The crystal structure of Z-(Aib)10-OH at 0.65 ? resolution: Three complete turns of 310-helix

Article abstract of DOI:10.1002/psc.2842

The synthetic peptide Z-(Aib)₁₀-OH was crystallized from hot methanol by slow evaporation. The crystal used for data collection reflected synchrotron radiation to sub-atomic resolution, where the bonding electron density becomes visible between the non-hydrogen atoms. Crystals belong to the centrosymmetric space group P 1. Both molecules in the asymmetric unit form regular 3₁₀-helices. All residues in each molecule possess the same handedness, which is in contrast to all other crystal structure determined to date of longer Aib-homopeptides. These other peptides are C-terminal protected by OtBu or OMe. In these cases, because of the missing ability of the C-terminal protection group to form a hydrogen bond to the residue i-3, the sense of the helix is reversed in the last residue. Here, the C-terminal OH-groups form hydrogen bonds to the residues i-3, in part mediated by water molecules. This makes Z-(Aib)₁₀-OH an Aib-homopeptide with three complete 3₁₀-helical turns in spite of the shorter length it has compared with Z-(Aib)₁₁-OtBu, the only homopeptide to date with three complete turns. The synthetic, achiral homo-decapeptide of α-amino-isobutyric acid (Aib) residues crystallizes with two molecules in the asymmetric unit. After refinement at sub-atomic resolution, there is electron density in different Fourier maps, which is clearly visible between non-hydrogen atoms. This is the bonding electron density that is not explained by the usual spheric atom model. In addition, there are second conformations of certain residues that become visible at this ultra-high resolution.

Full text of DOI:10.1002/psc.2842

Research Article
Received: 7 October 2015
Revised: 6 November 2015
Accepted: 11 November 2015
Published online in Wiley Online Library: 18 December 2015
(wileyonlinelibrary.com) DOI 10.1002/psc.2842
The crystal structure of Z-(Aib)10-OH at
0.65Å resolution: three complete turns of
310-helix
Renate Gessmann,a Hans Brücknerb and Kyriacos Petratosa*
The synthetic peptide Z-(Aib)10-OH was crystallized from hot methanol by slow evaporation. The crystal used for data collection
reflected synchrotron radiation to sub-atomic resolution, where the bonding electron density becomes visible between the
non-hydrogen atoms. Crystals belong to the centrosymmetric space group P1. Both molecules in the asymmetric unit form regular
310-helices. All residues in each molecule possess the same handedness, which is in contrast to all other crystal structure
determined to date of longer Aib-homopeptides. These other peptides are C-terminal protected by OtBu or OMe. In these cases,
because of the missing ability of the C-terminal protection group to form a hydrogen bond to the residue i-3, the sense of the helix
is reversed in the last residue. Here, the C-terminal OH-groups form hydrogen bonds to the residues i-3, in part mediated by water
molecules. This makes Z-(Aib)10-OH an Aib-homopeptide with three complete 310-helical turns in spite of the shorter length it has
compared with Z-(Aib)11-OtBu, the only homopeptide to date with three complete turns.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: α-aminoisobutyric acid; 310-helix; sub-atomic resolution; centrosymmetry; achiral peptide; left-handed helix; unprotected
peptide; three complete turns
in the long, C-unblocked Aib10. Furthermore, the only structure
available of a (Aib)10 peptide contains the heavy bromine (Z = 35)
Introduction
The conformational space available to α-amino-isobutyric acid (Aib)
residues is severely restricted by the second methyl group attached
to the Cα atom. This space comprises the left-handed and right-
handed helical region of the Ramachandran plot. Aib residues are
known as strong helix formers in peptides, also in the presence of
common amino acids [1–3]. Helical conformation is a prerequisite
for the formation of pores by self-association of naturally occurring
peptides rich in Aib-residues (e.g. peptaibols) in lipid bilayer mem-
branes [4]. Peptides consisting of only Aib residues show a clear
preference for left-handed and right-handed 310-helices in solution
and crystal structures. All crystal structures of Aib homopeptides
known to date [5–14] show the following common properties: cen-
trosymmetric space group, regular 310-helices with the maximum
number of hydrogen bonds involving also the N-terminal
protecting group, head-to-tail hydrogen-bonded columns and a re-
versal of the helical sense at the C-terminal residue. The latter was
not observed for Z-(Aib)3-OH anhydrate and monohydrate [5]. Apart
from these two peptides, only Z-(Aib)4-OH and Z-(Aib)5-OH [6] were
investigated by crystallography as C-unblocked homopeptides, and
in both the sense of the helix was found reversed at the last residue.
