How are 1,2,3-triazoles accommodated in helical secondary structures?

Article abstract of DOI:10.1039/c8ob00686e

1,4-Disubstituted-1,2,3-triazole (Tz) is widely used in peptides as a trans-amide bond mimic, despite having hazardous effects on the native peptide activity. The impact of amide bond substitution by Tz on peptide secondary structures is scarcely documented. We performed a Tz scan, by systematically replacing peptide bonds following the Aib residues with Tz on two model peptaibols: alamethicin F50/5 and bergofungin D, which adopt stable α- and 3₁₀ helices, respectively. We observed that the Tz insertion, whatever its position in the peptide sequences, abolished their antimicrobial activity. The structural consequences of this insertion were further investigated using CD, NMR and X-ray diffraction. Importantly, five crystal structures that were incorporated with Tz were solved, showing various degrees of alteration of the helical structures, from minor structural perturbation of the helix to partial disorder. Together, these results showed that Tz insertions impair helical secondary structures.

Full text of DOI:10.1039/c8ob00686e

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Inguimbert, Org. Biomol. Chem., 2018, DOI: 10.1039/C8OB00686E.
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DOI: 10.1039/C8OB00686E
Organic and Biomolecular Chemistry
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How are 1,2,3-Triazoles accommodated in helical secondary
structures?
Received 00th January 20xx,
Accepted 00th January 20xx
Khoubaib Ben Haj Salah,a Sanjit Das,a Nicolas Ruiz,b Vanessa Andreu,c Jean Martinez,d Emmanuel
Wenger,e Muriel Amblard,d Claude Didierjean,e Baptiste Legrand,*d and Nicolas inguimbert *c
DOI: 10.1039/x0xx00000x
1,4-disubstituted-1,2,3-triazole (Tz) is widely used in peptides as a trans-amide bond mimic, despite having hazardous
effects on the native peptide activity. The impact of amide bond substitution by Tz in peptide secondary structures is
scarcely documented. We performed a Tz scan, by systematically replacing peptide bonds following the Aib residues by Tz
on two model peptaibols: alamethicin F50/5 and bergofungin D, which adopt stable α- and 310 helices, respectively. We
observed that the Tz insertion, independently of its position in the peptide sequences, abolished their antimicrobial
activity. The structural consequences of this insertion were further investigated using CD, NMR and X-ray diffraction.
Importantly, five crystal structures that incorporated Tz were solved, showing various degrees of alteration of the helical
structures, from minor structural perturbation of the helix to partial disorder. Together, these results showed that Tz
insertions impair helical secondary structures.
www.rsc.org/
emerged and was extensively applied on cyclic peptides.18,19,20
The insertion of Tz within the backbones of linear biologically
active peptides such as bombesin21,22 or kisspeptin23 increased
activity by extending metabolic stability, whereas the activity
of enkephalin decreased.24 However, the impact of the Tz
incorporation on the three-dimensional structure of these
peptides has not been studied despite the close relationship
between their conformation and activity.25,26 To the best of
our knowledge, only two studies have monitored Tz insertion
within the sequence of alpha-helical peptides. In the first
Introduction
Since the pioneering work of Meldal and Sharpless, copper (I)-
catalyzed azide-alkyne cycloaddition (CuAAC) affording 1,4-
disubstituted-1,2,3-triazoles (Tz) have generated broad
interest throughout the field of chemistry.1,2,3 Peptide
chemistry is certainly one of the fields that has benefited most
from click chemistry.4,5,6,7 CuAAC was applied to the synthesis
of otherwise inaccessible cyclic peptidomimetics,8 to the
ligation of unprotected peptide fragments to yield Tz-
containing proteins.9,10 The incorporation of Tz into peptides
study, the pLI mutant of the 33 amino acid long
-helical
coiled coil GCN4 transcription factor was selected as a model.27
A comparison of the X-ray structure of the native peptide with
three analogues where the X-Leu (X: Lys or Glu) dipeptides
and proteins stabilizes discrete conformations such as
strand,11 and
-helix through side chain to side chain
cyclization.12,13,14 In several studies, the Tz ring was considered
-

