Báo cáo Y học: Conformationally constrained human calcitonin (hCt) analogues reveal a critical role of sequence 17–21 for the oligomerization state and bioactivity of hCt ppt

Conformationally constrained human calcitonin (hCt) analogues
reveal a critical role of sequence 17–21 for the oligomerization
state and bioactivity of hCt
Athanasios Kazantzis
1
, Michaela Waldner
1
, John W. Taylor
2
and Aphrodite Kapurniotu
1
1
Physiological-chemical Institute, Department of Physical Biochemistry, University of Tu
¨
bingen, Germany;
2
Rutgers University,
Department of Chemistry and Chemical Biology, Piscataway, NJ, USA
Calcitonin (Ct) is a 32-residue peptide hormone that is
mainly known for its hypocalcemic effect and the inhibition
of bone resorption. Our p revious studies have led to poten t,
side-chain lactam-bridged human Ct (hCt) analogues
[Kapurniotu, A. Kayed, R. , Taylor, J.W. & Voelter W.
(1999) Eur. J. Biochem. 265, 606–618; Kapurniotu, A. &
Taylor, J .W. (1995) J. Med. Chem. 38, 836–847]. We have
hypothesized that a possibly type I b turn/b sheet confor-
mation in the region 1 7–21 may play an important role in
hCt bioactivity. To investigate this hypothesis, analogues of
the potent hCt agonist cyclo17,21-[Asp17,Lys21]hCt (1)
bearing type I (and II¢)orIIb turn-promoting substituents at
positions 18 and 19 were designed, synthesized and their

solution conformations, human Ct receptor binding
affinities and in vivo hypocalcemic potencies were assessed.
The novel analogues include cyclo17,21-[Asp17,
D
-Phe19,
Lys21]hCt (2), cyclo17,21-[Asp17,Aib18,Lys21]hCt (3),
cyclo17,21-[Asp17,
D
-Lys18,Lys21]hCt (4), corresponding
partial sequence peptides containing the lactam-bridged
region 16–22, and nonbridged control peptides. Only 1
showed a higher Ct receptor binding affinity than hCt,
whereas a nalogues 2–4 had similar r eceptor affinities to hCt.
In the in vivo hypocalcemic assay, 3 and 4 were as potent as 1,
whereas 2 completely lost the high potency of 1, s uggesting
that type I ( and II¢) b turn-promoting substituents are fully
compatible with in vivo bioactivity. CD spectroscopy showed
that analog ues 1–4 were markedly bsheet-stabilized com-
pared to h Ct and indicated t he presence of distinct b turn
conformeric populations in each of the analogues.
Unexpectedly, the
D
-amino acid- o r Aib-containing cyclic
analogues 2–4 but not 1 or hCt self-associated i nto SDS
denaturation-stable dimers. Our results demonstrate a
crucial role of the conformational and topological features of
the r esidues in sequence 17–21 and in particular of residues
18 and 19 for human Ct receptor binding and in vivo
bioactivity and also for t he self association s tate of hCt.
These results may assist to delineate the structure-function

relationships of hCt and to design novel hCt agonists for the
treatment of osteoporosis and other bone-disorder-related
diseases.
Keywords: Human calcitonin; b turn/b sheet conformation;
dimerization; receptor binding; hypocalcemic activity.
Calcitonins (Ct) are peptide hormones of 32 amino-acid
residues that have been mainly known for their hypocalce-
mic effect and the inhibition of bone-resorption [1,2].
Calcitonins are u sed therapeutically for the treatment of
osteoporosis and other with bone disorder-related diseases
[1,2]. A marked species-specific differenc e in hypocalcemic
potencies is observed for the Cts. C ts of ultimobranchial
origin, i.e. salmon Ct ( sCt), are the most potent ones,
whereas the human hormone (hCt) h as a strongly reduced
potency [1,2]. Therefore, sCt is the main Ct to be applied
therapeutically to date. However, there is only a 50%
sequence homology between sCt and hCt, which is the cause
for immunogenic reactions in humans w hen t reated with
sCt [3]. Therefore, the development of hCt analogues
bearing high bioactivity and a close structural similarity to
the hCt sequence still remains an important task.
The b iologically active conformation of the Cts yet
remain to be identified. It has been long proposed that the
propensity of the Cts to f orm an amphiphilic a helix in the
region 8–22 might strongly correlate with their bioactivit ies
[4–10]. H owever, while several reports have suggested t hat
this might b e the case for sCt, no evidence h as been
presented for a direct l ink between helicity and bioactivity
for t he human sequence. In contrast, there is increasing
evidence, suggesting that other factors, including a b turn/

b sheet conformation in the middle region of h Ct, overall
conformational flexibility, tertiary structure i nteractions and
interactions of specific residues may be related to b ioactivity
[7,10–15]. NMR studies in nonhelix-inducing media
suggested short a ntiparallel b sh eets and b turns to b e
Correspondence to A. Kapurniotu, Physiological-chemical Institute,
University of Tu
¨
bingen, Hoppe-Seyler-Str. 4, D-72076 Tu
¨
bingen,
Germany, Fax: + 49 7071 2978781, Tel. + 49 7071 2978781,
E-mail:
Abbreviations: Aib, 2-aminoisobutyric acid; BOP, (benzotriazol-
1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; Ct,
calcitonin; DCM, dichloromethane; DMF, dimethylformamide;
DIEA, N,N¢-diisopropylethylamine; DMS, dimethylsulfide; EDT,
ethanedithiol; hCt, human calcitonin; MBHA, 4-methyl benzhydryl-
amine resin; Mtt, 4-methyltrityl; Pip, 2-phenylisopropyl;
OFm, fluoren-9-yl methylester; sCt, salmon calcitonin, TBTU,
O-benzotriazole-N,N,N¢,N¢-tetramethyluronium tetrafluoroborate;
tBu, tert butyl; TIS, t riisopropylsilan; TFE, 2,2,2-t rifluoroethanol;
TFMSA, trifluoromethanesulfonic acid; T rt, trityl.
(Received 15 A ugust 2001, revised 14 November 2001, accepted 19
November 2001)
Eur. J. Biochem. 269, 780–791 (2002) Ó FEBS 2002
present in the middle region of the Cts, while an ahelical
conformation was f ound to be significantly populated only
in alcohol-containing solvents [16–21].
As the Cts are short-sequence peptides of high c onfor-

