Interruption of a 310-helix by a single Gly residue in a poly-Aib motif: A crystallographic study

Article abstract of DOI:10.1002/bip.21535

The structural influence of a single Gly residue inserted into an Aib₁₆ homooligomer was studied in the solid state by X-ray crystallography. The peptides N₃Aib₈GlyAib₈PheNH₂ (1) and CbzPheAib₈

Full text of DOI:10.1002/bip.21535

Interruption of a 310-Helix by a Single Gly Residue in a Poly-Aib
Interruption of a 310-Helix by a Single Gly Residue in a Poly-Aib
Motif: A Crystallographic Study
Motif: A Crystallographic Study
`
Jordi Sola, Madeleine Helliwell, Jonathan Clayden
School of Chemistry, University of Manchester, Manchester M13 9PL, UK
Received 16 June 2010; revised 9 July 2010; accepted 16 July 2010
Published online 19 August 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21535
that even residues which typically break helices, such as Pro,
become incorporated into (albeit distorted) helical struc-
tures.4–6 Likewise, it is remarkable that analogues of tricogin
containing GlyGly adopt helical conformations:7 in the
sequence BocLeuAibValGlyGlyLeuAibValOMe, Aib drives he-
lix formation despite the tendency of Gly to favor b-turn
structures.8–12 Gly is found less frequently in natural occur-
ring a-helices, because its high-conformational flexibility
makes it entropically expensive to adopt the relatively con-
strained a-helical structure.8–12
Aib-containing helices1,2,13 may be a-helices (with a i 1 4
? i pattern of H-bonding and 3.6 amino acids per turn),
310-helices13 (with a i 1 3 ? i pattern of hydrogen bonding
and 3.0 amino acids per turn), or a mixture of a and 310-heli-
ces. 310-Helices are more tightly wound than a helices,
although the dihedral angles in a 310 helix are not greatly dif-
ferent from those in an a helix (/ and w typically 6578 and
6308 for the 310 helix and 6638 and 6428 for the a helix).3
One more intramolecular hydrogen bond is possible in a
stretch of 310-helix than an a-helix, but this stabilizing effect
must be set against the suboptimal hydrogen-bond geometry
and greater van der Waals interactions experienced in the
tighter 310-helix.14 For this reason, any preference for 310-hel-
ical conformation is greatest in short helices. For example,
oligomers of AibAla form helices whose hydrogen bonding
pattern is dependent on the chain length,15–17 with the
switch from 310 to a helix occurring once the length of the
(AibAla)n oligomer exceeds eight residues. Regular 310 helices
with more than two turns rarely occur in proteins,18,19 but
short 310-helical stretches are relatively common.18
ABSTRACT:
The structural influence of a single Gly residue inserted
into an Aib16 homooligomer was studied in the solid state
by X-ray crystallography. The peptides
N3Aib8GlyAib8PheNH2 (1) and CbzPheAib8GlyAib8 (2)
were found to adopt well-defined helical structures, which
are broadly 310 helical. Indeed, 2 is the longest
crystallographic 310 helix thus far reported. However, in
the region of the central Gly residue, a loosening of the
310 structure is observed in both peptides, with 1 clearly
showing local adoption of an a-helical structure in the
#
region of residues 7–9. 2010 Wiley Periodicals, Inc.
Biopolymers 95: 62–69, 2011.
This article was originally published online as an accepted
preprint. The ‘‘Published Online’’ date corresponds to the
preprint version. You can request a copy of the preprint by
emailing the Biopolymers editorial office at biopolymers@wiley.
com
INTRODUCTION
he presence of aminoisobutyric acid (Aib) in peptides
stabilizes helical structures1,2 by virtue of the ‘‘confor-
mational Thorpe-Ingold effect’’:3 conformations in
which the peptide chain bends back on itself are sta-
In 1971, Marshall proposed that the gem-dimethyl group
of an Aib residue would restrict the conformations available
to an Aib containing peptide such that the 310 helix becomes
favored,20 and 310-helical conformations are commonly
observed in peptides with high frequencies of quaternary
amino acids,1–3 such as the peptaibols.21 Thus, while peptides
with a low proportion of Aib residues may adopt almost per-
T
bilized relative to chain-extended conformations,
which incur steric crowding of Aib’s gem-dimethyl group.
