43]) that gave an
apparent association constant of 3200 M–1.
In order to detect spatial vicinities between MET and
Copyright © 2011 SciRes. PP
Anticushing Drug Metyrapone Exhibits Specific Interactions with Serine Containing Systems.
326 A Possible Molecular Target?
(a) (b)
Figure 2. (a) Metyrapone (MET) formula and proton no-
menclature (b) Job-Plots built from concentration-weighted
chemical shift variations (mM*Hz) as a function of molar
fraction (F), for H5 (7.356 ppm, ), H4 (7.82 ppm, ), H16
(7.47 ppm, ) protons of MET or H2” proton of phosphos-
erine (4.19 ppm, ), 298 K D2O. The total concentrations
were kept at 5 mM. Bottom traces with proton numbering
below (from bottom to top): aromatic part of the 1H-NMR
spectra of MET (bottom trace), and in the presence of
O-phosphoserine (corresponding molar ratios MET/OPS of
1:0, 9/1; 3/2 and 1/1, respectively.
OPS, nOesy experiments (mixing time of 250 ms) were
then recorded on the 1/1 mixture. Hence weak dipolar
correlations were found between one of methylenic pro-
tons (4.22 ppm) of OPS and H16, H15 and H13 protons
of MET, and also between CHα, (4.15 ppm) and H15 not
shown). However, molecular dynamics simulations could
not lead to propose any stable conformation.
Diacylglycerol backbone. In order to observe chains
interaction without any polar head contribution, disper-
sions of dimyristin and dipalmitin were prepared as for
phospholipid MLV. As the 1H spectra recorded on the
dispersions containing MET (R = 1/1 M/M) were the
single sum of the spectra of pure dispersions and MET
recorded separately under the same conditions, it was
concluded that no interaction occurred at the molecular
level (not shown).
3.3. ESR Experiments
Spin label experiments were then realised to investigate
the membrane fluidity in different temperature conditions
at the chain level. Two probes were separately used, 5
NS gate information about superficial membrane fluidity,
while 16 NS concerned the inner membrane region. The
overall result (Figures 3(a) and (c)) shows an increase in
the mobility of the two probes contribution in the two
groups with the temperature increase. As previously de-
scribed in SUV DMPS model [47,49], the phase transi-
tion in control groups occurs near 313 K.
The ESR results were found in full agreement with the
experiments recorded by NMR: hence, no interaction
with phospholipids and sphingolipids except DMPS was
found. In this latter case, an increase in the mobility of
the two probes contribution was found when MET was
added to DMPS solution.
5 NS results are drawn Figure 3(a). An overhall in-
crease of the membrane fluidity and a shift of the phase
transition, 5 degrees lower, could be observed in the
MET group.
In the inner compartment, 16NS results (Figure 3(c))
show a major effect of MET with a total vanishing of
phase transition and a strong increase of the membrane
between 303 and 313 K. At the highest temperature
(above 315 K) the rotational correlation time of the con-
trol group reached the same value as MET group.
However, a true interaction between free serine and
MET was very probable. Under a biological point of
view, it was important to know if MET could also have
interactions with a serine group engaged in a peptide
structure. This was tested by using a reference decapep-
tide NASDSDGQDL as target.
3.4. Interactions with the Model Peptide
After purity control and attribution of peptide protons
using classical NMR methods [50], the same method as
before was used on equimolar mixtures of MET/peptide.
By comparison with the spectrum of pure MET (Figure
4(a)) significant chemical shift variations were observed
on the MET/peptide sample, as shown on Figure 4(a),
similar as with OPS/MET reparations. Moreover, the p
Copyright © 2011 SciRes. PP
Anticushing Drug Metyrapone Exhibits Specific Interactions with Serine Containing Systems.
A Possible Molecular Target?