mational flexibility, their b ioactive conformation may b e
completely different from t hat observed in the media used i n
the N MR studies [22]. Introduction of conformational
constraints has been often proven to be a necessary strategy
towards ÔlockingÕ a p eptide into a bioactive conform ation
[22,23]. The (i,i + 4) side chain-to-side ch ain c yclization
approach has been successfully used for the stabilization o f
bioactive a helical conformations of several medium-size
peptide hormones [ 24,25]. We have previously applied this
approach to constrain t he potentially bioactive, a helical
conformation of hCt [14]. These studies unexpectedly led to
the discovery of the potent but nonhelical hCt agonist,
cyclo17,21-[Asp17,Lys21]hCt (1). Base d on our structure-
activity results and previously published NMR data [19], we
have suggested that a type I bturn/ b sheet conformation
between residues 17 and 21, t hat might have been stabilize d
by the introduced lactam bridge, may play an essential role
in hCt bioactivity [14]. T his b turn could be centered at
amino acids Lys18 and Phe19 a ccording t o the NMR data
[14,19].
To investigate t he importance o f the conformational and
topological features of the region between amino-acid
residues 17 and 21 for hCt bioactivity and the b turn
hypothesis, we have followed two strategies: in the first one,
we prepared a s eries of ring-size analogues of 1 to study the
effect of ring-size o n b turn/ b sheet stabilization a nd hCt
bioactivity [ 15]. T hese studies led v ery r ecently to the
discovery of the superpotent b turn/b sheet-stabilized, hCt-
agonist cyclo17,21-[Asp17,Orn21]hCt [15]. In the second
strategy, w hich we present in this r eport, we designed and

synthesized a nalogues of 1 bearing t ype I- ( and II¢-) and type
II-stabilizing amino acid substitutions for Lys18 and Phe19,
corresponding partial sequence p eptides containing the
lactam-bridged r egion 16–22, and also nonbridged control
peptides (Scheme 1) and studied the e ffect o f these substi-
tutions on conformation, self-assembly state, hCt receptor
binding affinity, and in vivo hypocalcemic activity.
MATERIALS AND METHODS
Materials
Protected amino acids, r esins for pep tide synthesis, BOP,
and TBTU were purchased from Bachem, Novab iochem
and Rapp P olymere. Solvents a nd miscellaneous chemicals
for syntheses, HPLC purifications, SDS/PAGE, and CD
studies were from Merck a nd Aldrich, and were of the
highest purity grade available [15]. S ynthetic hCt and sCt for
CD and bioactivity studies were from Novabiochem. The
saline solution ( 0.9%, w/v) for the hypocalcemic assay w as
from Delta P harma a nd BSA (99%) from Sigma. Medium
and a ll reagents f or cell culture were from G ibco BRL.
Insulin and h ydrocortisone for t he cell culture were from
Sigma (tissue culture grade). Salmon Tyr22
125
I-labelled
calcitonin (
125
I-labelled sCt) w as from Amersham Pharma-
cia Biotech.
Peptide synthesis, purification, and characterization
Solid phase peptide synthesis of 1–6 was performed as
recently described on MBHA with N

a
-Boc-protected amino
acids [14,15,25]. F ollowing deprotection and cleavage from
the resin using HF and scavengers a ccording to our recently
published procedure disulfide bridge formation was
achieved by air oxidation of the crude peptides at 10
)4
M
in 0.1
M
NH
4
CO
3
[15] in the presence of 0 .5–1
M
GdnHCl to
improve solubilities and oxidation yields and its c ompletion
was followed b y HPLC. Cru de, oxidized peptides were
purified by reverse phase HPLC on a C
18
Nucleosil 250/8
column (Grom) with a length of 25 cm, an internal diameter
of 8 mm and a 7-lm particle size. The flow rate was
2.0 mLÆmin
)1
and e luting buffers were: A, 0 .058% (v/v)
trifluoroacetic acid in water and B , 0.05% (v/v) trifluoro-
acetic acid in 90% (v/v) C H
3

CN and w ater. T he elution
program was: 7 min at 30% B, followed by a gradient from
30% to 60% B over 3 0 min.
Peptides 2–6 were also synthesized by the Fmoc/tBu
strategy on Rink–MBHA resin with N
a
-Fmoc-protected
amino acids and standard protection of the side-chains
[Asp(OtBu), Glu(OtBu), G ln(Trt), Cys( Trt), H is(Trt),
Lys(Boc), Tyr(tBu) and Thr(tBu)], with the exception of
residues Lys21 and Asp17 of the cyclic peptides 2– 4,for
which the Mtt, respectively, the P ip groups were applied.
For the side chain-to-side chain cyclization, th ese g roups
were selectively cleaved following treatment o f the peptide
resin with a mixture of 1% t rifluoroacetic acid and 5%
triisopropylsilan (TIS; v/v) in dichloromethane ( DCM;
2 · 2min and 6· 10 min) [26]. Cyclizations were
performed with fourfold excess (benzotriazol-1-yloxy)-tris-
Scheme 1. Amin o-acid sequences of h Ct and analogues 1–6. Ami no-
acid residues are presented with th e one letter code except for Asn17
and T hr21 and th e introduced substitut es. Numbers above the hCt
sequence indicate positions of the sub stituted residues. Th e amino
termini and the C -terminal amide groups o f hCt and the a nalogues are
not shown.
Ó FEBS 2002 Conformationally constrained hCt analogues (Eur. J. Biochem. 269) 781
(dimethylamino)phosphonium hexafluorophosphate (BOP)
and N,N¢-diisopropylethylamine (DIEA) [ 15], and were
usually performed twice (1 · 4hand1· overnight). Pep-
tide resins were then acetylated. P rotected amino acids
(fourfold exces s) were c oupled using O-benzotriazole-

N,N,N¢,N¢-tetramethyluronium tetrafluoroborate (TBTU;
fourfold excess) and DIEA (sixfold excess). The final
cleavage of the peptide and t he side chain p rotecting groups
from the resin was performed with trifluoroacetic a cid/H
2
O/
thioanisol/EDT/phenol (10/0.5/0.5/0.25/0.5) (v/v w ith the
exception o f p henol) [ 27]. F ormation of the d isulfide bridges
of the crude peptides and HPLC purification were
performed as described above.
Identity of the HPLC purified synthetic peptides 1–6 was
verified by matrix a ssisted laser desorption ionization mass
spectrometry (MALDI-MS) with a Kratos Compact
MALDI I (Shimadzu Europe, D uisburg, Germany) and
a-cyano-4-hydroxycinnamic acid as matrix. Purity of the
HPLC purified peptides were also confirmed by analytical
HPLC analyses. The fo llowing results of MALDI-MS
were obtained for the synthesized peptides by the Boc-
and the Fmoc- protection strategy, respectively:
Cyclo17,21-[Asp17,Lys21]hCt (1): MH+ of 3427.5 (calcu-
lated 3428.9); cyclo17, 21-[Asp17,
D
-Phe19,Lys21]hCt (2):
MH+ o f 3427.1 (3428.1, respectively) (calculated 3428.9);
cyclo17, 21-[ Asp17,Aib18,Lys21]hCt (3): MH+ of 3387.3
(3386.5, respectively) (calculated 3386.9); cyclo17,
21-[Asp17,
D
-Lys18,Lys21]hCt (4): MH+ of 3427.0 (3449.3
(Na+ adduct), respectively) (calculated MH+ 3428.9 and