were replaced by Gly[Tz]Leu 3a showed different outcomes
as
a non-classical amide bond bioisostere that could
ranging from partially disordered to fully conserved structures.
As the native sequence was not entirely conserved, to
distinguish the effect of Tz incorporation from that of the
substitution of the Lys or Glu residues by the flexible Gly
residue was not an insignificant task. In a recent study, a
accommodate any peptide secondary structure.15,16,17 Its
hydrogen bonding capacity and its planar character have
raised considerable interest for the use of the 1,4-
disubstituted Tz as a trans peptide bond surrogate (Figure 1).
Consequently, the concept of Tz as an amide bond mimic has
structured helical
-aminoisobutyric acid (Aib) oligomer with
the sequence Cbz-Phe-(Aib)4-Aib6-(Aib)4-Gly-NH was fully
2
a.USR 3278 CRIOBE, PSL Research University, EPHE-UPVD-CNRS, Université de
Perpignan Via Domitia, Laboratoire d’Excellence « CORAIL ». Bâtiment T, 58
avenue P. Alduy, 66860 Perpignan, France. E-mail: nicolas.inguimbert@univ-
perp.fr
destabilized in solution by replacing Aib6-Aib7 dipeptide with
Gly[
Tz]Aib.28 Different results were reported in response to
differences in peptide sequence, position and the number of
Tz substitutions. However, no clear trend emerged, prompting
researchers to anticipate Tz conformational effects through
molecular modelling.29 The specific properties of Tz compared
b.Laboratoire Mer Molécules Santé. Université de Nantes, UFR de Sciences
pharmaceutiques et biologiques, 9 rue Bias - BP 61112, 44035 Nantes, France.
c. Akinao, 58 avenue P. Alduy, 66860 Perpignan, France.
d.Institut des Biomolécules Max Mousseron (IBMM), Univ Montpellier, CNRS, UM,
ENSCM, Montpellier, France. E-mail: baptiste.legrand@umontpellier.fr
e. CRM2, UMR UL-CNRS 7036, Faculté des Sciences et Technologies, Université de
Lorraine, 70239 Boulevard des Aiguillettes, 54506 Vandoeuvre-lès-Nancy, France.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
with the amide bond (stretching of the CCdistance of ~1
Å, higher dipole moment and two nitrogen atoms as hydrogen
bond acceptors, Figure 1), do not appear to be fully compatible
with peptide helical structures. In this study we investigated
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the effects of Tz substitution on the activity and structures of
two stable helical peptaibols: the 20-mer alamethicin F50/5
(Alm) 4a and the 14 amino acid residues of bergofungin D
Table 1. Yields and CD spectroscopy data for peptaibols 4 and 5
DOI: 10.1039/C8OB00686E
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Yield[a] λmin (nm) [θ]202-208 [θ]220-231
(deg cm² (deg cm² [θ]222/[θ]208
dmol-1) dmol-1)
R=
HC[b]
(BergD) 5a 30,31
.
These antibacterial peptides contain high
Cpd
Tz
position
proportions of Aib (25-50%)32,33 That promote
- and 310-
helices, and that are stabilized by (i,i+4) and (i,i+3) C=O⋅⋅⋅H-N
hydrogen bonding patterns, respectively.30,34 The Alm
structure is broken by a proline in position 14 delineating two
helices (Figure 3). It can multimerize to form voltage-gated
channels enabling its biological effect.35,36
Alamethicin sequence 4a: AcUP2UA4UA6QU8VU10GL12UP14VU16UQ18QFol [c]
4a
4b
4c
4d
4e
4f
45
28
34
20
42
45
28
48
37
38
208-221 -14971
208-220 -12566
-12018
-8558
-2957
-5182
-7411
-8369
-12094
-11108
-6225
-2487
0.8
0.7
31
22
>1
13
19
22
31
28
nh
nh
3-4
5-6
207-225
207-224
-4595
-7592
0.6
0.7
0.7
0.7
0.7
0.8
-
8-9
Results and Discussion
Synthesis and biological activity of Alm and BergD analogues.
10-11
16-17
17-18
13-14[d]
multiple[
207-224 -10033
208-220 -11798
208-223 -15976
208-225 -13571
4g
4h
4i
We previously showed that Fmoc protected Tz-containing
dipeptides 3b (Figure 1) are attractive precursors because they
are easily obtained in a few steps and are fully compatible with
solid-phase peptide synthesis (SPPS).37,38 The Aib-Xaa
dipeptides, evenly distributed in both peptaibol sequences,
201-231
-2542
4j
mulet]iple[
201-231
-3753
-
f]
Bergofungin D sequence 5a: AcVU2UV4GL6UU8OQ10UO12UFol [c]
allowed for a Tz scan using Aib[Tz]Xaa surrogates all along
5a
5b
5c
5d
5e
5f
63
56
34
55
54
54
208-222
-7065
-3338
-1018
-668
0.5
9
the Alm and BergD sequences (Table 1). We have synthesized
nine and five analogues of Alm and BergD, respectively,
containing single or multiple Tz substitutions. The analogues
containing multiple Tz insertions results from the combination
of all single substitutions in one peptide. As Alm and Berg D
contained a AibAibXaa sequence two substitution possibilities
2-3
3-4
207-234 -435.7
-
-
-
-
-
nh
nh
nh
nh
nh
208-234
204-234
203-237
202-234
2210
-2565
-656
7-8
-2224
-887
2-3, 7-8
3-4, 7-8
1423
-2286
existed: Aib[
peptide to two possibilities 4i