All other structures of Aib-homopeptides are protected at the
C-terminal with OMe (methoxy) or OtBu (tert-butoxy) groups, which
interrupt the helical hydrogen bonding scheme by replacing the
hypothetical following NH-group with oxygen. The latter replace-
ment results in unfavourable short distance between the oxygen
of the protecting group and the C =O group of residue i-3, and it
is avoided by reversing the backbone in the opposite direction.
These findings lead us to investigate the C-terminal conformation
incorporated at the N-terminus, which was found to affect the
crystal packing and hence the resulting structure. This renders the
latter structure not comparable with the other homopeptides.
Finally, the easily tunable wavelength at the microfocus beamline
I-24 at the British synchrotron (Diamond) enabled data collection
in the sub-atomic resolution of 0.65 Å.
Materials and methods
Synthesis and crystallization
The protected decapeptide Z-(Aib)10-OtBu was synthesized by
heating of Z-Aib5-Ox (5.38 g, 9.61 mmol) (Ox refers to the oxazolone
formed from the C-terminal Aib of the Z-protected pentapeptide
on reaction with acetic anhydride for 1.5 h at 100 °C) with H-(Aib)5-
OtBu (4.80 g, 9.61 mmol) in 150 ml butyronitrile for 26 h at 100 °C.
The solvent was evaporated in vacuo; the remaining residue
*
Correspondence to: Kyriacos Petratos, IMBB/FORTH, 70013 Iraklion Crete, Greece.
E-mail: petratos@imbb.forth.gr
a IMBB/FORTH, 70013, Iraklion Crete, Greece
b Department of Food Sciences, Interdisciplinary Research Center, Justus-Liebig-
University of Giessen, 35392, Giessen, Germany
Abbreviations: Z, benzyloxycarbonyl; OMe, methoxy; OtBu, tert butoxy; Aib,
α-aminoisobutyric acid; Ox, oxazonon.
J. Pept. Sci. 2016; 22: 76–81
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

CRYSTAL STRUCTURE OF Z-(AIB)10-OH
dissolved in 250 ml chloroform, and washed three times with 200 ml
KHSO4 (5%), KHCO3 and water. The organic phase was dried with
Na2SO4, and evaporated to dryness. The peptide was dissolved in
hot chloroform and crystallized by addition of light petroleum (b.p.
40–60 °C). Yield 7.74 g (76%), m.p. 252–255 °C, rF = 0.27 (mobile phase
ethyl acetate, distance start to solvent front 17.5 cm, pre-coated silica
plate from Merck, 20 × 20 cm). Positive mode electrospray ionization
mass spectrum: m/z 1081.46 [M + Na]+, 1059.13 [M + H]+, and
regular series of acylium fragment ions differing by 85.1 mass units
(Aib - H2O) ranging from m/z 304.87 (b2) to m/z 985.29 (b10) [15,16].
To Z-(Aib)10OtBu (4.30 g, 4.06 mol) in dichloro-methane (10 ml)
trifluoroacetic acid (80 ml) was added and the mixture stirred at room
temperature for 2 h. Solvents were removed in vacuo, the remaining
residue dissolved in MeOH and the decapeptide carboxylic acid
precipitated by addition of diethyl ether. rF = 0.62 (mobile phase
chloroform/methanol 85:15, v/v, distance start to solvent front
7.5 cm, pre-coated silica plate from Merck, 5 × 10 cm). Z-(Aib)10-OH
was dissolved in hot methanol and crystallized by slow evaporation.
Crystals were visible after a few days.