calculated for M+ N a+ 3450.9); [
D
-Phe19]hCt (5): MH+
of 3416 .8 (3418.9, respectively) (calculated 3418.9);
[
D
-Lys18]hCt (6): MH+ of 3 418.8 (3417.9, respectively)
(calculated 3418.9).
Solid phase peptide synthesis of the partial sequence
peptides 1a, 1b , 2a,and3a, their cleavages from the resin,
and HPLC purifications were performed as recently
described on MBHA with N
a
-Boc-protected amino acids
[15]. T he correct masses of HPLC purified peptides
were assessed b y F AB-MS: cyclo17,21-[Asp17,Lys21]hCt
(16–22)-NH
2
(1a): MH+ o f 949.4 (calculated ¼ 949.5);
[Asp17,Lys21]hCt(16–22)-NH
2
(1b): MH+ of 967.5 (cal-
culated 967.15); cyclo17,21-[Asp17,
D
-Phe19,Lys21]hCt(16–
22)-NH
2
(2a): MH+ of 9 49.4 (calculated 949.5); cyc lo17,
21-[Asp17,Aib19,Lys21]hCt(16–22)-NH
2

(3a): MH+ of
906.4 (calculated 906.5).
Far-UV CD spectropolarimetry
CD spectra were obtained with a J-720 spectropolarimeter
(JASCO) at room temperature. Spectra were measured at
0.2 intervals (0.5 n m for the p artial sequence peptides), with
a spectral ban d width of 1 nm, a scan speed of 20 nmÆmin
)1
(50 nmÆmin
)1
for the partial sequence peptides), a response
time of 4 s (8 s for the partial peptides), and represent the
average of three s cans in the range of 195–250 nm (185–
250 nm for the p artial sequence p eptides). Spectra were
measured in 10 m
M
aqueous sodium phosphate buffer
(pH 7.4) and in 10 m
M
sodium phosphate buffer (pH 7.4)
diluted 1/1 (v/v) with TFE and peptides were diluted directly
from their stock solutions into the buffer at the indicated
concentrations. UV a bsorbance a t 274.5 nm was used to
exactly determine the concentrations of the stock solutions
of analogues 1–6 ( 500 l
M
)in1m
M
HCl, using
e

274.5
¼ 1440
M
)1
Æcm
)1
[15]. Stock solutions of the partial
sequence analogues 1a, 1b, 2a,and3a (10 m
M
)were
prepared in 10 m
M
HCl. The spectra are presented as plots
of the m ean residue e llipticity ([h]) vs. the wavelength with
the spectra of the buffer solution alone already subtracted.
Analysis of secondary structure contents
Secondary structure analyses o f the spectra were performed
by multilinear r egression analysis using the program
LINCOMB
and t he r eference spectra of Brahms & Brahms
[28] and Perczel et al. [29].
SDS/PAGE
SDS/PAGE was performed with 18% homogeneous poly-
acrylamide gels using the MINI-PROTEAN II electro-
phoresis system (Bio-Rad) a s previously described [30]. To
obtain comparable results to the CD concentration depen-
dence studies, p eptide solutions were prepared by the same
procedures as for C D, and assembly states were investigated
at final peptide concentrations of 50 l
M

. For SDS/PAGE,
peptide s tock solutions (500 l
M
in 1 m
M
HCl ( see under
CD part) were diluted i nto 1 0 m
M
sodium phosphate
buffer, pH 7.4, at a concentration of 100 l
M
,aswasalso
done for the CD experiments. The peptide solutions were
then diluted with sample buffer [ 30] to a final concentration
of 50 l
M
, boiled for 5 min, and electrophoresed.
Cell culture
T47D cells were obtained f rom the American Tissue Culture
Collection and were cultured in RPMI 1640 containing
10% heat inactivated fetal bovine serum, 1% streptomycin/
penicillin, 0.1 l
M
insulin, and 0.1 l
M
hydrocortisone in 5%
CO
2
and 37 °C. The latter hormones were omitted from the
medium when subculturing cells that were to be used for the

receptor binding assay 1–3 days later. Subculturing was
performed with t rypsin/EDTA as described [31,32] and for
the binding experiments cells were subcultured in 12-well
dishes. Receptor b inding experiments w ere performed when
cells reached 90% confluence (1–3 d ays after s ubculture).
Receptor binding assay
The assay was performed based on previously established
protocols [14,31–33]. Briefly, cells in the 12-well dishes were
washed with NaCl/P
i
(1 mL) at ambient temperature and
then prewarmed (37 °C) assay buffer that consisted of
RPMI 1640 and 0.1% (w/v) BSA w as added to t he
cells (930 lL).
125
I-labelled s Ct (5 lCi, specific activity
2000 lCiÆmmol
)1
) in i ts lyo philized form was reconstituted
in 100 m
M
HCl (200 lL), aliquoted at 4 °C in e ppendorf
tubes (15 lL each), that w ere thereafter kept at )20 °C, and
for e ach 12-well plate one tube was thawed a t room
temperature, diluted with a ssay buffer ( 245 lL) and used
immediately. Twenty microliters of the
125
I-labelled sCt
solution(14.4pmol)werethenaddedtoeachwell,andthe
wells were mixed b y gentle shaking. Thereafter, solutions