The helix-forming influence of Aib is sufficiently powerful
Correspondence to: Jonathan Clayden; e-mail: clayden@man.ac.uk
Contract grant sponsor: EPSRC
C
VV
2010 Wiley Periodicals, Inc.
fect
a
helices (BocValAlaLeuAibValAlaLeuValAlaLeuAib-
62
Biopolymers Volume 95 / Number 1

Interruption of 310-Helix by Single Gly Residue
63
ValAlaLeuAibOMe is an example7), all homopeptides of Aib
from the trimer to the CbzAib11Ot-Bu22 form 310 helices.
Of course, homopeptides of Aib have no intrinsic enantio-
meric preference and must form both left and right handed
helices: chemical exchange between diastereotopic methyl
groups in such systems has been used to establish that
the free enthalpy of activation for helix inversion in
CbzAib10Ot-Bu is 46 kJ mol21 at 265 K.23 However, it has
been shown for even just one chiral amino acid in an oligo-
Aib chain is enough to induce a preferred helix sense, both in
short (pentapeptide)24,25 and long (10–20-mer)26,27 helices.
Even a single Aib residue is enough to induce helicity in a
heptapeptide.4,28 Yet the reverse effect—how significantly a sin-
gle non-Aib unit destabilizes the 310 helix when inserted into a
long run of Aib residues—has apparently never been studied.
We showed by NMR that the peptaibol-like21 11-mer
CbzPheAib4GlyAib4AibCH2OH adopts a 310-helical structure
in solution despite the presence of the medial Gly residue. Mo-
lecular dynamics studies suggest that incorporation of Gly in
Aib oligomers increases the a-helical propensity of the latter
due to the reduction of side-chain contacts.29 We now report a
crystallographic study of more extended oligopeptides consist-
ing of two runs of eight Aib residues located either side of a Gly
residue. The compounds were originally made as part of an
NMR study of foldamer conformation,27 but they turned out
not only to be crystalline but also to illuminate in more detail
the extent of the destabilizing effect of this single Gly on the
310-helical structure in the crystalline state. In both structures
studied, a partial shift from the 310 toward an a-helical confor-
mation is seen just to the N-terminal side of the Gly residue.
MATERIALS AND METHODS
Peptide Synthesis
The peptides studied, N3Aib8GlyAib8PheNH2 1 and CbzPheAib8-
GlyAib8Ot-Bu 2, were made by the route shown in Scheme 1. Tet-
ramer 319 was obtained from the tert-butyl ester protected Aib by an
iterative procedure of acylation with 1-azidoisobutyryl chloride
AzibCl30 followed by hydrogenation of the corresponding azides.
From tetramer 3, either hydrogenation or ester hydrolysis liberated
the N terminus (in 4) or C terminus (in 5), respectively, ready for fur-
ther coupling. Treatment of 5 with Ac2O at 1208C forms the corre-
sponding azlactone31,32 that was opened with 4 by refluxing both
components in acetonitrile to furnish the octapeptide 6. Again, hy-
drogenation of 6 gave the free amino octamer 7 that was coupled
with a Cbz-protected amino acid (Gly or Phe) using standard
reagents to obtain CbzPheAib8OtBu 8a or CbzGlyAib8OtBu 8b. 8b
was hydrolyzed to the free acid 9, which was converted to its
azlactone and opened using L-phenylalaninamide to obtain
CbzGlyAib8PheNH2 10. Similar ester hydrolysis of 6 or 8a to form
the corresponding acids 11 and 12, followed by azlactone formation
and opening with the appropriate deprotected counterparts (by
hydrogenolytic cleavage of the Cbz group from 10 or 8b), gave the
oligomers N3Aib8GlyAib8PheNH2 1 and CbzPheAib8GlyAib8Ot-Bu 2.
Peptides 1 and 2 were obtained as white powders precipitated
from the reaction mixture and after isolation by filtration. No fur-
ther purification was required.
