Influence of the chirality of short peptide supramolecular hydrogels in protein crystallogenesis

Article abstract of DOI:10.1039/c4cc09024a

For the first time the influence of the chirality of the gel fibers in protein crystallogenesis has been studied. Enantiomeric hydrogels 1 and 2 were tested with model proteins lysozyme and glucose isomerase and a formamidase extracted from B. cereus. Crystallization behaviour and crystal quality of these proteins in both hydrogels are presented and compared.

Full text of DOI:10.1039/c4cc09024a

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Influence of the Chirality of Short Peptide
Supramolecular Hydrogels in Protein Crystallogenesis
Cite this: DOI: 10.1039/x0xx00000x
Mayte ConejeroꢀMuriel,a José A. Gavira,*a Estela PinedaꢀMolina,a Adam
Belsom,b Mark Bradley,b Mónica Moral,c Juan de Dios GarcíaꢀLópez Durán,c
Angélica Luque González,d Juan J. DíazꢀMochón,*d Rafael ContrerasꢀMontoya,e
Ángela MartínezꢀPeragón,e Juan M. Cuerva,e and Luis Álvarez de Cienfuegos*e
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
For the first time the influence of the chirality of the gel fibers
in protein crystallogenesis has been studied. Enantiomeric
hydrogels 1 and 2 were tested with model proteins lysozyme
(polyacrylamide) or are constituted by the natural chiral product
(agarose).
To test this hypothesis we selected two cysteineꢀbased peptides,
and glucose isomerase and a formamidase from B. cereus
.
compound
1
(
N,
N’ꢀdi(benzoyl)ꢀ
Lꢀcysteine diamide) and its
Crystallization behaviour and crystal quality of these proteins in
both hydrogels are presented and compared.
corresponding
D
enantiomer
2
(
N,
N’ꢀdi(benzoyl)ꢀ
D
ꢀcysteine
diamide) (Figure 1) due to their capacity to selfꢀassembly in
neat water to give the corresponding hydrogels.7 They were
tested with two model proteins, chicken HEWL lysozyme and
glucose isomerase, and one target protein, a formamidase from
B. cereus being the first time that supramolecular peptide based
hydrogels are used for this purpose. Additionally, the results
were compared with crystal growth in agarose gel to investigate
their potential in protein crystallization.
Chirality is ubiquitous in nature and has major implications in
cell interactions and biological processes. Within this topic,
chiral discrimination on chiral surfaces has significant effects in
protein adsorption and cellular adhesion and proliferation.1 It
would be expected that similar chiral discrimination were
relevant in 3Dꢀenviroments, such as in protein crystallogenesis.
Remarkably, the expected influence of the chirality in protein
crystallogenesis remains unexplored probably owing to the
difficulty to obtain a pair of enantiomeric environments suitable
for crystallization. In this sense, the use of stereochemically
wellꢀdefined fiber hydrogels can be an ideal media to study the
influence of the chirality in the process of crystal growth.
Recently, peptide hydrogels have found useful applications in
biology due to their biocompatible and biodegradable nature.2 It
has been shown that these supramolecular fibrous aggregates
interact selectively with proteins3 and also reduce denaturation.4
Considering this, we wondered if supramolecular hydrogels
based on the selfꢀassembly of short peptides could be
compatible with the process of protein crystallization since gels
are used as an excellent media for crystallization.5 It is known
that peptides hydrogels are able to form stereochemically well
defined 3D ordered structures6 and, therefore, the inherent
chirality of peptides can interact diastereoisomerically with
proteins, thus expecting different behaviors. It is worth noting
that the structural simplicity of short peptides is essential for
this purpose, allowing an easy access of their corresponding
enantiomers. This property is not feasible with other common
macromolecular hydrogels, which either have no stereocenters
Figure 1. Characterization of hydrogels
circular dichroism and rheology. TEM images of negatively stained
dried hydrogels at 3 mM: A) ; B) . Scale bar at 0.2 µm. C) CD
spectra and D) rheology of hydrogels and
1 and 2 by TEM microscopy,
1
2
1
2.
Compounds and were selected from a family of 22 cysteine
derivatives7 based on its capacity to form a stable and
completely transparent hydrogel. TEM images of hydrogel
1
2
1
showed selfꢀassembled nanofibers of diameters ranging from
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DOI: 10.1039J/oC4uCrnC0a9l0N2a4Ame
10 to 20 nm and lengths that go up to µm in size (Figure 1).8
Fibrous aggregates coꢀexist with regions where fibers are more
dispersed. Hydrogel
2 presented fibers of the same aspect. To
gain insight into the molecular arrangement of these hydrogels
circular dichroism (CD) spectra were measured (Figure 1).