Tz]Aib or Aib[

-
Tz]Xaa thus leading for each
for Alm and 5e for BergD.
j
-f
[a] % after HPLC purification ; [b] Helical content; nh: non characteristic helix
signature; [c] Ac: acetyl; Fol: Phenylalaninol; U: Aib; O: Hydroxyproline. [d] Pro14 was
additionally mutated by an Aib residue in the peptide 4h. [e] in positions 3-4, 5-6, 8-9,
10-11, 16-17 and [f] 3-4, 5-6, 8-9, 10-11, 17-18.
These peptides were assembled on 2-chlorotrityl resin
preloaded with phenylalaninol according to the general
Fmoc/tBu microwave-assisted SPPS procedure. Stepwise
synthesis of the peptaibols analogues 4-5 at a 0.1 mmol scale
using single coupling steps for amino acids or for dipeptides 3b
proceeded smoothly, and the final products were obtained
with correct yields after purification (Table 1).39,40 Compound
First, biological activity of the Alm analogues was evaluated at
a concentration of 50 µg/mL, a level ten-fold higher than the
MIC determined for Alm 4a. Interestingly, in contrast to the
native Alm, all analogues were found inactive in a growth
inhibition assay on gram positive bacteria (Bacillus subtilis), a
primary target of peptaibols. On human KB cell lines, only
compounds 4b (IC50: 189 ± 24 µg/ml) and 4f (IC50: 85 ± 18
µg/mL) are somewhat cytotoxic. BergD and its analogues were
all inactive as it was recently observed for the closely related
bergofungin A (BergA).34 Thus, we showed that Tz insertion
abolished the antimicrobial activity of Alm regardless of the
position of the mutation and remained slightly cytotoxic for 4b
1
structures and purity were ascertained by LC-MS, H, 13C and
15N NMR (Supporting information S3-S18 and S30-56).
and 4f
.
Structure of Alm and BergD analogues.
Far-UV CD spectroscopy allowed for the helical propensity of
the various analogues in solution to be assessed (Figure 2). As
expected, the CD spectra of the native Alm 4a and BergD 5a
displayed the characteristic profile of a helix with a positive
maximum around 195 nm and two negative maxima around
209 and 228 nm. The helical content (HC) was 31% for Alm and
9% for BergD.41 Even if controversial,42,43,44 the [
ellipticity ratio was 0.80 for Alm and 0.47 for BergD indicating
]222/[]
208
Figure 1. Synthesis of Fmoc-1,4-disubstituted 1,2,3-triazolodipeptides
3 and
a predominant
for BergD.
-helix for Alm and most likely a 310 type helix
comparison of trans-amide bond with 1,4-disubstituted 1,2,3-triazole . Distance
3
between Cα-Cα, value of the dipole moment and triazole numbering are given.
2 | J. Name., 2012, 00, 1-3
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4h. Very close CD profiles were recorded forVie4waArtaicnledOnl4inhe
indicating that their helix structures exhibDiOteI:d10s.1im03i9la/Cr8sOtaBb0i0li6t8y6.E
We further investigated the structural impact of such Tz
insertions using high-resolution techniques such as X-ray
crystallography and NMR spectroscopy.
To our knowledge, only the work of Ghadiri et al.27 reported
crystal structures of helical peptides containing Tz in their
backbones (with resolutions around 2.2 Å). Fortunately, we
obtained single crystals suitable for X-ray diffraction studies of