Crystallization and X-Ray Collection
Crystals suitable for X-ray analysis were obtained by slow evapora-
tion from an initial 1:1 acetonitrile–dichloromethane mixture. Crys-
tals of peptide 1 are monoclinic, space group C2, with unit-cell
˚
dimensions a 5 41.064 (8), b 5 8.6671 (17), c 5 27.132 (5) A, and
b 5 92.826 (4) 8 and those of peptide 2 are orthorhombic, with
space group P212121 and unit cell dimensions a 5 16.340 (3), b 5
SCHEME 1 Synthesis of peptides 1 and 2. a H2, Pd/C, EtOH; b CF3CO2H:CH2Cl2 1:1; c Ac2O, 1208C
2 h; d MeCN, reflux 48–72 h; e CbzPheOH, PyBOP, DIPEA, CH2Cl2 overnight; f CbzGlyOH, EDC,
HOBt, CH2Cl2, 48 h. N3AibX is used to mean the azido derivative of Aib, i.e. N2¼¼N-CMe2C(5 O)-X.
Biopolymers

`
J. Sola et al.
64
˚
17.121 (3), and c 5 36.538 (7) A. The crystal data and refinement ordered dichloromethane molecules, one at 0.25 occupancy. In both
details are summarized in Table I. structures, the absolute configuration was known, but in 1, it was
Data collection for both 1 and 2 was carried out on a Bruker also confirmed by anomalous dispersion of chlorine, arising from
SMART APEX CCD diffractometer with graphite monochromated the presence of the solvent dichloromethane. In each case, the crys-
Mo Ka radiation; cryocooling to 100 K was achieved with an Oxford tals diffracted weakly at high resolution, and so the data were cut at
˚
Cryosystems 700 Series Cryostream. The structures were solved by 0.9 and 1.0 A resolution for 1 and 2, respectively. The crystal data
direct methods33 and refined by full-matrix least squares against for 1 and 2 have been deposited with the Cambridge Crystallo-
F2.33 Most nonhydrogen atoms were refined anisotropically, except graphic Data Centre, deposition numbers 780,358 and 762,200,
some solvent atoms at partial occupancy in 1 (see below). Hydrogen respectively.
atoms bonded to carbon were included in calculated positions using
the riding method. Those bonded to nitrogen and oxygen were
found by difference Fourier methods; in peptide 1, these hydrogen
atoms were refined isotropically with restraints on the bond lengths
RESULTS AND DISCUSSION
to the parent atoms, and with Ueq values 1.2 (nitrogen) or 1.5 (oxy-
The crystal structures of 1 and 2 showed that both peptides
gen) times those of the parent atoms; for peptide 2, free isotropic
adopt a helical conformation in the crystalline state—a right
refinement of these hydrogen atoms was carried out. In 1, several
solvent molecules were found in the crystal lattice, including an ace-
tonitrile molecule at 0.5 occupancy, a water molecule, and two dis-
handed helix in the case of 1 and a left-handed helix in the
case of 2 (Figures 1 and 2). Both the octadecapeptide 1 and
Table I Crystal Data and Structure Refinement for 1 and 2
Identification code
1
2
Empirical formula
C
75 H125 N21 O18, 0.5(C2 H3 N),
1.25(CH2Cl2), H2 O
1753.66
C87 H140 N18 O21
Formula weight
Temperature (K)
˚
Wavelength (A)
1774.17
100 (2)
0.71073
100 (2)
0.71073
Crystal system
Space group
Monoclinic
C2
Orthorhombic
P212121
˚
Unit cell dimensions (A, 8)
a 5 41.064 (8)
b 5 8.6671 (17)
c 5 27.132 (5)
a 5 90
a 5 16.340 (3)
b 5 17.121 (3)
c 5 36.538 (7)
a 5 90
b 5 92.826 (4)
c 5 90
9,645 (3)
b 5 90
c 5 90
10,222 (3)
3
˚
Volume (A )
Z
4
1.208
4
1.153
Density (calculated) (mg/m3)
Absorption coefficient (mm21
F(000)
)
0.154
3,758
0.083
3,824
Crystal size (mm)
Theta range (8)
Index ranges
0.45 3 0.25 3 0.08
1.76–23.26
245 \5 h \5 45,
29 \5 k \5 9,
229\5 l\5 30
29,777
0.45 3 0.40 3 0.15
1.72–20.81
216 \5 h \5 16,
217\5 k\5 17,
236\5 l\5 36
49,807
Reflections collected
Independent/observed [I [ 2r(I)] reflections
Rint
13,710/11,484
0.0889
None
10,692/10,099
0.0610
None
Absorption correction
Refinement method
Full-matrix least-squares on F2
80/1,205
1.147
Full-matrix least-squares on F2
1/1,224
1.155
Restraints/parameters
Goodness-of-fit on F2
Final R indices [I [ 2 sigma(I)]
R indices (all data)
R1 5 0.0882, wR2 5 0.1944
R1 5 0.1056, wR2 5 0.2039
20.08 (17)
R1 5 0.0725, wR2 5 0.1474
R1 5 0.0767, wR2 5 0.1493
–
Absolute structure parameter
Largest diff. peak and hole (e A
23
˚
)
0.805 and 20.455
0.268 and 20.209
Values from Table I (y axis can be read as pm for hydrogen bond lengths or 8 for dihedral angles) plotted against residue number.