(50 lL) of different concentrations of the peptides in assay
buffer were added to t he cells, and following gentle mixing
cells were incubated for 1 h at room temperature. Peptide
solutions were freshly m ade p rior to each experiment by
782 A. Kazantzis et al. (Eur. J. Biochem. 269) Ó FEBS 2002
diluting peptide s tocks ( 500 l
M
in 1 m
M
HCl [14]) in
assay buffer. Binding was terminated by aspiration of the
medium and w ashing of the cells with NaCl/P
i
three times.
Cells were then removed from the wells by s hort treatment
(1 min) with 0.5
M
NaOH (2 · 0.5 mL) and bound radio-
activity was assessed by c-counting (counter efficiency
 70%). Nonspecific binding was determined a s t he binding
of 100 n
M
sCt. This was assessed from 13 independent
experiments to b e 12.94% ( ± 3.59). Specific binding was
the difference between total binding (tracer alone) and
nonspecific binding.
In vivo
hypocalcemic assay
The in vivo hypocalcemic assay in mice w as performed as
described previously [14,15]. Hypocalcemic activities are

plotted as percent reduction of [Ca
+2
] (mean ± SEM of 3–
10 mice) relative to control (3–8 mice). Basal [Ca
+2
]
(mean ± SEM of 84 mice) was 1 0.11 ± 0 .06 mgÆdL
)1
.In
good agreement with our previous findings [14,15],
maximum hypocalcemic e ffect w as caused by 2 lgofhCt
and D[Ca
2+
] ¼ )2.01 mgÆdL
)1
([Ca
2+
] ¼ 8.10 ±
0.15 mgÆdL
)1
), which corresponded to a [Ca
2+
] reduction
of 19.90% ± 0 .81 (mean ± S EM of eight mice treated
with peptide vs. 7 control mice). Statistical significance of the
hypocalcemic effects o f the analogue s vs. the effects of the
respective doses of hCt was assessed using
ANOVA
. S ignifi-
cant statistical significance (P < 0.05) was found for the

effects of 1, 3, and 4 at doses of 10–0.1 ng that corresponded
to the linear parts of the curves as compared to t he respective
hCt effects, whereas the effects o f 2, 5, and 6 were very
similar t o h Ct. O f note, the low maximum hypocalcemic
effects of 2 and 5 also differed significantly from the
maximum effect of hCt (P < 0.05 and < 0.01, respectively).
In addition, the effects of the 100 ng doses of 2 and 5 also
differed significantly from the effect of hCt (P <0.05).
Effective concentrations at 50% o f the maximal e ffect
(EC
50
) were estimated by nonlinear regression analyses of
the data using the software
PRISM
(GraphPad Software, Inc.)
RESULTS AND DISCUSSION
Design of the analogues
A b turn conformation strongly depends on the n ature a nd
chirality of the amino-acid residues a t its corner positions
and even small changes of these residues may dramatically
affect the type a nd stability o f the turn [34,35].
To investigate the importance of t he type of the
postulated turn for hCt b ioactivity, the i +1turn-residue
L
-Phe19 of 1 was r eplaced by
D
-Phe19 t o giv e cy clo17,21-
[Asp17,
D
-Phe19,Lys21]hCt (2) ( Scheme 1). This substitu-

tion was expected to stab ilize a type II btur n conformation
[34,35] which, according to our hypothesis [14,15], should
have a negative effect on bioactivity. Next, cyclo17,21-
[Asp17,Aib18,Lys21]hCt (3) was designed (Scheme 1 ). The
substitute Aib18 for Lys18 w as chosen. Due to its s trong
conformational space restriction [34], t he Aib residue should
favor the postulated type I b turn conformation centered
at the Lys–Phe bond and was expected to result in a
bioactive analogue [34,36–38]. Next, cyclo17,21-[Asp17,
D
-Lys 18,Lys21]hCt (4) was designed in which the chirality
of the putative i + 1 turn-residue of 1, or Lys18, was
inversed (Scheme 1). T his substitution was expected to
stabilize a type II¢ b turn, which places the side chains of the
corner residues at roughly th e same position a s a type I
b turn [34,35,39]. Thus, this substitution was expe cted to
maintain or increase the bioactivity of (1) [34]. Enhancement
of potency through s tabilization o f a type II ¢ turn has been
previously reported f or somatostatin [34,40]. Importan tly,
studying t he structure–activity relationships of a type I I¢-
stabilized analogue of 1 would offer direct information
about the role of the topographical features of the side
chains of the residues i n region 17–21 for receptor binding
and in vivo bioactivity.
Residues Lys18 and Phe19, that were elected to be
substituted, had not previously been known to be important
for hCt bioactivity or i ts overall co nformation. However, to
be able to s eparately evaluate effects of the substitutes alone
vs. the introduced confo rmational restrictions, the nonbrid-
ged peptides [

D
-Phe19]hCt (5)and[
D
-Lys18]hCt (6)were
also synthesized and s tudied (Scheme 1).
CD spectroscopy describes the average conformation
of polypeptides and the contribution o f a local confor-
mational feature such as a f our-residue b turn to the CD
spectrum of a polypeptide of 32 amino acids will usually
remain unrecognised due to o ther secondary s tructure
elements [41]. Therefore, t o be able to obtain more detailed
information about a potential bturn stabilization we also
synthesized a nd studied the conformation of cyclo17,21-
[Asp17,Lys21]hCt(16–22)-NH
2
(1a), cyclo17,21-[Asp17,
D
-Phe19,Lys21]hCt(16–22)-NH
2
(2a), cyclo17,21-[Asp17,
Aib19, Lys21]hCt(16–22)-NH
2
(3a), and [Asp17,Lys21]
hCt(16–22)-NH
2
(1b) that comprise mainly the lactam
bridge-containing region 16–2 2 of analogues 1, 2 and 3,
respectively, and als o a linear control peptide for 1a ,
analogue 1b.
Conformational analyses by CD: studies of hCt

and the analogues in aqueous buffer, pH 7.4
CD spectra of hCt and analogues 1–6 (Fig . 1A) were
measured at concentrations of 5 l
M
where all peptides were
found in preliminary CD concentration-dependence studies
to be in a monomeric state (data not shown) [14,15]. Visual
inspection of the spectra indicated that all bicyclic analogues
had s imilar o verall conformations. Secondary structure
analyses of the spectra with the reference spectra of Brahms
& Brahms [ 28] suggested b sheet contents of about 40% for
1–4, the rest being predominantly random coil. hCt
contained 27% bs heet, the r est consisting mainly of random
coil. These results indicated an about 50% increase of
b sheet contents in 1–4 compared to hCt. Since the peptides
were monomers, t his fi nding suggested they were b turn
stabilized. O n t he other hand, the similarity of the spectra of
2–4 to the spectrum o f 1 suggested that the introduced
substitutes d id not affect the o verall conformation of 1.
Interestingly, also 5 and 6 contained 50% more b sheet t han
hCt, which suggested that nature and c hirality of residues 18
and 19 are strongly associated with b sheet stabilization
of hCt.
The CD s pectra of 1a , 1b,and3a exhibited a strong
negative b and between 185 and 190 nm that is characteristic
for both turn-types (type I and II) [42]. T he spectrum of 2a
did not exhibit such a minimum. Its shape indicated t hat the
peptide was in a c onformeric e quilibrium state and that it
Ó FEBS 2002 Conformationally constrained hCt analogues (Eur. J. Biochem. 269) 783
contained contributions of three previously repo rted bturn

