Structural features and molecular aggregations of designed triple-stranded β-sheets in single crystals

Article abstract of DOI:10.1039/c6cc00127k

Design, synthesis and single-crystal conformations of hybrid triple-stranded β-sheets composed of E-vinylogous residues are reported. Restricting conformational flexibility of β-strands through the insertion of carbon-carbon double bonds at facing positio

Full text of DOI:10.1039/c6cc00127k

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DOI: 10.1039/C6CC00127K
Journal Name
COMMUNICATION
Structural Features and Molecular Aggregations of Designed
Triple-Stranded β-Sheets in Single Crystals
Received 00th January 20xx,
Accepted 00th January 20xx
Anupam Bandyopadhyay, Rajkumar Misra, and Hosahudya N. Gopi*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Design, synthesis and single crystal conformations of hybrid triple-
stranded β-sheets composed of E-vinylogous residues are
reported. Restricting conformational flexibility of β-strands
through the insertion of carbon-carbon double bonds at facing
positions leads to the increased peptide crystallinity, which
allowed unambiguous structural characterization of three
stranded β-sheets. This strategy can be further explored for the
design of functional β-sheets.
I
II
Scheme 1: Stable N-C-Cβ=C (I) and H-C-Cβ=C(II) eclipsed conformations of
E-vinylogous amino acid residues.
β-Sheets are remain beguiling even after sixty years from their
discovery by Pauling and Corey.1 Interest in the design of β-
sheets has greatly increased after the recognition of their
involvement in many neurodegenerative diseases.2 β-Hairpin
is a simple super secondary structure consists of anti-parallel
β-strands and a reverse turn. The registry of β-strands in β-
hairpin also offers a basis for understanding the stability of
anti-parallel -sheets in protein structures. Extensive efforts
have been made in the literature towards the design and
characterization of short peptides that readily fold into stable
β-hairpins in solution.3 Besides the natural β-turn inducing
sequence Asn-Gly, the D-Pro-Xxx (D-Pro-Xxx, where Xxx is Gly,
Ala, Pro, Aib etc.) motif discovered by Gellman4 and Balaram5
group’s has been extensively utilized to design a variety of β-
Scheme 2: Sequences and schematic representation of the designed three-
stranded β-sheets P1 and P2 (dgL, dgV and dgF are denoted as ,β-dehydro -
Leu, , β-dehydro --Val and ,β-dehydro--Phe, respectively).
hairpin mimetics. In addition to the -hairpins, the D-Pro-Xxx
turn motif, which is known to induce either type I’ or type II’
β-turns6 has also been employed for the construction of three,
four and five-stranded sheets.7 Along with the
-turns,
other organic templates,11 β- and
cation-π14 interactions have also been explored to design
stable -hairpin mimetics. Besides understanding their
conformations, the success of the de novo design of -hairpins
has been extensively utilized to derive the inhibitors for
protein-protein interactions,15 amyloid fibril formation,16 and
anti-angiogenic candidates,17 protein epitope mimetics,18 self-
assembled biomaterials.19

-amino acids,12 π-π,13
significant efforts have also been made in the literature
towards the design of β-sheet mimetics.8 Nowick and
colleagues have shown the utilization of substituted aromatic


templates in the design of 
-hairpin mimetics.9 Eisenberg and
Nowick further explored β-sheet inducing aromatic amino acid
templates to study the structure and self-aggregation
properties of amyloid oligomers.10 Additionally, a variety of
We have been interested in the utilization of naturally
occurring non-ribosomal

,β-unsaturated

-amino acids (E-
vinylogous amino acids or

,β-dehydro-
-amino acids) in the
design of foldamers.20 Though these amino acids have been
widely present in many biological active peptide natural
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products,21 however, little is known regarding their potential
utility in the peptide design. Nevertheless, Schreiber et al
demonstrated that the extended sheet type of structures in
(E)-vinylogous dipeptides.22 Further, Hofmann and colleagues
predicted the helical conformations of homooligomers of (E)-
vinlogous amino acids through theoretical studies.23 Careful
inspection of the single crystal conformations of various (E)-
vinylogous residues reported in the literature20, 22revealed that
they adopt mostly H-C-Cβ=C eclipsed conformations instead
of N-C-Cβ=C eclipsed conformations (Scheme 1) even though
they encompass with 1,3 allyic strain. In addition, the
vinylogous amides preferred local s-cis conformation to avoid
the steric clash along the peptide backbone, which promotes
them to adopt extended conformations. Based on these
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DOI: 10.1039/C6CC00127K
observations, we predicted that these
, β unsaturated -
amino acids may serve as ideal candidates for the construction
of β-sheet structures. Recently, we reported the single crystal
conformation of an octapeptide β-hairpin by incorporating (E)-
vinylogous residues at the facing positions of the anti-parallel
β-strands20c as well as miniature β-meander mimetics.24 In
continuation, we sought investigate whether the remarkable
structural features of E-vinylogous residues can be utilized to
design multistranded β-sheets. Herein, we are reporting the
single crystal conformations of two 15 residue hybrid three-
stranded β-sheets, P1 and P2, by incorporating guest (E)-
P1
P2
Figure 1: X-ray structures of three-stranded β-sheets P1 and P2.
NOEs between the
CH CβH) suggesting the well folded triple
conformations of P1 in solution. The sequential dNN NOEs
observed between the Gly5 Leu6 as well as Gly10 Leu11