Biopolymers

Interruption of 310-Helix by Single Gly Residue
65
FIGURE 1 X-ray crystal structure of N3Aib8GlyAib8PheNH2 1.
the nonadecapeptide complete just over five turns of the Intermolecular contacts in the crystals of 1 lead to antiparal-
helix. The helical structure of 1 continues right through to lel sheets, with adjacent molecules in each sheet making head-
its amide C-terminus, whereas 2’s helicity is interrupted at to-tail hydrogen-bonded interactions through encapsulated
˚
its esterified C-terminus by a characteristic Schellmann water molecules (which receive a hydrogen bond [2.01 A] from
motif.34
the NH group of residue 2 and donate hydrogen bonds [1.82
˚
The adoption of a left-handed helical structure by 2 is sur- and 1.98 A] to two of the C terminal C¼¼O groups of adjacent
prising,35 as typically, peptides containing L-amino acids molecules) and tail-to-tail interactions with the adjacent sheet
adopt right-handed helical structures in solution,36,37 even through hydrogen bonded acetonitrile molecules. There is also
when the majority of the peptide consists of achiral Aib resi- a direct hydrogen-bond between the NꢀꢀH of residue 1 and the
dues.26 We assume therefore that the left-handed helicity of 2 C-terminal C¼¼O of an adjacent molecule (2.32 A). These
˚
arises as a result of crystal packing interactions. Inspection of looser, solvent-mediated contacts presumably permit the struc-
the N terminus of 2 reveals that the benzyl group of the Phe ture of 1 to relax into a more typical right handed helix.
residue unusually lies in such as way as to eclipse its carbonyl
Detailed analysis of the geometric parameters associated
group unfavorably (w 5 139.18, where typically for a right with the crystal structures is presented in Tables II and III.
handed helix w would lie in the region of 230 to 2408). As a These data allow some degree of insight into the effect of the
result, however, the two N-terminal NꢀꢀH bonds are suffi- medial Gly residue on the nature of the helix adopted in each
ciently exposed to form hydrogen bonds with the two C-ter- case. Each table presents the following parameters:
minal C¼¼O groups of the next helix in the crystal (hydrogen
(a) / and w (for simplicity, reported as positive values).
Although a and 310 helices display similar values of /
(characteristically 638 and 578, respectively), the value
of w in a 310 helix tends to lie close to 308, while in an
a-helix values of w are closer to 428. In tables, values
of these parameters, suggesting a local a-helical struc-
ture (/ [608 or w[ 368), are highlighted in bold.
˚
bond lengths of 1.91 and 2.16 A). The C-terminal t-Bu and
the N-terminal Bn groups avoid the close interactions, which
would result from a negative value of w. The structure of 2
consists of a series of staggered antiparallel head-to-tail
chains, members of the chains linked by this pair of hydrogen
bonds.
FIGURE 2 X-ray crystal structure of CbzPheAib8GlyAib8Ot-Bu 2.