reference s pectra: one class C CD spectrum [42] (a negative
band between 200 and 210 nm, a weak negative b and at
about 220 nm, and a possitive band between 180 and
195 nm), one spectrum correponding to an op en or ÔZÕ
conformation (one minimum at 195–200 nm) [43], and the
third component could be the type I and II bturn spe ctrum
according t o Brahms a nd Brahms [28] that exhibits a
characteristic minimum at a bout 225 nm and a maximum
at 210–220 nm.
The spectra of 1a and 1b were very similar to each other.
The spectra of both peptides showed positive bands at
about 220 nm (Fig. 1B) th at most likely arise from coupling
between the phenylalanyl (there are three P he residues in
sequence 1 6–22) and the amide chromophores [44,45]. This
suggestion w as further s upported by t he observation t hat
the intensity of the 220 band, that most likely corresponds to
the phenylalanyl La band [42,45], was significantly le ss in 1b
and its maximum was blue shifted compared to 1a [44,45].
The similarity between the CD curves of 1a and 1b
suggested that the lactam bridge of 1a did not significantly
constrain t he aqueous conformation of 1b . However, the
intense positive band of 1a at 220 nm suggest ed that
cyclization may have resulted in topological changes of the
side chain of one or more Phe r esidue(s). Interestingly, these
positive bands were not present in 2a and 3a suggesting a
significant effect of residues
D
-Phe19 and Aib18 on back-
bone conformation and t opography of the Phe side chain(s)
in sequence 16–22. The s pectrum of 3a had, except for t he

minimum at 185–190 nm, a lso a strong maximum at about
198 nm and a marked negative band at a bout 212 nm. This
shape is i ndicative of an equilibrium of the t wo forms o f
type I b turn conformers that have been shown to be
populated by cyclic bturn model peptides [43].
Together, the results of C D s pectroscopy under aqueous
conditions, pH 7.4, suggested that all c yclic peptides had a
stabilized b turn/b sheet structure as c ompared to hCt and
that distinct bturn populations and their mixtures were
present in each of them. This latter finding su ggested that
the lactam bridge did not completely restrict the confor-
mational flexibility of the analogues and was consistent with
results o f s everal NMR and CD studies o n cyclic model
peptides [42,43,46].
Conformational analyses by CD: studies of hCt
and the analogues in 50% TFE in aqueous buffer, pH 7.4
Fifty percent aqueous TFE is a solvent system that is
applied as a structure-inducing agent to short polypeptide
sequences that usually exhibit strong conformational flex-
ibility in pure aqueous buffers [23,47–49]. The conformeric
states that are stabilized und er these conditions have been
often related to bioactive, i.e. receptor-bound, con forma-
tions [50]. In a ddition, TFE is a solvent that is able to
stabilize the a helical conformation in a flexible polypeptide
chain that has an a helical propensity [51].
Fig. 1. Far -UV CD spectroscopy of hCt and analogues 1–6 (A,C) and 1a, 1b, 2a, and 3a (B,D) in aqueous buffer (A,B) and in 50% aqueous TFE (C,D).
The spectra were recorded in 10 m
M
aqueous sodium phosphate buffer, pH 7 .4 (A,B) and in 50% TFE in aqueous phosphate buffer, pH 7.4 (C,D)
at a peptide concentration of 5 l

M
(for hCt and 1–6)and1m
M
(for 1a, 1b, 2a ,and3a) and at room temperature.
784 A. Kazantzis et al. (Eur. J. Biochem. 269) Ó FEBS 2002
As shown in F ig. 1C, TFE had a strong structuring and
a helix inducing effect on hCt and the nonbridged peptides 5
and 6 that conta ined 40–50% a helical components accord-
ing to s econdary structure a nalysis by t he reference s pectra
of Brahms and Brahms [28]. This was consistent with the
long described a helix-forming propensity of the middle
region of the calcitonin sequence [4,16,52,53]. In contrast,
nearly no a helical contents were found for all bridged
analogues w hich contained i nstead 40–50% b sheet struc-
ture, t he rest being mainly r andom coil. Thu s, it appears
that the a helix-inducing e ffect of TFE on 1–4 was not as
strong as on hCt, 5, and 6, most likely b ecause 1–4 were
already significantly constrained by t he lactam bridges. Of
note, the CD spectra of 1–4 were very similar to each other.
Taken together, the results in 50% TFE were consistent
to the ones under pure aqueous conditions and suggested
that the introduction of the substituents at positions 18 and
19 of analogue 1 did not affect its o verall con formation. In
addition, the studies in 50% TFE confirmed our earlier
observations [14] that the 20- membered Asp17 t o Lys21
lactam bridge had an a helix-destabilizing a nd a bsheet-
stabilizing effect on hCt. It has been reported that (i,i +4)
Asp/Lys bridges may result in both stabilization [ 54] and
destabilization [55] of a helices. Together with t hese reports,
our results suggest that the effect of such bridges on a helix

stabilization strongly depends on the particular peptide
sequence the bridges a re being i ntroduced into [14].
CD spectra of 1a , 1b, 2a,and3a in 50% TFE were
measured next (Fig. 1D). All three cyclic analogues, but not
the line ar 1b, exhibited a marked positive p-p*bandat
about 195 nm that has been observed in type I and type II¢
b turn models [42,43]. The CD spectra of these t hree
peptides differed strongly from each other, however, in the
region between 200 and 250 nm: the spectrum of 1a had a
clear minimum at about 208 nm and its shape was
reminiscent of a class C spectrum (see above), or a type I
b turn, as has been found for cyclic model peptides [42,43].
The spectrum of 2a had a clear minimum at 225 nm.
Together with the maximum a t  195 nm, this minimum
suggested an conformeric equilibrium between a type I
b turn [28] with another b turn population [ 35]. The
spectrum of 3a had a pronounced minimum a t  215 nm
and a shoulder at  225 nm that, together with the
maximum a t 195 nm, indicated t he presence of the two
type I b turn conformeric forms that have been described by
Perczel et al. [43]. I mportantly, the 50% TFE solvent system
allowed for a clear distinction between the conformation of
1a vs. 1b which showed only weak CD b ands. Such
distinction was not possible under pure aqueous conditions
(see above). Together with the results in aqueous buffer, the
TFE-data suggested that the lactam b ridge stabilized a
specific conformeric population in 1a, which, h owever,
retained also a h igh degre e of flexibility.
Oligomerization studies by CD and SDS/PAGE analysis
For the ab ove d escribed CD studies , peptide concentrations