-strand residues (NH

NH, NH

CH and
-sheet




vinylogous residues at the facing positions of antiparallel -
are consistent with either type I’ or type II’ β-turn
conformations of DPro-Gly segments. Similar types of NOEs are
also observed in peptide P2 (data not shown). Further, the CD
analysis also hints the β-sheet signature of these peptides in
methanol. Peptide P1 showed CD negative maxima at 228 nm,
while peptide P2 displayed CD negative maxima at around 250
nm (See ESI Fig. 6 and 7). We speculated that the observed
red shift in P2 is probably due to the interactions of juxtaposed
aromatic side-chains of dgF2 and dgF14 (Scheme 2).
strands (Scheme 2) and their structural analogy with protein
structures.
The hybrid three stranded β-sheets P1 and P2 were
designed based on the earlier reported octapeptide
(
-hairpin
P3, Boc-Val-dgL-Val-DPro-Gly-Leu-dgV-Val-CONH2).20c In P1
,
three (E)-vinylogous residues dgL2, dgV7, and dgL14 were
incorporated at the facing positions of anti-parallel β-strands
(Scheme 2). Similarly, P2 was designed by incorporating dgF2,
dgL7 and dgF14 at the facing positions of the anti-parallel β-
D
The X-ray quality single crystals of P1 and P2 were obtained
from the slow evaporation of aqueous methanol solution and
their crystal structures are shown in Figure 1. Both peptides
stands. Similar to P3, we used Pro-Gly dipeptide segment to
nucleate either type I′ or type II′ β-turns in P1 and P2. Valine
was incorporated at the position i of the first β-turn to induce
trans-amide bond geometry with proline at the i+1 position,
while Ala was chosen to incorporate at the position i of the
second β-turn to avoid any steric clash with the N-terminal
acetyl group. To complement the exposed NH and carbonyl
groups of the central strand, the C-terminal tetrapeptide β-
strands were designed. Peptides were synthesized by solid
phase method on Rink amide resin using standard Fmoc-
chemistry. The final peptides were purified using reverse
adopted well-defined three-stranded β-sheet structures in
single crystals. Though the conformations of multi-stranded β-
sheets have been extensively investigated in solution, to the
best of our knowledge, this is the first report depicting the
single crystal conformations of designed three-stranded β-
sheets. Instructively, the structures are stabilized by nine
intramolecular H-bonds, existed between the anti-parallel β-
strands. In contrast to the 10 and 14 membered H-bonds of
regular anti-parallel β-sheets, these structures are stabilized by
14 and 18 membered intermolecular H-bonds along with the
10 membered H-bonds. Further, all exposed NHs and the
carbonyl groups are involved in the intermolecular H-bonding
with other parallelly arranged three-stranded β-sheets in the
crystal packing. In addition to the standard nine NH---OC H-
bonds, the structures are also stabilized by the week CH---O H-
bonds existed between the CH and CO groups of anti-parallel
β-strands (See ESI). The parameters of CH---O H-bonds are
found to be within the standard limits of the values reported in
the literature.25 It is noteworthy to mention that the average
1
phase HPLC on a C18 column and subjected to H NMR, CD and
single crystal X-ray crystallography studies. The wide
dispersion of amide NHs, vinyl protons and C-H chemical
shifts of

- and (E)-vinylogous residues observed in the 1H
NMR (CD3OH) of P1 and P2 indicate the well-defined
structures in solution. Fully assigned 1H NMR spectra are
shown in the supporting information. The peaks were assigned
using 2D NMR (TOCSY, ROESY and COSY). The critical NOEs
observed in the ROESY spectrum of P1 are illustrated in the
supporting information. The strong sequential NOEs between
CH (i)
NH (i+1) and cross-strand long range
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
i+1 = 30°

i+2 = 90° and

i+2 = 0°).6 Similar to P1, the structural
View Article Online
DOI: 10.1039/C6CC00127K
analysis of P2 also reveals that it adopted a three stranded -
sheet conformation with type I' -turns. All three vinylogous
residues dgF2, dgL7 and dgF14 are nicely accommodated into
the anti-parallel β-strands without any compromise in overall
β-hairpin conformation. Further, the
Val15 in P2 are also adopted semi-extended conformations by
displaying values -90 and -98°, respectively. In addition, Val
13 also showed the value -98°. Careful analysis of the P2
-residues Lue12 and


structure reveals the side-chains of juxtaposed aromatic
residues dgF2 and dgF14 are separated by 3.86 Å (head to
head). The H-bond parameters and torsion angles of P1 and P2
are tabulated in the supporting information (Tables S1 and S2).
In hindsight, the hybrid octapeptide β-hairpin P3 (Fig. 2a)
with a similar sequence of the N-terminal β-hairpin of P1
(a)
(b)
(c)
(residues 1-8) showed the type II
angles are i+1= 60° i+1= -120° i+2 = -80° and i+2 = 0)
conformation in single crystals (Fig. 2a).20c Though the
individual hairpins with type I and type II β-turns have been
′ β-turn (idealized torsion