Biopolymers

`
J. Sola et al.
66
Table II Structural Parameters for N3Aib8GlyAib8PheNH2 (1)
i 2 1 C¼¼O to i
i 2 2 C¼¼O to i
i 2 1 C¼¼O to i
1 2 HꢀꢀN
i 2 2 C¼¼O to i
1 2 HꢀꢀN ff
1 2 HꢀꢀN hydrogen
1 2 HꢀꢀN hydrogen
˚
bonding distance (A)
˚
bonding distance (A)
Residue
/ (8)
w (8)
ff C¼¼O. . .H (8)
C¼¼O. . .H (8)
1
–
19.2
42.2
41.3
24.4
32.0
45.4
53.9
46.3
45.1
36.5
26.8
28.7
28.2
30.8
34.9
39.6
30.7
5.1
–
–
–
–
2
50.5
50.6
57.6
51.4
51.7
54.3
60.1
66.7
57.7
57.6
51.9
51.9
50.6
49.9
49.1
57.3
89.1
2.17
2.08
2.12
2.23
2.51
2.80
3.08
2.80
2.25
2.02
2.15
2.09
2.12
2.18
2.09
2.31
–
–
155
169
171
144
128
128
106
129
170
165
153
174
172
159
147
150
–
–
3
3.56
3.91
3.90
3.17
2.25
2.12
2.42
3.61
3.97
4.01
4.24
4.24
4.03
3.58
3.40
–
123
125
146
143
147
170
149
109
130
138
126
128
135
140
97
4
5
6
7
8
9 Gly
10
11
12
13
14
15
16
17
18 PheNH2
–
(b) Lengths of the possible hydrogen-bonded contacts
are located in the table such that residue i lies approx-
imately at the mid-point of the 10- or 13-membered
cyclic structure created by each hydrogen bond.
between (i) residue i and residue i 1 3 (typical of a
310 helix) and (ii) residue i and residue i 1 4 (typical
of an a helix). The shorter of these two alternative
hydrogen bonds is in each case shown in bold. Values
(c) Angles of the possible hydrogen-bonded contacts
between (i) residue i and residue i 1 3 (typical of a 310
Table III Structural parameters for CbzPheAib8GlyAib8Ot-Bu 2
i 2 1 C¼¼O to i
i 2 2 C¼¼O to i
i 2 1 C¼¼O to i
1 2 HꢀꢀN ff
i 2 2 C¼¼O to i
1 2 HꢀꢀN ff
1 2 HꢀꢀN hydrogen
1 2 HꢀꢀN hydrogen
˚
bonding distance (A)
˚
bonding distance (A)
Residue
/ (8)
w (8)
C¼¼O. . .H (8)
C¼¼O. . .H (8)
0 Cbz
1 Phe
–
–
–
–
–
–
54.8
57.9
58.0
50.0
51.5
50.6
44.6
50.7
53.3
58.8
60.0
53.3
53.3
54.2
51.4
47.2
57.9
42.4
39.1
30.4
21.2
30.6
29.2
30.3
40.6
38.8
41.7
32.7
23.0
32.6
27.9
27.5
26.6
41.1
36.6
52.9
2.34
2.12
2.12
2.08
2.03
2.17
2.16
1.91
2.32
2.25
2.22
2.07
2.10
1.89
2.21
2.15
–
–
159
172
175
174
172
163
163
174
137
154
167
170
174
155
163
160
–
–
2
3.92
4.23
4.32
4.07
4.19
4.17
3.73
3.18
3.45
4.10
4.15
4.13
3.98
4.17
3.93
–
120
123
125
126
133
129
114
147
134
118
123
118
137
130
126
–
3
4
5
6
7
8
9
10 Gly
11
12
13
14
15
16
17
18 AibOt-Bu
–
–
–
–
Biopolymers

Interruption of 310-Helix by Single Gly Residue
67
FIGURE 3 Structural parameters for N3Aib8GlyAib8PheNH2 1. Values from Table I (y axis can be
read as pm for hydrogen bond lengths or 8 for dihedral angles) plotted against residue number.
helix) and (ii) residue i and residue i 1 4 (typical of turns, with the central Gly having a weak effect on its regular-
an a helix), reported as the angle subtended by the ity—an effect which is also evident in the slight kink in the
carbonyl carbon and the NH hydrogen at the car- middle of the helix (see Figure 2).