as low a s 5 l
M
were applied which are clo se to physio-
logically relevant concentrations. Confirming previous
findings [14,15], no con centration dependence of t he CD
spectra or aggregation was found between 5 and 100 l
M
for
hCt and also for 1 in aq ueous buffer, pH 7.4. This suggested
that the conformations observed by CD were adopted by
monomeric peptides. However, there was a striking con-
centration dependence of the CD spectra o f 2–4 between
5 and 100 l
M
(Fig. 2 A), that was indicative of peptide self
association [5]. The mean residue ellipticities at 202 nm
([h]
202
), that corresponded t o the minima of the C D spectra,
Fig. 2. Studies on t he oligomerization propensity o f h Ct and 1–6.
(A) CD concentration dependence studies: the concent ration depen-
dence ( 5–100 l
M
) of t he mean residue ellipticity at 202 nm ([ h]
202
)
for analogues 2, 3,and4 in aqueous buffer is shown. In the inset
the line ar regression analysis of the data points of 2 th at were intro-
duced to t he eq uation [([ h]
202(observed)

) [h]
202(mono)
)/[analogue]]
1/2
¼
[2/K
d
([h]
202(dimer)
) [h]
202(mono)
)]
1/2
([h]
202(dimer)
) [h]
202(observed)
)ispre-
sented. CD measurements at various analogue concentrations ([ana-
logue]) were perfo rmed in 10 m
M
sodium phosphate buffer, pH 7.4,
and at room t emperature. (B) and (C) SDS/PAGE analysis and silver
staining of hCt and an alogues 1–6. Molec ular mass markers (in kDa,
lane 1). (B) Lane 2, hCt; lane 3, 6;lane4,2;lane5,3;lane6,4;lane7,1,
and (C) lane 1, molecular mass markers; lane 2, hCt; lane 3, 5;lane4,2.
100 l
M
solutions of hCt and the analogu es in 10 m
M

phosphate
buffer, pH 7.4 w ere diluted 1 : 1 with sample buffer containing 2 %
SDS, boiled and electrophoresed as described under Materials and
methods.
Ó FEBS 2002 Conformationally constrained hCt analogues (Eur. J. Biochem. 269) 785
decreased with increasing concentrations, s uggesting that
the peptides b ecame more ordered during s elf association
[42]. Plateau values were reached at 50 l
M
(Fig. 2 A). The
change of [h]
202
was b est fitted to an equation describing
peptide cooperative dimerization [5].
Dimerization was also confirmed by SDS/PAGE
(see below). [h]
202
for the monome rs ([h]
202(mono)
)were
obtained at 5 or 1 l
M
, where all analogues were essentially
monome ric, and were )6059 (2), ) 6802 (3)and) 7680
deg.cm
2
/dmol (4). Plots of the observed [ h]
202(obs)
vs.
{([h]

202(obs)
) [h]
202(mono)
)/[analogue]}
1/2
(i.e. Figure 2A,
inset) gave dissociation constants o f 1 .44 · 10
)5
,1.10 · 10
)5
and 2.47 · 10
)5
M
for the dime rs of 2, 3, and 4, respectively.
Human Ct belongs to the family of aggregating and
amyloid-forming polypeptides [56,57]. Fibrillation of aque-
ous hCt solutions strongly hampers its therapeutic use for
the t reatment of bone-disorder-related diseases [ 58]. Sec-
ondary structure analysis of spectra corresponding to
monomeric and dimeric popu lations suggested that dimer-
ization occured at the expense o f unordered structures a nd
was accompanied b y a s ignificant antiparallel b sheet
stabilization. The l atter one was most likely due to
intermolecular, structure-stabilizing interactions [59,60].
According t o the reference spectra of Perczel et al.[29],
dimerization of 2–4 was accompanied by increases in
antiparallel b sheet contents of 18%, 14%, and 20%,
respectively. These results are consistent with a recently
proposed model of hCt aggregation at pH 7.4 into fibrils via
formation and stacking of antiparallel b sheets [57].

As observ ed for hCt a nd 1, CD concentration depen-
dence studies of 5 and 6 showed that these analogues also
did not aggregate. This suggested that self-assembly of 2, 3,
and 4 was related to both the confor mational r estriction, i.e.
the b turn/b sheet stabilization that had been achieved by
the lactam bridge, and the topological features of the side
chains of residues 18 a nd 19. It has b een previously
suggested that several hydrophobic residues, that may
occupy the one face of the putative a helical region 8–21 of
hCt, participate in the initial helix–helix association step
[61]. This s tep i s then followed by f ormation of b sheet
aggregates [61]. Thus, a reason for the increased b sheet
formation and oligomerization propensity of 2–4 could be
the changed topogr aphy of the side chains of r esidues 18
and 19 in 2–4. This may have led to formation of an
hydrophobic face in the lac tam bridge-stabilized b sheet a n d
an increase d dimerization and oligomerization propensity
[55,62]. Association of b sheets i nto multimers and fibrils
would be consistent with models of hCt fibrils [56,57].
To further study the self-assembly states of 2–4,50l
M
solutions of hCt and the analogues were next subjected to
SDS/PAGE analysis (Fig. 2B,C). B ased on the results of the
CD studies, 2, 3, and 4 were expected to predominantly
consist o f (noncovalent) dimers. Stability of the dimers
towards S DS treatment conditions (2% S DS, 100 m
M
2-mercaptoethanol, 100 °C f or 5 min [30]) was not known.
In fact, mixtures of peptide monomers and dimers at a ratio
of about 40/60 were observed in 2, 3, and 4 (Fig. 2B),

whereas hCt, 1, 5, and 6 mainly consisted of monomers
(Fig. 2C). These results were in good agreement with the
dimerization propensity of 2–4 as observed by CD. O f note,
the dimeric forms o f these analogues were resistent to the
denaturating SDS/PAGE conditions, indicating an unusu-
ally strong self association potential. Such strong aggrega-
tion potential has been described for other amyloid
polypeptides, including hu man islet amyloid polypeptide
(IAPP), which shares a receptor and hypocalce mic activity
with hCt [30,63–66].
Receptor binding affinities
hCt exerts its biological effe cts v ia binding to a receptor that
belongs to the family of seven-tran smembrane G -protein
coupled re ceptors [10]. C t r eceptors are localiz ed in bone
and k indney and also in the central nervous system, i.e. the
brain [10]. In addition, specific high affinity receptors for Ct
have been found in several cancer cell lines including the
human breast cancer cell line T47D [10,31].
We have used the T47D cell line t o assess human receptor
binding affinities of the synthetic analogues as compared to
sCt, which is the strongest known naturally occuring Ct
ligand, and h Ct which is a weak ligand [33]. Binding
affinities were assessed via the competitive i nhibition of the
Fig. 3. Hu man Ct receptor binding of hCt, sCt
and analogues 1–6 to T47D cells assessed via
displacement o f bound
125
I-labelled sCt. Cells
were prepared and incubated with
125