Alm analogues 4g, 4h, and, of BergD 5a and analogues 5b, 5d
and 5f at near atomic resolution. Except for compound 5f, all
of the analogues adopted fully helical structures with the N2
and C5-H components of the Tz involved in hydrogen bonds.
These data showed that estimates of helicities with CD
spectroscopy were largely underestimated due to the
presence of numerous achiral Aib residues as previously
reported.47,48 We first compared the crystal structure of the
Alm (PDB entry 1amt) with those of 4g and 4h which were the
analogues that retained helical contents similar to those of the
native Alm (~30%) (Figure 3). The N-terminal sequences (from
residue 1 to 16) of both compounds 4g and Alm were closely
related with a root mean square deviation (rmsd) of 0.32 Å on
C atoms (Figure S2-1). In 4g, the sequence following the Tz
insertion (17-18) was presumed to be unfolded because no
electron density was observed for this region in the Fourier-
difference map. In contrast, 4h was completely folded but the
Tz incorporation in position 13-14 associated with the
substitution of Pro14 by Aib induced a large 30° bend of the C-
terminal moiety in the opposite direction from that of the
native structure, although the first twelve residues were
Figure 2: CD spectra of Alm F50/5, BergD and analogues 4b-j and 5b
methanol at 20°C.
-f in
These results were fully consistent with those previously
obtained for the Alm while BergD probably adopted a 310 helix
structure similar to that of BergA.30,34 The maxima of the signal
intensities for the analogues generally decreased and in some
cases, the CD spectra did not display a helix profile. First, we
observed that the CD spectra were similar for Alm 4a and the
analogue 4g when the Tz was incorporated at the C-terminal
extremity in position (17-18). A decrease of the signal by one
third (HC decreased from 31% to ~20%) was observed when
the Tz insertion was localized at the N- or C-terminal part of
the peptide 4b (3-4), and 4f (16-17), respectively or at the
center of the sequence 4e (10-11), near the Alm hinge. When
the Tz was introduced in the middle of the N-terminal helix,
the helicity dropped to around 13% for 4d (8-9), and was
totally lost in the case of 4c (5-6). Importantly, a single Tz
substitution regardless of its position in the BergD analogues
5b-d strongly affected the 310-helix CD signature (Figure 2).
superimposable (rmsd of 0.27 Å on C atoms) (Figures 3A and
S2-1). In Alm and 4g, the hydrogen bond network was
disrupted by Pro14, making Aib10 and Gly11 carbonyl oxygen
atoms solvent-accessible. In 4h, the Pro14Aib substitution plus
the Tz insertion allowed for the formation of a complete
network of interactions, including a weak Tz-C5-H….O=C-Aib10
(dC5H-O = 2.74 Å), a strong Val15-NH…O=C-Gly11 (dNH-O = 2.28 Å)
and Aib17-N-H….N2-Tz, (dNH-N2 = 2.51 Å) hydrogen bonds, which
affected the native Alm helix shape and altered the
distribution of the polar groups on its surface, thereby masking
the Aib10 and Gly11 carbonyl oxygens from the solvent (Figure
2A and table S2-3).30 We observed notable differences in the
torsion angle values of Leu12 and the