Hydrogel
1
exhibited a negative band near 218 nm (nπ*
* disulfide band
transition), a positive band near 250 nm (n
σ
and/or ππ* of the aryl groups) and a negative band near 280 nm
(ππ* of the aryl groups) in good agreement with the CD
spectrum from other oligopeptides hydrogels9 identified as the
β
ꢀsheet conformation. The CD spectrum of hydrogel
specular image of hydrogel showing that the chirality of the
peptides is conserved and transferred supramolecularly to the
fibers. Hydrogels and were also analyzed using a regime of
2 is the
1
1
2
simple oscillatory shear stress through frequency (see
Supporting Information) and amplitude sweep tests. Both
hydrogels showed values of G’ (elastic modulus) higher than
G’’ (viscous modulus) indicating the predominant elastic
behavior of the gels (Figure 1). Based on the magnitude of the
Figure 2. A) Crystallization of lysozyme in hydrogel
1 using the 2L
configuration of the counterdiffusion technique setꢀup in Eppendorf
tubes schematically represented. The evolution of the supersaturation is
easily followed from top to bottom as an increase of crystal size and the
reduction of the nucleation density (Right: optical microscopic picture
under polarized light). B) and C) show the average values of standard
quality indicators of lysozyme and glucose isomerase crystals,
values of G’, G’’ and σc (G’
can conclude that these hydrogels are extremely weak. The
similar values of G’ and G’’ for hydrogels and confirm that
≈ 30 Pa; G” ≈ 4 Pa; σc < 1 Pa) we
respectively, grown in hydrogels 1, 2 and agarose from cryoꢀprotected
1
2
crystals diffracted at Xaloc beamꢀline (ALBA). Data sets were collected
at 100 K keeping constant all data acquisition configuration setꢀup and
analysis parameters.
both enantiomeric hydrogels have the same rheological
properties. VTꢀNMR studies for both hydrogels showed that the
signals of the aromatic region were sifted 0.5 ppm to downfield
as the temperature increased from 25 ºC to 70 ºC. The small
Lysozyme crystals grown in hydrogel
2 were better than those
obtained in hydrogel , reaching the resolution limit of the
1
values of T2 (1.84 s for 7.74 ppm signal) and the negative
detector (0.95 Å) and having the best quality indicators in terms
of I/σ(I), Rmerge and mosaicity (Figure 2B). It is also worth
mentioning the excellent reproducibility of crystals grown in
1
values of n
O
e indicated that the HꢀNMR signals corresponded
to aggregates of the diꢀpeptide which are not higher enough to
be NMRꢀsilent.10 The stability of these aggregates indicated that
Tgel should be higher than 70 ºC. In fact at 75/25 H2O/DMSO
hydrogel
2 (Figure S1 and Table S2). Moreover crystal quality
indicators of crystals grown in agarose (able to produce protein
crystals of high quality5a, d) were similar than those obtained in
mixture, the reported value of Tgel for
1
was above 90 ºC.8 DSC
did not show any
scans in both directions of hydrogels9c 1 and
2
hydrogel
1 but still inferior to hydrogel 2. Agarose gel has been
proven to facilitate the soaking of different compounds5a
including cryoꢀprotectants12. In order to evaluate the possible
cryoꢀprotection capacity of these new hydrogels we compared
the crystal quality of cryoꢀprotected and “naked” (flash frozen
endothermic or exothermic peaks. These results indicated
continuous gel to sol and sol to gel transitions (see SI).
The two enantiomeric hydrogels
1 and 2 were then tested as the
supramolecular medium for protein crystallization. Following
the procedure described in SI, protein was allowed to diffuse in
the gel prior to the addition of the precipitant. Figure 2A shows
a typical counterꢀdiffusion pattern of lysozyme crystals grown
without cryoꢀprotectant) lysozyme crystals grown in hydrogel
1
and agarose from two data sets collected at beamꢀlines ID23
and BM30 (ESRF). It is important to notice that the polymeric
in hydrogel 1. The effect of hydrogels 1 and 2 on the quality of
nature of both agarose and hydrogel
1 allowed us to collect full
lysozyme crystals was evaluated from a set of lysozyme
crystals measured at beamꢀlines Xaloc (ALBA) following the
standard quality criterion.11
data sets without needing to use any additive (Figure S2, S3 and
Table S3).
A similar analysis was performed with glucose isomerase
crystals grown in hydrogels
crystals obtained in hydrogel
1
,
2
1
and agarose. In this case
and agarose were more
homogeneous and of better quality than those grown in
hydrogel (Figure 2C, Table S4 and Figure S4). However, a
second crystal form (P21212) was identified only in the
experiments run with hydrogel that coꢀexist with the other
2
2
polymorph (I222), the form normally found under similar
crystallization conditions. Dauter and coꢀworkers have
described a new crystal form (
have solved its structure (PDB ID. 1OAD).13 Remarkably, the
polymorph found in hydrogel presents different unit cell
P21212) of glucose isomerase and
2
parameters (Å): 86.00, 93.68, 99.22 versus 98.45, 129.59, 78.33
for a, b and c respectively (Figure S4). To date there is only
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one PDB entry (PDB ID. 1O1H) with similar unit cell and
space group but obtained under different crystallization
conditions.