I-labelled
sCt as described under Materials and meth-
ods. Specific r adioligand binding is plotted vs.
the concentration of c ompeting sCt, hCt, and
1–6 as indicated. Data for 1–6 represent the
mean ± SD for three to five independen t
experiments and data for h Ct and sCt are t he
mean of 13 and 1 4, respectively, assays.
786 A. Kazantzis et al. (Eur. J. Biochem. 269) Ó FEBS 2002
specific binding of the r adioligand
125
I-labelled sCt that
binds with high affinity and selectivity to the Ct receptors of
this cell line [33]. As shown in Fig. 3, 1 sh owed increased
binding affinity compared to hCt. Receptor binding affinity
of 1 (IC
50
¼ 2n
M
) was threefold lower than the a ffinity of
sCt (IC
50
¼ 630 p
M
), that was about 6 times more potent
than hCt ( IC
50
¼ 4n
M
) [33]. The higher binding affinity of

1, compared to hCt, was consistent with both its high
binding affinity to the rat brain Ct receptor and its increased
in vivo hypocalcemic potency compared to hCt [14,15].
Analogues 2–4 had nearly i ndistinguishable binding
isotherms to hCt (IC
50
¼ 4n
M
) suggesting that chirality
of residues 1 8 and 19 plays a crucial role for hCt binding
affinity to t he T47D receptors. This result was c onfirmed by
the results of the binding studies o f 5 and 6; 5 showed a
significantly reduced binding affinity (IC
50
¼ 18 n
M
)as
compared to hCt (IC
50
¼ 4n
M
), while 6 showed almost
no binding.
Together, the obtained receptor binding d ata showed that
(a) the introduction of the Asp17,Lys21-lactam bridge in
hCt resulted in a significant increase in human Ct receptor
binding affinity (b) r esidues 18 and 19 of hCt and their
chirality are strongly associated with receptor binding
affinity (c) inversement of the chirality of residues 18 and
19 strongly reduces the binding affinity of hCt and (d) the

Asp17,Lys21-lactam br idge leads to a partial inversement of
the latter effect.
In vivo
hypocalcemic activities
To directly assess the biological relevance of the introduced
substitutions, we n ext studied in vivo hyp ocalcemic potencies
in mice [1–3,14,15]. Analogues 3 and 4 exhib ited identical
bioactivity to 1 [14], which was 5 times more potent than
hCt (Fig. 4). However, 2 was co mpletely devoid of the
increased bioactivity of 1.Analogue2 hadanEC
50
of 25 ng
that corresponded to a hypocalcemic potency that was even
lower than t he potency of hCt (EC
50
, 20 ng). Of note, 2 was
unable to reach the maximum hypocalcemic effect o f hCt
(20%) ( caused by 2 lg hCt), even when 10-fold higher doses
were applied. The maximum effect of 2 was caused by the
20-lg dose a nd was at 16.1%. A nalogue 5 had t he same
dose–response c urve as 2. This i ndicated that t he confor-
mational restriction was not capable of r eversing the
negative e ffect of the inversement of ch irality of Phe19 on
in vivo bioactivity of 1. In contrast, 6 had the same potency
and maximum effe ct as hCt which suggested that inverse-
ment of chirality of L ys18 was w ell tolerated.
Correlation of solution conformations with receptor
binding affinities and
in vivo
bioactivities of hCt

and analogues 1–6
The results of the CD studies and the studies on receptor
binding affinities and hypocalcemic potencies in vivo of hCt,
sCt, analogues 1–6 and/or 1a, 1b, 2a,and3a are summa-
rized in Table 1. Our findings that 3 and 4, that contain type
IandII¢ b-turn-promoting substitutes, had the same
hypocalcemic potency as 1,whereas2, that contains the
type II b-turn-promoting substitute, lost t he high potency of
1 supported the suggestion that a type I b turn/ b sheet i n the
region 17–21 of hCt may play an important role in in vivo
bioactivity [14,15]. Because 3 and 4 were also designed to
contain the same side-chain topology in the turn-corner
residues as 1, the above findings also indicated t hat a type I
b turn side-chain topography in region 17–21, might be fully
compatible with in vivo bioactivity.
Our CD studies showed that the overall secondary
structure of 2 was very s imilar to the ones of 1, 3,and4.In
contrast, the CD studies of the partial sequence analogues
showed that the Asp17,Lys21-lactam bridge and the
substitutes r esulted i n a stabilization o f d istinct bturn
conformeric populations in each one of the s hort a nalogues.
In particular, t he studies in 50% TFE indicated that type I
b turn conformers were the mostly populated b turn
conformers in 1a and 3a, whereas confo rmeric e quilibria
of several turn-types w ere p redominant under pure a queous
conditions. Importantly, t he CD studies both under pure
aqueous and in 50% TFE indicated that 2a populated
distinct bturn conformeric states compared to 1a,and3a.
Fig. 4. Hyp ocalcemic potencies of hCt and the
analogues 1–6. Serum calcium levels were

measured in gro ups of 3–10 mice pe r dose and
3–8 control mice 1 h a fter subcutane ous
injection of the peptide s olution or vehicle
alone. Hypocalce mic activities of ea ch dose
are expressed as percent reduction of calcium
(mean ± S EM) caused by the p eptide relative
to control.
Ó FEBS 2002 Conformationally constrained hCt analogues (Eur. J. Biochem. 269) 787
Furthermore, 2 was t he only analogue with reduced in vivo
activity as compared to hCt, suggesting a crucial role of the
topological features of the side chain of Phe19 i n confor-
mation and in vivo bioactivity o f hCt.
In the T47D receptor bindin g studies, only 1 showed a
higher binding affinity than hCt, whereas 2–4 were equally
potent t o hCt. Analogues 5 and 6 showed decreased binding
affinities as compared to hCt. Thus, t hese studies demon-
strated the cr ucial role of residues 18 a nd 19 and their
chirality for human receptor binding. M oreover, these
findings suggested that the side chains of residues 18 and 19
and/or of other residues i n r egion 1 7–21 may be d irectly
involved in receptor binding. T hese results we re consistent
with a recent model of ligand–Ct-receptor interaction and
activation. According to this model, all three regions of the
Ct sequence, including the N-terminal l oop 1–7, the
potential ahelica l region 8 –22, and t he C-terminal region
22–32 interact with distinct domains of the Ct receptor
[10,67]. Accordingly, even small or local changes in confor-
mational and topographical features in Ct, may r esult i n
dramatic changes in binding affinities an d efficacies [10].
Taken together, our CD and bioactivity studies suggested