Figure S1). Diffraction data were collected at 100 K on the microfocus
beamline I24 [17] of Diamond Light Source in Didcot, England using
a Pilatus 6 M detector (Dectris Ltd, Baden, Switzerland). A dataset of
1800 images covering 360° of rotation was collected from a single
crystal. The data were integrated using the software package XDS
[18] and scaled with AIMLESS [19] implemented in the CCP4 suite [20].
The dataset statistics are summarized in Table 1. Although the
data were collected to a nominal resolution of 0.63Å, the processed
data were limited to 0.65 Å resolution because of the very low com-
pleteness beyond 0.65 Å.
Structure solution and refinement
The structure was solved by direct methods with SHELXS86 [21]. All
142 non-hydrogen atoms of two peptide molecules termed hereaf-
ter mol A and mol B, and one water oxygen atom could be located
in the first electron density map as highest peaks. The structure was
refined using the program SHELXL [21].
During refinement, one disorder of the N-terminus of mol A com-
prising seven non-hydrogen atoms, disorders of the last residue of
both molecules and a disorder of one methyl group of Aib 5 in mol
B were detected, which were described as the second components
in different conformation. Interestingly, the next high peaks in the
electron density maps of the initial structure solution proved to
be the oxygen atoms of the second C-terminal conformation of
mol B. Several restraints were introduced to improve the geometry
and the thermal parameters of these disordered residues. Both
Data collection
One single plate with the smallest dimension of about 25 μm was
mounted on a cryoloop without cryoprotectant and kept in place
with a minimal amount of vacuum grease (Supporting Information
Table 1. Data collection and processing. Values in parentheses are for
the outermost resolution shell
Crystal data
Chemical formula
Mr
C48O13.57N10H78.9
1013.31
Crystal system, space group
Temperature (K)
a, b, c (Å)
Triclinic, P 1
100
15.784 (8), 18.424 (7), 20.999 (12)
α, β, γ (°)
107.768 (6), 91.626 (4), 103.158 (15)
V (Å3)
5631 (5)
Z
4
Wavelength (Å)
Resolution range (Å)
λ = 0.59039
19.9–0.65 (0.7–0.65)
0.03
μ (mmÀ1
)
Crystal size (mm)
No. of measured reflections
No. of unique reflections
Mean I/σ(I)
0.12 × 0.12 × 0.025
107 673 (4186)
31 118 (1749)
9.6(4.3)
Completeness (%)
Rpim$ (%)
72.5(20.5)
4.4(15.4)
8.2(21.3)
9.04, 8.23
10.13, 9.25
31 118
Rmeas* (%)
Final Rcryst, Rcrys Fo > 4σFo (%)
Final Rfree, Rfree Fo > 4σFo (%)
No. of all unique reflections
No. of reflections for Rfree
No. of parameters
1568
1504
No. of restraints
371
Average B-factor# (Å2)
mol A main/side chain
mol B main/side chain
3.2/4.8
2.9/4.2
Rpim = Σ(1/(N-1))1/2 · |I - < I > |/ΣI, N = number of symmetry related
$
Figure 1. Wall-eyed stereo view of the superposition of mol B (blue) on mol
A (red), the water molecule is shown as a green sphere. Hydrogen bonds
appear in orange for mol A and light blue for mol B. N and C denote the
amino-termini and carboxyl-termini of the molecules. For clarity, only the
main conformations are shown.
reflections.
* Rmeas = Σ(N/(N-1))1/2 · |I - < I > |/ΣI.
#
Non-hydrogen atoms.
J. Pept. Sci. 2016; 22: 76–81
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

Gessmann, Brückner and Petratos
equally occupied conformations of the Z-protection group of mol A
were refined to be almost at the same position after the application
of anisotropic refinement. There was also a clear appearance of
negative electron density at the atom ring positions. The latter
observations obliged us to refine these regions isotropically.