angles of Pro14 and
Aib14 in 4a
(

= -28°) and 4h = -64°), respectively (Table S2-
(
2). The partial unfolding of 4g and the structural alteration of
4h, both of which affected the C-terminal part of the peptide,
could explain the loss of biological activity of analogues 4g and
Also, multiple Tz insertions in both peptaibol analogues 4i, 4j,
5e and 5f shared weak CD signals with negative maxima
shifted around 200 nm and 230 nm, and the positive maxima
around 195 nm could no longer be observed. Thus, a single Tz
mutation in the BergD as well as multiple Tz insertions in both
peptaibol sequences may prevent the helix formation. In
addition, it has been shown that the Pro14Ala or Pro14Aib
mutations restored the continuity of the hydrogen bond
network and stabilized the Alm helix structure.45,46 Here, we
4h
. These results highlighted the role played by the
Gly11(Xaa)3Pro14 hinge and the less organized C-terminal part
of Alm in the bundle formation required for biological
activity.45,49 Moreover, 4g and 4h crystal packings contain
voids most likely filled with disordered solvent. These voids
formed very different channels from those proposed for Alm in
the lipid membrane. In the 4g and 4h crystals, the helix and
channel axes were perpendicular, while for Alm, they were
expected to be parallel within the membrane (Figure S2-2).
replaced the Aib13Pro14 dipeptide with Aib13[ Tz]Aib14 to yield

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Figure 3. Crystal structures of the alamethicin, bergofungin D and their analogues with Tz insertions. A. Portion of the crystal structures of alamethicin (in cyan, PDB
entry 1amt) and its analogues 4g (in green) and 4h (in magenta). Superimposition of their backbones in cartoon representation highlights the differences in C-terminal
parts (bent orientations and bent amplitudes). B. Superimposition of the backbone in cartoon representation of the bergofungin A (in forest, PDB entry 5mas) and D
(
5a, in grey). C. Superimposition of the backbones in cartoon representation of the bergofungin D ( 5a, in grey) and analogs 5b (in orange), 5d (in green) and 5f (in
magenta). Hydrogen bond networks are shown as yellow dotted sticks while the hydrogen bonds involving the Tz rings are shown as red dotted sticks. Hydrogen
atoms were omitted for clarity, except the amide protons and hydrogen atoms engaged in hydrogen bo nds
We also solved the crystal structure of the BergD 5a, which S2-4). Remarkably, in both structures, the Tz mimicked the
exhibited a kinked 310 helix similar to those of the BergA amide bond that formed near complete hydrogen bond
recently reported,34 and other natural analogues (Figure S2- networks all along the helices (Figure 3C, Table S2-5). The N2
3).50 BergD is one residue shorter than BergA and the residue atom of the Tz was engaged in a hydrogen bond with the NH
preceding the second hydroxyproline is an Aib residue instead amide of Leu6 and Gln10 for 5b and 5d, respectively (dNH-N2
=
of an isovaline (Figure S2-3). The 14 BergD residues fit well 2.37 and 2.31 Å, respectively). The Tz-C5-H of compound 5d
with the last 14 residues of BergA with an rmsd of 0.22 Å on participated in a weak hydrogen bond with the carbonyl
C
atoms (Figure 3B). Worth noting is that analogues 5b and oxygen of Val4 (dC5H-O = 2.12 Å). In 5b, the Tz-C5-H at the N-
5d with Tz insertions in position 2-3 and 7-8 respectively, terminal extremity could not be engaged in any intramolecular
adopted similar conformations to BergD with rmsd of 0.84 Å hydrogen bond but it formed a hydrogen bond with an
and 0.63 Å on C atoms (Figures 3C and S2-4). We only noticed acetonitrile molecule.
a small deviation of the 5b N-terminus. When the first four Importantly, we also succeeded in solving the structure of
residues were omitted, the rmsd decreased to 0.59 Å (Figure compound 5f which contained a double Tz insertion in
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positions 3-4 and 7-8. In contrast to compounds 5b and 5d While the backbone chemical shifts are widely used to identify
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with a single Tz insertion, only the last seven residues retained the type and location of the secondary DstOrIu: c10tu.1r0e3s9/iCn8OprBo0t0e6i8n6sE,
a helical conformation. The N-terminal moiety was rather we used them, here, to assess to what extent the Tz insertions
extended containing a type 2
residues ( 1 = -81°, 1 = 141°,