that both r eceptor binding affinity and in vivo bioactivity of
hCt are associated with specific local conformational
features of the backbone and with topological features of
side chains of residues within the region 17–21. Previous
studies have shown that replacement of Phe19 by Leu does
not affect the in vivo hyp ocalcemic potency of hCt [1]. The
aromatic rings of the three P he residues Phe16, Phe19, and
Phe22 in hCt have been suggested to occupy the hydro-
phobic side o f the potential amphiphilic a helical region of
hCt [5,18]. Based on this model, inversement of chirality of
Phe19 of hCt, as performed in 2, would disrupt the
hydrophobic face of the putative b ioactive a helical confor-
mation. This could be one plausible explanation for the
observed strong decrease of in vivo bioactivity in 2 as
compared to 1 [5,18]. It is noteworthy that the CD spectrum
of 1a indicated interactions between phenylalanyl and
amide chromophores, whereas no such interactions were
observed in the spectrum of 2a.
For analogues 1 and 2, we observed a clear correlation
between hypocalcemic activity and receptor binding affinity.
In contrast, n o such correlation was observed for 3 and 4.
These latter a nalogues had lost the high receptor binding
affinity of 1 and w ere similarity potent t o hCt, whereas they
maintained the increased in vivo hypocalcemic potency of 1.
Similarly, 5 and 6 had reduced binding affinity to the T47D
receptor compared to hCt and the same hypocalcemic
potency to hCt. It is believed that the hypocalcemic activity
of the Cts is the result of a receptor-me diated inhibition of
bone resorption via a direct effect of Ct on osteoclasts and of
the c alciuretic effect of Ct on kidney [ 10]. Therefore, in vivo

bioactivity of the Cts is d etermined by many different
factors including receptor binding, signal transduction,
receptor regulation, as also bioavailability and biodegrada-
bility of the ligand [9,10,68]. The analogues 2–6 presented
here differ from 1 and hCt only in the chirality o f residues 19
and/or 18 and/or the p resence of Aib instead of Lys18.
Thus, these analogues are expected to have in vivo ahigher
proteolytic stability t han 1 and hCt [69]. Therefore, the
Table 1. S ummary of the results of the CD s tudies, the receptor binding affinities, and the hypocalcemic potencies in vivo of hCt, sCt, analogues 1–6
and/or the partial sequence analogues 1a, 1b, 2a, and 3a. The CD data of the partial sequence analogues 1a, 1b, 2a,and3a are presented, because
there w ere no differences between the spectra of the respective complete s equence peptides. CD spectra were measured in 1 0 m
M
phosphate buffer,
pH 7.4 and in 50% TFE in 10 m
M
phosphate buffer, pH 7.4, at room temperature. Peptide concentrations were 1 m
M
(for 1a, 1b, 2a,and3a)and
5 l
M
(for hCt, sCt, and 1–6). Exp. turn, expected stabilized turn based o n the analogue design strategy. Min., minimum of CD spectrum; max.,
maximumofCDspectrum.
Analogue
(exp. turn)
Conformational analysis of partial sequence peptides by CD
Receptor binding
affinities (in vivo)
Hypocalcemic potencies
In aqueous solution In 50% aqueous TFE
1 Min., 190 nm; max., 220 nm:

type I and II b turn conformers;
Min., 208 nm; max.,
195 nm:
Threefold higher than
hCt
Fivefold more potent than
hCt
interactions of phenylalanyl with type I b turn
amide chromophores
2 (type II) Max., 185 nm;
min., 208 nm and 222–224 nm:
type I and II b turn conformers
Min., 225 nm; max.,
195 nm: equilibrium:
type I b-with another
b turn conformer
Same as hCt Less potent than hCt; has
80% of hCt maximum
effect
3 (type I) Min., 185–190 and 212 nm;
max., 198 nm:
Min., 215 nm; max.,
195 nm: equilibrium
two type I conformers of two type I
conformers
Same as hCt Same as hCt
4 (type II¢) ND ND Same as hCt Same as hCt
5 ND (5 random coil as hCt) ND (5 a helix as hCt) Fivefold lower than hCt As 2
6 ND (6 random coil as hCt) ND (6 a helix as hCt) Nearly no binding As hCt
hCt Spectrum of 1b: similar to 1a;

less maximum at 220 than in 1a
Spectrum of 1b: very
weak bands
Sixfold lower than sCt the strongest potency;
sCt sCt: mainly random coil
a
sCt: a helix
(more than hCt)
a
The strongest binding
(IC
50
¼ 630 p
M
)
95-fold lower than sCt;
95-fold higher than hCt [15]
a
The sCt data were not shown in this work (see also references [4,12]).
788 A. Kazantzis et al. (Eur. J. Biochem. 269) Ó FEBS 2002
results of our studies support the notion that the in vivo
hypocalcemic potency of the C ts is directly associated to a
distinct bioactive con formeric population r ather than t o
differences in proteolytic degradation rates [1,7,14,15,18,
67,70–72].
In conclusion, our structure activity studies supported the
suggestion that a type I bturn/ b sheet conformation in the
region 17–21 may play a n important role in hCt b ioactivity
and showed that the conformation and the topological
features of the side chains o f amino acid residues 18 a nd 19

are strongly associated with the self-assembly state, the
human receptor binding affinity and the in vivo hypocalce-
mic potency of hCt.
ACKNOWLEDGEMENTS
We are grateful to J. Bernhagen for his he lp with the receptor binding
and the hypocalcemic assay. We thank H. R. Rackwitz and
M. Schno
¨
lzer for help with the HF cleavage and D. Finkelmeir f or
excellent technical assistance with the cell culture and the receptor
binding assay. We thank N. Greenfield for the CD programs. We thank
S. Stoeva and and her group for the the MALDI-MS and
H. Bart holoma
¨
and R. Mu
¨
ller for FAB-MS. We thank K. T enidis,
R. Kayed , and K. Sweimeh for their contributions to c ertain
experimental parts of this work. We thank W. V oelter for supporting
this work . This w ork was supported by the Deutsch e Forschungs-
gemeinschaft (DFG) grant numbers Ka 979/2-1 an d -2.
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