Hydrogens were added in calculated positions and refined as riding
with the thermal displacement parameters fixed to 1.5 (for methyl
hydrogens) and 1.2 (for all others) times of the isotropic displace-
ment parameter of the parent atom. The torsion angles of the
C-terminal hydrogens were allowed to refine, and because of the
low occupancy and the resulting estimated high standard
deviations the maximum shift/error of 1.36 remains for the second
conformations in the last cycles of refinement. Experimental details
are listed in Table 1. Xtalview [22], Swiss PDBViewer [23], Coot [24],
Ortep-3 [25], Pymol [26] and POV-Ray [27] were used for geometric
analysis, visualization and for the production of the figures.
1430000. These data can be obtained from The Cambridge Crystal-
lographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Results and discussion
Two independent molecules (mol A and mol B) were located in the
crystal’s asymmetric unit. Figure 1 shows a superposition of the two
independent molecules (main conformations only). The rms-
deviation of all atoms is 0.96 Å with the maximum of 3.2Å at the
C-termini, while the rms-deviation of main chain atoms is 0.57 Å
with a maximum of 2.4Å. Both molecules possess a higher flexibility
at both ends. This is reflected by the second conformations and
Table 3. Hydrogen-bond geometry (Å,°)
Accession number
D–H···A
H···A
D···A
D–H···A
N···O = C
The coordinates and structure factors have been deposited with
the Cambridge Data Bank [28] under accession code CCDC
intramolecular, mol A
N_3–H_3···O(Z)
N_4–H_4···O_1
2.15
2.14
2.16
2.07
2.11
2.11
2.23
2.09
2.24
2.968(2)
2.951(2)
3.014(2)
2.931(2)
2.925(2)
2.925(2)
3.032(3)
2.913(3)
3.098(20)
154.5
152.6
163.0
165.7
154.3
154.3
151.0
155.8
166.6
130.5(1)
131.2(1)
129.4(1)
132.2(1)
129.0(1)
131.7(1)
126.9(1)
128.9(1)
129.3(3)
Table 2. Backbone torsion angles for the right handed molecules
N_5–H_5···O_2
N_6–H_6···O_3
Angle(°)
Mol A
2nd conf.
Mol B
2nd conf.
N_7–H_7···O_4
ζ2(Ζ)
ζ3(Ζ)
ω(Ζ)
φ(1)
ψ(1)
ω(1)
φ(2)
ψ(2)
ω(2)
φ(3)
ψ(3)
ω(3)
φ(4)
ψ(4)
ω(4)
φ(5)
ψ(5)
ω(5)
φ(6)
ψ(6)
ω(6)
φ(7)
ψ(7)
ω(7)
φ(8)
ψ(8)
ω(8)
φ(9)
ψ(9)
ω(9)
φ(10)
ψ(10)
151.2(7)
À166.0(7)
À177.3(2)
À57.1(2)
À30.5(2)
À179.6(2)
À47.9(2)
À40.1(2)
À173.1(1)
À56.7(2)
À30.2(2)
À177.8(1)
À58.6(2)
À19.0(2)
À173.6(1)
À48.9(2)
À34.1(2)
À176.8(1)
À52.6(2)
À32.7(2)
À178.6(1)
À53.3(2)
À34.2(2)
À175.5(1)
À53.7(2)
À32.8(2)
À177.5(1)
À64.2(3)
À16.3(3)
À157.8(2)
À51.7(3)
À49.5(3)
148.1(4)
À130.3(2)
À23.3(3)
À177.3(2)
À56.5(2)
À29.8(2)
À178.1(2)
À50.7(2)
À35.8(2)
À174.8(1)
À54.2(2)
À31.9(2)
À177.8(1)
À55.6(2)
À24.5(2)
175.1(2)
—
N_8–H_8···O_5
À174.8(3)
—
N_9–H_9···O_6
À178.5(2)
—
N_101–H_101···O_7
N_102–H_102···O_7
mol B
—
—
—
—
—
—
N_3–H_3···O(Z)
N_4–H_4···O_1
2.18
2.14
2.13
2.10
2.14
2.25
2.05
2.10
2.21
2.06
1.96
3.021(2)
2.961(2)
2.960(2)
2.960(2)
2.986(2)