-turn involving the two first alter the Alm and BergD helices. We noticed that the backbone
2 = 60° and 2 = 27° (Figure atoms resonances were primarily altered around Tz rings for



3C). Thus in a double insertion system, the peptide bond all analogues. Atoms in close vicinity were those with the
substitution between Aib3 and Val4 unwound the N-terminal highest variations solely due to the proximity of the Tz.
3
part of the BergD helix, while the central Tz insertion between In general, the J(HNHα) values were very close to those of the
Aib7 and Aib8 was well tolerated. Interestingly, we observed in native peptide even if slight variations occurred near the Tz
compound 5f that the Tz-N3 atom was also engaged in a insertion. Thus, single insertions may not impact the overall
backbone side chain hydrogen bond with the NH
of Gln10 (dNH- structure of Alm and BergD. In contrast, atomic resonances
N3 = 2.16 Å). As expected, while the CD spectroscopy allowed varied significantly along the entire backbone for cases with
us to quickly determine the helix propensity for all compounds multiple Tz incorporations such as for 4i and 4j, but we could
in solution, we noticed discrepancies with the solid-state not discriminate whether they arose from current effects of Tz
structures. The Alm analogues 4a, 4g and 4h shared similar rings or from large structural alterations of these analogues.
helicities whereas the C-terminal moiety was disordered in the Nevertheless, few NOEs were observed on the ROESY spectra
crystal for 4g. In addition, despite fully helical folds for for both compounds and NMR structure calculations did not
compounds 5b and 5d in solid-state, we observed that both Tz converge indicating that they were most likely not folded.
insertions strongly affected the 310 helix signature of BergD. In Thus, we then focused on the other analogues and
this context, we further investigated the behavior of the investigated the backbone geometry around the Tz using
analogues in solution via extensive NMR studies.
seven diagnostic NOE correlations identified from the crystal
structure of the Tz-containing GCN4-pLI -helical coiled coils
1
The NMR signals were well dispersed and assignment of H, 13
C
and 15N chemical shifts were carried out combining (pdb ID 1U9F, Figure 4).27 The presence of unambiguous NOE
homonuclear COSY, TOCSY, ROESY and heteronuclear 13C-, 15N- correlations between the HψTz(i) proton and the HN(i-1),
HSQC and 13C-HMBC experiments (Tables S4). The Alm HN(i+1), HN(i-2), H
(i-2), H(i-3), H(i-4) protons and Hα(i-3)-
3J(HNH
) coupling constants were in the 4-6 Hz range from Hβ(i) were characteristic of local Tz-containing helix turns
Ala4 to Val9 (Table S6-1). These values were characteristic of (Figure 3A). Solution NMR structures were then calculated
helical conformation and were similar to those reported by using complete NOE sets as distance restraints. Although the
Esposito et al.51 We first monitored the chemical shift HψTz(i)-HN(i-2) and
H