experiments we knew that agarose used in combination with
capillary counterdiffusion did not improve or even fully
impeded crystallization. Crystals grown in three different
crystallization conditions (Table S7) were evaluated. Only
The quality of this primitive orthorhombic crystal was the
highest of all glucose isomerase crystals tested, with the
structure determined at 1.20 Å and refined to R/Rfree values of
10.73/13.22 (%) (Table S5). When compared with the model
used for molecular replacement (PDB. ID 1OAD) the deviation
of the main chain is minimal (rmsd of 0.3). It is worth
mentioning that this new polymorph has only been obtained in
free solution using other precipitant, as in the case of the PDB
ID. 1O1H that crystallized using MPD, or in the presence of
high agarose concentration.5a In our case, under identical
cryoꢀprotected crystals obtained in hydrogel
2 using the
condition C18 of the GSK crystallization screening kit (Triana
S&T, Granada, Spain) produced the best crystals to date (1.4 Å
resolution limit, Table S6). On the other hand hydrogel
1
inhibited nucleation while crystal grown in agarose were of low
quality proving the different behavior of these hydrogels.
To sum up, we present the novel use of supramolecular
hydrogels based on shortꢀpeptides in neat water for protein
crystallization. These hydrogels, made of symmetrical
L and D
crystallization condition, the
P21212 polymorph was obtained
diꢀcysteine derivatives, were used to grow protein crystals of
high quality. Differences in crystal quality and packing for the
three proteins have been observed using enantiomeric
only with hydrogel and not in agarose at 0.5% (w/v), our
2
control, suggesting a novel interaction between the chiral gel
fibers and the protein at the nucleation stage. It has been
previously demonstrated that the nature of the gel fibers, such
as agarose15 and silica,16 affect protein nucleation density
(promoted and inhibited respectively) with no effect on the
final crystal packing i.e. formation of new polymorph. Up to
date, the correlation between the proteinꢀfiber interaction and
polymorphism is unknown. Taking into account that both,
chemical composition and the physical properties of the media
hydrogels
1 and 2, suggesting a significant influence of the
fibers’ chirality in the crystallization process. The formation of
the orthorhombic polymorph of glucose isomerase and the
growth of the highest quality crystals of formamidase
exclusively in hydrogel 2, support this hypothesis. This fact
highlights the relevance that chirality may have in protein
crystallogenesis for Xꢀray structural determination. Moreover
these supramolecular peptide based hydrogels are excellent
mediators for protein crystallization producing lysozyme and
glucose isomerase and formamidase crystals with excellent
quality indicators. Peptide based hydrogels also acted as a cryoꢀ
protectant avoiding the use of additives. Hence, these novel
chiral gels would expand the field of protein crystallogenesis.
are identical (hydrogels
1 and 2), we hypothesized that the
formation of a different polymorph has to be an effect of the
chirality at the nucleation state in terms of stabilization or
induction of the second polymorph.
It is also known that the incorporation of hydrogel fibers in the
protein crystal lattice occurs with agarose, silica and PEG
hydrogels.5a,15a,16a,c,17 To prove that the same effect also
happens with peptide hydrogels, dissolution experiments of
Notes and references
This research was funded by the MICINN (Spain) projects BIO2010ꢀ
16800 (JAG), CTQꢀ2011.22455 (LAC & JMC), CTQ2012ꢀ34778 (JJDM
& ALG), “Factoría Española de Cristalización” ConsoliderꢀIngenio 2010
(JAG & MCM) and EDRF Funds (JAG, LAC & JMC), P12ꢀFQMꢀ2721
(LAC) Junta de Andalucía. JJDM thanks MICINN for a R&C Fellowship
and MCM thanks CSIC for her JAE Fellowship. We would like to thank
Dr. S. MartinezꢀRodriguez for providing the plasmids of formamidase
and B. Morel and E. SánchezꢀCobos for their help with the CD studies.
We are very grateful to the staff at beamꢀline BM30, ID29 (ESRF) and
Xaloc (ALBA) for support during Xꢀray data collection.
lysozyme crystals grown in hydrogel
1 were carried out (Figure
3). This result showed that effectively the peptide fibers are
incorporated within the protein crystals. The unꢀcorrelated
behavior of incorporation and nucleation effects opens a
question on the different roles that gel nature can have in the
whole process of protein crystallogenesis.
a
Laboratorio de Estudios Cristalográficos, Instituto Andaluz de Ciencias
de la Tierra (CSICꢀUGR), Av. de las Palmeras 4, 18100 Armilla,
b
Granada, Spain. Eꢀmail: jgavira@iact.ugrꢀcsic.es, School of Chemistry,
University of Edinburgh, West Mains Road, EH9 3JJ Edinburgh, United
Kingdom. c Dpt. de Física Aplicada, Facultad de Ciencias (UGR), 18071ꢀ
Granada, Spain. d Facultad de Farmacia, Dpt. de Química Farmacéutica y
Orgánica (UGR). Centre for Genomics and Oncological Research:
Pfizer/UGR/Andalusian Regional Government, PTS Granada, Av. de la
Ilustración 114, 18016 Granada, Spain. Email: juandiaz@ugr.es, e Dpt. de
Química Orgánica, Facultad de Ciencias (UGR). Eꢀmail: lac@ugr.es,
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Dissolution sequence of a lysozyme crystal grown in
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maximum resolution of 1.78 Å (Table S6). From previous
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