3.094(8)
2.901(23)
2.960(2)
3.062(5)
2.916(2)
2.755(3)
159.1
154.0
157.4
166.4
160.7
161.1
164.0
165.0
162.5
163.8
157.3
129.3(1)
131.7(1)
131.4(1)
132.0(1)
130.1(1)
132.1(5)
132.1(16)
132.8(1)
133.7(2)
130.9(4)
136.2(1)
—
—
—
—
N_5–H_5···O_2
—
—
N_6–H_6···O_3
—
—
—
N_7–H_7···O_4
—
N_83–H_83···O_5
N_84–H_84···O_5
N_9–H_9···O_6
—
—
—
—
—
—
N_103–H_103···O_7
N_104–H_104···O_7
O_104–H_104···O_8
intermolecular A to B
N_1–H_1···O_9i
B to A
N_1–H_1···O_9ii,1
N_1–H_1···O_92
N_2–H_2···O_101
mol A to water 1
H2O11–H21···O_8
OH_101–HH_101···H2O11
mol B to water 1
H2O11–H11···O_103
mol B to water 2
HOH22···O_8
—
À175.1(7)
À62.4(15)
À21.6(22)
177.0(10)
À49.7(13)
—
—
À48.0(5)
À34.8(8)
À174.7(4)
À55.4(5)
À29.6(2)
À177.3(1)
À51.1(2)
À32.4(2)
À177.8(1)
À54.5(2)
À25.5(2)
À179.9(1)
À50.6(2)
À29.3(3)
174.8(3)
—
—
2.03
2.903(2)
173.2
167.0(2)
—
—
—
1.91
2.01
2.25
2.777(2)
2.862(16)
3.108(3)
169.4
161.6
164.5
160.8(2)
143.9(16)
109.4(2)
—
—
—
—
—
—
—
1.83
1.84
2.871(3)
2.610(1)
170.1
151.1
129.3(1)
127.3(1)
—
—
—
—
—
—
2.24
3.118(4)
174.8
130.8(3)
À56.9(20)
À10.5(47)
160.33(35)
À50.9(44)
À39.5(33)
—
À40.5(10)
À151.5(12)
À33.1(22)
À59.9(20)
—
2.881(23)
2.627(84)
—
127.4(6)
OH_102–HH_102···H2O22
mol A to water 2
H2O22···O_10iii,2
1.88
146.9
119.1(17)
À51.4(5)
À33.8(4)
—
3.072(35)
—
133.2(31)
* The angles ζ2(Z), ζ3(Z) and ω(Z) are defined as C1-C7-O7-C, C7-O7-C-
N(1) and O7-C-N(1)-Cα(1), respectively, and ψ(10) is the angle N(10)-CA
(10)-C(10)-O (of group OH 10).
The numbers denote disordered atoms with occupancies (1) 0.9; (2) 0.1;
(3) 0.75; (4) 0.25.
Symmetry codes: (i)Àx,Ày + 1,Àz + 2; (ii)Àx + 2,Ày,Àz + 1; (iii) xÀ1, y, z + 1.
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2016; 22: 76–81
wileyonlinelibrary.com/journal/jpepsci

CRYSTAL STRUCTURE OF Z-(AIB)10-OH
higher thermal motion. Figure S2 in the Supporting Information
shows the thermal ellipsoids.
The disorder at the termini of the molecules is more pronounced
including the high resolution data (Supporting Information
Figure S3).
Both chains adopt a regular 310-helical conformation stabilized
by intramolecular hydrogen bonds with their torsion angles listed
in Table 2. Eight consecutive H-bonds are formed starting from
the carbonyl-group of the protection group and ending at the
amino-group of Aib10 (Table 3).
In the crystal, one right-handed molecule A is head-to-tail hydro-
gen bonded to two left-handed molecules B, at the N-terminus with
one H-bond (N1(A) to O9(B)) and at the C-terminus with two (N1(B)
to O9(A) and N2(B) to O10(A)) (Table 3). Columns in the opposite
crystal direction are formed between the left-handed molecules A
and right-handed molecules B. The disordered 90% occupied water
at the C-terminus of molecule A connects these columns via hydro-
gen bonds in such a way that always two antiparallel columns share
the waters.