(i-3)-H
, and 13CO backbone atoms detected in the ROESY spectra for a few compounds, the other
upon the Tz insertions in the various analogues (Tables S5) and probes were generally observed in the ROESY spectra for all
compared their 3J(HNHα) coupling constants (Table S6-1). analogues, except 5f
(i) correlations were only
1
1
variations of HN, 15N, H 13C
, 
.
Figure 4. A. Expected backbone NOE correlations surrounding the 1,2,3-triazole in Tz-containing helical template based on the crystal structure of GCN4-pLI coiled
coils (pdb ID 1U9F). Strong, medium and weak NOEs are represented by green, light green and orange arrows, respectively. B. NOEs observe d in the canonical α-helix
but not expected in the analogues around the triazoles. C. Survey on the helix-consistent NOE correlations in the ROESY spectra of the analogues. a: ambiguous due
to strong overlaps; nd: not detected
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Figure 5: NMR structures of compounds A. 4a, 4c, 4g and 4h ; B. 5a, 5d, 5e and 5f
These results were in agreement with the set of crystal As expected, whole analogues displayed helix structures in
structures described above which were mostly helical while solution while the N-terminal extremity of 5f was disordered.
the N-terminal moiety of 5f was extended. We calculated the Through the use of NMR, we showed that the single Tz
NMR solution structures of 4c
,
4g,
4h
,
5d
,
5e and 5f (Figure 5). insertions within 4b-h and 5b-d did not alter the overall native
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helical fold. However, the variations in CD signal intensities sequences. The specific geometry of the Tz did not strictly
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DOI: 10.1039/C8OB00686E
around 208 and 222 nm upon the incorporation of Tz in the substitute the amide bond and more importantly abolished the
Alm analogues reflected a loss of stability within the helices at antimicrobial activity of Alm even when very slight
varying degrees depending on the Tz position. The Alm helix conformational variations occurred. In this context, one should
was more prone to destabilization when the Tz were inserted cautiously use the Tz as a trans peptide bond surrogate in
in the center of the main 1-14 helix region, i.e, in positions 5-6 helical peptides, especially when they exhibit close structure-
and 8-9 (Table 1, Figure S1). We also noticed that the activity relationships. Even if the overall peptide structure is
variations of the BergD CD signature in response to Tz conserved at the point of the Tz insertion, its stability and
insertions (Figure 2) were not related to a complete loss of dynamics may be greatly impaired, resulting in the cessation of
structure, for example 5b and 5d displayed helix crystal its biological activity.
structures despite non-characteristic canonical CD profile.
While the single Tz insertions in 5b (2-3), and 5d (7-8) did not
affect their overall folds, the double insertions 2-3, 7-8 in 5e
Conflicts of interest
and 3-4, 7-8 in 5f led to various results. In the first case, the
double insertion did not impact the helical structure, while in
the latter the helix was partially unwound. Such results could
“There are no conflicts to declare”.
be explained by the fact that 5c should be less stable than 5b
.
Acknowledgements
According to the general trend observed for the Alm
analogues, the 2-3 substitution was less invasive than the 3-4.
Therefore, 5e could accommodate both the 2-3 and 7-8
substitutions while 5d was only folded around the Tz rings in
position 7-8.
We thank: Comité des Pyrénées Orientales de la Ligue
Nationale Contre le Cancer and the Bonus Qualité Recherche
of Perpignan University, MESR for financial support. The
spectroscopic experiments have been performed at the
“Laboratoire de mesures physiques” (LMP) at the University of
Montpellier and using the Biodiversité et Biotechnologies
Marines
facilities
(Bio2Mar,
http://bio2mar.obs-
banyuls.fr/fr/index.html) at the University of Perpignan, and
the ‘Plateforme de mesures de diffractionX’ of the Université
de Lorraine is appreciated. We are grateful to the staff
members of the BM30A beamline at ESRF (Grenoble, France)
and to our American colleague J Almany for comments and
carefully proofreading of the manuscript.
Conclusions
Finally, in addition to the number and the position of Tz
insertions in the peptide sequences, its nature and more
specifically the types of amino acids surrounding the Tz ring
should also be considered. Since the amino acids displayed
various propensities for helix, coil and -strand structures, one
can anticipate that Tz dipeptide insertions should also affect
the secondary structures of peptides and proteins. From our
data, we first noticed that we obtained fully helical crystal
Notes and references
1
2
3
4
5
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