Copyright © 2011 SciRes. PP
Figure 3. ESR spin labeling experiment: (a) temperature dependence of the order parameter (5 NS) for pure DMPS (black
diamond full line) and in the presence of MET 2 mg/ml (black square, dashed line); (b) typical 5 NS spectrum parameter
used for order parameter estimation are inner (2T) and outer hyperfine (2T//) splitting; (c) temperature dependence of the
rotational correlation time (16 NS) for pure DMPC and in the presence of MET 2 mg/ml; (d) typical 16 NS spectrum, pa-
rameter used for rotational correlation time was central peak intensity H0, High field peak intensity and the width of the
mid-field line W0.
nOesy experiment then recorded also showed weak di-
polar connectivities, mainly between H15, H6 and sev-
eral peptide protons, suggesting vicinities in the 4 - 6 Å.
However, whereas MET well exhibited some vicinities
with serine groups, others of more strong intensity were
also present with Gly and Gln aminoacids.
4. Discussion
The present work primarily investigated the interactions
of MET with membrane components, phospholipids,
sphingolipids or cholesterol, as evoqued in the literature
[51]. The use of multibilayers (dispersions prepared by
the freezing-thawing method [52]) combined with solid-
state 31P-NMR spectroscopy allows to investigate both
dynamics and structural collective properties of the bi-
layers. Such a method allows to easily obtain a screening
the specificity of interactions with a given phospholipid
system. In the present case, as preliminary tests performed
Anticushing Drug Metyrapone Exhibits Specific Interactions with Serine Containing Systems.
328 A Possible Molecular Target?
Figure 4. 1H-NMR spectrum of pure MET, 5 mM, 298 K,
D2O; (b) same conditions as (a), on a NASDDGQDL/MET
preparation (1/1 M/M); Bars indicate dipolar correlations
between labelled groups. Inset shows a partial 2D-nOeSY
map recorded on the same sample.
on “standard” lecithin or DMPC small unilamellar vesi-
cles [53], or permeabilization tests on large unilamellar
vesicles [54] had given no arguments, an investigation a
possible specificity was required. Among the main lipid
species tested, MET interactions with DMPS systems
were evidenced (from a simple fluidization for low
amounts of MET to a drastic detergent effect for molar
ratios exceeding 1/7 M/M), and could be precised by
studying separately the interactions of MET with lipid
building blocks: while the lipidic p art seemed not to play
any role in the interaction with MET, dramatic spectral
modifications were noted in the presence of phosphoser-
ine; moreover, spatial connectivities were identified be-
tween aromatic protons of the second cycle of MET (see
nomenclature Figure 2) and serine groups, even if the
fast exchange mode of interaction—that allowed job-plot
constructions [55]—cou ld not allow to determine a stab le
conformation via molecular dynamics calculations. By
comparing 31P-NMR and ESR results, one can observe
that MET induces dramatic changes at the polar head
level (31P), whereas similar liquefying effects were found
part of the chain (5 NS), while the same observation
could be done at high temperature—at the deep part level
of the layer (16 NS). It can thus been proposed that the
target for MET interaction is mainly located at the polar
head level, were serine groups are present, and that the
overall membrane destructuration would result from dy-
namics perturbation and sterical hindrance, as observed
for cholesterol-phospholipid interactions [56]. Neverthe-
less this interaction with a given amino acid—serine—
would also be consistant with the recognition by MET of
a signaling site of the target protein guanylate cyclase—a
serine containing protein [31]. Thus an attempt was made
to test the interaction of MET with a serine containing
peptide, i.e. to test the conseque nces for interactions with
MET to use a serine engaged in a peptide bound. Despite
the presence of true interactions with MET (close to
those observed with phosphoserine), and the observation
of intermolecular nOes, it was not possible to ascertain
any specificity in this case. Especially, dipolar connec-
tivities were also noted with Gly and Gln, suggesting that
MET interactions with aminoacids would be at least not
specific of serine moieties. However, it must be kept in
mind that the peptide used is of small size, hydrophilic
and structureless, that means not favorable for any stable
conformation or molecular associations found in pro-
Hence, the future experiments now on course will have
to deal with a more realistic model, i.e. a “serin-pocket”
containing peptide with more stable supramolecular or-
5. Acknowledgements
Thanks to Will Sorin for helpful discu ssions and Mrs. C.
Bigname for fruitful collaboration.
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