A one-molecule-layer of these planes built by the antiparallel
columns is shown in Figure 2 viewed approximately along the
diagonal [1 1 1]. Parallel to one layer (e.g. above and below the
plane shown in Figure 2) are layers of columns of molecules of
the same handedness consisting of the other molecules. These
layers exhibit a shift of about three residues or one helical turn
along the projection to the molecules in the layer in Figure 2.
The rings of the Z-protection group of both molecules are
staggered in the crystal with average angle between the two planes
of 10.6° for the first conformation of mol A and with 5.5° for the
second conformation of molecule A. The average was performed
between different atoms in both rings defining the plane (Table S1
of the Supporting Information).
In mol B, an additional H-bond is formed between the carboxyl-
group of Aib10 and the C = O-group of Aib8, which leads to the
formation of an oxy-analogue of a β-turn [29]. In the β-turn
oxy-analogue of mol A, this H-bond is mediated by a co-crystallized,
disordered water (OH-group of Aib10 to water 1 and water 1 to
C = O-group of Aib8, Figure 1). This water molecule also forms an
H-bond to the major component of the C = O-group of a symmetry
related mol B. This can be seen in Table 2 and Figure 2. Water with
smaller occupancy is found in the vicinity of Aib10 of mol B, 4.25 Å
away from the first water molecule. This water is, similar to the first
water, hydrogen bonded twice to mol B, namely to the C = O-group
of Aib8 and to the minor component of the OH-group of Aib10. A
third H-bond is formed with the symmetry related minor compo-
nent of the C = O group of Aib10 of mol A. The helical parameters
of the main backbone conformation of both molecules A and B
were determined with the program HELANAL [30]. The average
number of residues per turn is 3.22 and 3.15; the average rise per
residue is 1.89 and 1.96 Å, and the average helical twist per residue
is 111.7 and 114.4°, thus showing that molecule B is slightly more
tightly wound than helix A.
The valence symmetry around the Cα atom is asymmetric for the
Aib residues (Table 4 and Table S2). If one designates as CL and CR
the atoms which occupy the same position as Cβ and α-hydrogen
in L-amino acids respectively, the bond angles N-Cα-CL and C-Cα-
CL are significantly smaller than the N-Cα-CR and the C-Cα-CR.
This observation is in excellent agreement with theoretical calcula-
tions and with the bond angles of other right-handed 310-helical
Aib-peptides [12,14].
The influence of the high resolution to the electron density maps
was also investigated. The data were limited to a high resolution of
1.0 and 0.8Å in the last cycles of refinement. The result is shown in
the difference Fo-Fc maps in Figure 3. By decreasing the resolution
limit, more details become visible, which are not explained by the
usual spheric model of the atoms in the refinement program. At
the sub-atomic resolution of 0.65 Å, the bonding electron density
becomes visible between the non-hydrogen atoms.
Table 5 lists the helical parameters of the two herein described
molecules (main conformations only) together with the closely
Figure 2. Crystal packing of Z-(Aib)10-OH viewed approximately down the diagonal showing the infinitely long, antiparallel helical columns of alternating
right-handed and left-handed molecules. Right-handed helices from molecules A are depicted in red, left-handed molecules A in orange; dark and light
blue are the right-handed and left-handed helices of molecules B, respectively. The water molecules are shown as green spheres and the intermolecular
hydrogen bonds as green dashed lines. For clarity, only the main conformations are shown.
J. Pept. Sci. 2016; 22: 76–81
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

Gessmann, Brückner and Petratos
Figure 3. Electron density (cyan) around Aib7 of mol B. The Fo-Fc maps are contoured at 3σ (0.273 e ÅÀ3). The high resolution limit cut-off of the data is 1 Å
(A), 0.8 Å (B) and 0.65 Å (C).
which seems to stabilize the internal structure. This is confirmed by
thin layer chromatography experiments: in ethyl acetate (100%)
and butyl acetate–methanol (98 : 2) the homopeptides with full
Table 4. Angles (°) defining the valence geometry around the Cα-
atoms, average values of the main conformations
molecule
N-Cα-CL
C′-Cα-CL
N-Cα-CR
C′-Cα-CR
turns (n = 5, 8, 11) and N-terminal Z protection and C-terminal OtBu
protection show relatively higher retention factor values compared
with the homopeptides, which have one residue more or less [15].