bonyl oxygen. The more linear of these two alterna-
In case of 1, the effect of the central Gly is more marked.
tive hydrogen-bonds is in each case shown in bold. The i 1 4 ? i hydrogen bond is shorter than the i 1 3 ? i
hydrogen bond across residues 7–9, whereas the i 1 4 ? i
hydrogen bond has a more favorable angle across residue 5–
˚
9. Across residue 8, there is a short [2.12(2) A] and almost
Values are again located in the table such that residue
i lies approximately at the mid-point of the 10- or
13-membered cyclic structure created by each hydro-
gen bond.
linear i 1 4 ? i hydrogen bond. Values of w indicative of an
a helix are also evident at residues 6–10 (as well as 2, 3, and
16). Residues 8 and 9 also have higher values of /. Overall,
the 310-helical structure of 1 is significantly perturbed by the
Gly residue, especially just to its N-terminal side, where sev-
eral parameters point to local adoption of an a-helical struc-
ture.
All three groups of parameters indicate that the general
structure of these peptides, especially in the case of 2, is a 310
helix, and in the case of 2 that the 310 helix extends essentially
the full length of the peptide, bar the C-terminal ester. No i
1 4 ? i hydrogen bond is shorter than the equivalent i 1 3
? i hydrogen bond, although a loosening of the 310 helix is
nonetheless evident in the vicinity of the central Gly in the
drift to higher values of w around residues 7–9 and higher
values of / at residues 10 and 11. The angle of the i 1 4 ? i
hydrogen bond is also marginally more favorable than the i
1 3 ? i hydrogen bond around residue 9. We therefore con-
A graphical indication of the effect of the Gly residues in
the two peptides can be obtained from the plots shown in
Figures 3 and 4, in which the parameters of Tables I and II
(values in either 8 or pm) are plotted against the residues’
position in the peptide. The distortion of the 310-helical
clude that for 2, the 310 helix continues for a remarkable five structure in the region of residues 7–9 is clearly evident.
Biopolymers

`
J. Sola et al.
68
FIGURE 4 Structural parameters for CbzPheAib8GlyAib8Ot-Bu 2. Values from Table I (y
axis can be read as pm for hydrogen bond lengths or 8 for dihedral angles) plotted against residue
number.
3. Toniolo, C.; Crisma, M.; Formaggio, F.; Peggion, C. Biopoly-
mers 2001, 60, 396–419.
CONCLUSION
Insertion of a single Gly residue into a run of 16 Aib residues dis-
places the crystallographic conformational preference of two
related oligomers from a 310 helix toward an a-helix in the vicin-
ity, and particularly to the N-terminal side, of the Gly residue.
This displacement toward the a-helical structure is evident in a
lengthening an distortion of the i 1 3 ? i hydrogen-bonding
contacts and a shortening and straightening of the i 1 4 ? i
hydrogen-bonding contacts, along with a shift in the dihedral
angles toward those typical of an a helix. In one of the oligomers,
the distortion nonetheless fails to fully break the 310-helical struc-
ture, making CbzPheAib8GlyAib8Ot-Bu the longest reported
crystallographically characterized 310 helix yet observed, with 18
residues making over five full turns of the helix.
4. Karle, I. L.; Flippen-Anderson, J. L.; Uma, K.; Sukumar, M.;
Balaram, P. Biopolymers 1990, 29, 1433–1442.
5. Fox, R. O.; Richards, F. M. Nature (London) 1982, 300, 325–
330.
6. Karle, I. L.; Flippen-Anderson, J. L.; Agarwalla, S.; Balaram, P.
Biopolymers 1994, 34, 721–735.
7. Karle, I. L.; Flippen-Anderson, J. L.; Uma, K.; Sukumar, M.;
Balaram, P. J Am Chem Soc 1990, 112, 9350–9356.
8. Chou, P. Y.; Fasman, G. D. Biochemistry 1974, 13, 222–245.
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´
We are grateful to the Departament d’Innovacio, Universitats i Empresa
de la Generalitat de Catalunya for a postdoctoral fellowship (to JS).
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69
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Biopolymers