′
′
studied with designed β-hairpins and in natural protein
sequences, however, the same hairpin sequence adopting
both type I
′
and II
′
β-turns, is rarely observed. The type II
′
adopted by the P3 and type I
′
adopted by the P1 and P2 serve
as high resolution model examples. The overlay of P1 and P3 is
shown in Fig. 2b. The conformational exchange between the β-
turns in P1 and P3 involves an approximate 180° flip in the
central DPro-Gly peptide unit. The preference for type I' β-
(d)
(e)
turns in the three-stranded -sheets over the type II' turns are
consistent with the results reported by Sibanda and Thornton
in their survey of two residue turns in high resolution protein
structures.26 In contrast to the flat β-hairpin conformation of
the octapeptide P3, a right handed twist has been observed in
the type I
in P1 P2 and P3 reveal that the H-bond distances between the
anti-parallel -strands in the type I β-hairpin structure is
relatively shorter than that of corresponding β-hairpin with
type II β-turns (Tables S3-S8). These results are consistent
with the Richardson’s prediction of on the β-hairpins with type
turns.27 Further, we sought to investigate the resemblance of
naturally occurring type I containing β-hairpins with P1 in the
′ β-turn hairpins. The analysis of H-bond parameters
,

′
(f)
′
Figure 2:a) X-ray structure of P3; b) Superposition of β-hairpin P3 with type II'
over P1; c) Superposition of P1 over -cobra toxin; d) Overlay of P1 over the
type I' -hairpins from the protein structures; e) Lateral assembly of three
stranded β-sheets through intermolecular H-bonds; f) Vertical stacking of -
sheets.
I′
′
context of whether the right handed twist observed in P1 is
analogous to the natural β-hairpins (Table S9). The overlay of
ten type I′ β-hairpin structures derived from the protein crystal
structures with P1 is shown in Fig. 2d (also Fig. S5). A good
correlation was observed between the natural β-hairpins from
protein structures and the designed β-hairpin structure. In
intramolecular CH---O bond distances are shorter than the
intermolecular C-H---O H-bonds. Further, the structural
analysis of P1 reveals that, except Leu12 and Val15, all other
addition, the superposition of the three stranded
over the randomly selected
-cobratoxin (PDB 2CTX),28
suggested that these designed hybrid -sheets can be used to
-sheet P1

-residues in the anti-parallel β-strands adopted extended
conformations. Residues Leu12 and Val 15 adopted semi-
extended conformations with the values -80 and -98°,



mimic multiple β-sheets structure (Fig. 2c) of proteins. Overall,
these results suggested that naturally occurring (E)-vinylogous
amino acids can be utilized to construct stable multi-stranded
β-sheet structures without deviating overall folding of the
molecule.
As self-aggregation of β-sheets plays a major role in many
neurological disorders,2 we further investigated the
aggregation patterns of P1 and P2 in crystal packing. We
respectively. Inspection of the torsion angles of vinylogous
residues dgL2, dgV7 and dgL14 revealed that they are well
accommodated into the
characteristic backbone conformations. The
vinylogous residues were found to be similar to that of
values of -residues. Both D-Pro-Gly segments adopt a type

-sheets with very similar
and 1 values of
and




I′ beta-turn conformation (idealized torsion angles are i+1= 60°
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Am. Chem. Soc., 2001, 123, 8667; c) S. H. Gellman, Curr.
realized, besides the lateral aggregations of β-strands through
intermolecular H-bonding of solvent exposed amide groups,
the crystal structure also provided evidence of vertical β-sheet
stacking through hydrophobic interactions of amino acid side-
chains along with the additional support from β-turn amide NH
H-bonds. Similar to that of β-strand amino acid side chains, the
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-turn amide group is positioned vertical to the plane. Both
lateral and vertical stacking of three-stranded β-sheets are
shown in Fig. 2e and 2f, respectively. The intermolecular H-
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amide (NH and CO) is involved in the H-bonding with solvent
water molecules (Fig. S8), while in P1 and P2 the turn amide
NH is directly involved in the intermolecular H-bonding with
another β-sheet molecule. These crystal structures may serve
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natural systems.
-sheet aggregations in
In conclusion, we have presented the utilization of
naturally occurring unsaturated -amino acids in the design of
multiple -hairpin structures. The insertion of (E)-alkenyl

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structural analysis suggested that the -strands in type I'
hairpins are more closely packed compared to the -hairpin
with the type II' -turn. The work presented here may serve as
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-

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‡ CCDC Numbers of P1 and P2 are 1435239 and 1032408,
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