In agreement, their melting point also shows higher values than the
neighbouring peptides. Figure 4 shows a superposition of these five
peptides. One can see that the three OtBu protected molecules
show a reversed backbone in the last residue, reflected by the op-
posite sign of the φ/ψ torsion angles. Z-(Aib)10-OH does not reverse
the backbone in the last residue and has the same sign of the φ/ψ
torsion angles as the preceding residues (Table 2) and is therefore
the only long Aib-homopeptide without a reversed helical sense
in the last residue. Furthermore, counting the number of intramo-
lecular hydrogen bonds, there are nine in mol B, and in mol A the
ninth hydrogen bond is mediated by one water molecule. Nine is
exactly the number of intramolecular hydrogen bonds in Z-(Aib)
11-OtBu, and therefore, the shorter Z-(Aib)10-OH has also three com-
plete 310-helical turns.
mol A
mol B
107.75(14)
107.51(16)
107.12(15)
107.15(16)
110.87(20)
110.76(16)
110.56(19)
109.90(16)
Table 5. Helical parameters for Z-(Aib)9-OtBu, Z-(Aib)10-OH and Z-(Aib)11-
OtBu. The parameters are derived from helices comprising all Aib-residues
Helix
Average number Average rise Average helical
of residues/
turn
per residue
[Å]
twist per
residue [°]
Z-(Aib)9-OtBu mol A
Z-(Aib)9-OtBu mol B
Z-(Aib)10-OH mol A
Z-(Aib)10-OH mol B
Z-(Aib)11-OtBu
3.21(11)
3.19(10)
3.22(13)
3.15(08)
3.06(06)
1.93(5)
1.94(5)
1.89(8)
1.96(5)
1.97(4)
112.0(3.9)
112.8(3.3)
111.7(4.6)
114.4(2.9)
117.8(2.5)
Crystallization of partially protected peptides is facilitated by the
possibility to form additional intermolecular hydrogen bonds as
herein described. This makes the deprotection of peptides a
method of choice in cases, where a fully protected peptide resists
to form crystals suitable for X-ray analysis. Attempts are in progress
in our lab to crystallize deprotected peptides derived from
protected counterparts, which have never formed crystals to date.
related molecule Z-(Aib)11-OtBu [14] and the two independent mol-
ecules of Z-(Aib)9-OtBu (main conformation only) [12]. All of them
deviate from the theoretical 310-helical values, which are 3.0 as av-
erage number of residues per turn and 120° as average helical twist
per residue. The closest to the theoretical values are exhibited by
the undecapeptide. This peptide possesses three complete turns,
Figure 4. Least-squares superposition of the molecular structures of Z-(Aib)11-OtBu appear in green, Z-(Aib)10-OH in red (mol A) and blue (mol B) and
Z-(Aib)9-OtBu in yellow (mol A) and cyan (molB). For clarity, only the main conformations are shown.
wileyonlinelibrary.com/journal/jpepsci
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2016; 22: 76–81

CRYSTAL STRUCTURE OF Z-(AIB)10-OH
13 Toniolo C, Crisma M, Bonora GM, Benedetti E, Di Blasio B, Pavone V,
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Acknowledgements
We are grateful to Dr Danny Axford, for assistance at the beamline
I24 during data collection. The research leading to these results has
received funding from the European Community’s Seventh
Framework Programme (FP7/2007-2013) under BioStruct-X (grant
agreement N° 283570). This work was partly performed/conducted
in the framework of the BIOSYS research project, Action KRIPIS,
project no MIS-448301 (2013SE01380036) that was funded by
the General Secretariat for Research and Technology, Ministry of
Education, Greece, and the European Regional Development Fund
(Sectoral Operational Programme: Competitiveness and Entrepre-
neurship, NSRF 2007–2013)/European Commission.
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J. Pept. Sci. 2016; 22: 76–81
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