University of Groningen
Thermotropic liquid crystals from engineered polypeptides
Pesce, Diego
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to
cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2015
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Pesce, D. (2015). Thermotropic liquid crystals from engineered polypeptides [Groningen]: University of
Groningen
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 16-06-2017
Chapter 1
Protein and Peptide Liquid Crystals
Abstract
Liquid Crystals (LCs) can be found in various contexts ranging from DNA
and cell membranes to sensor technology and soaps, providing a truly
multidisciplinary environment for research at the interfaces of chemistry,
biology, physics and engineering. Their physical behavior classifies them as a
fourth state of matter, lying between the crystalline solid and disordered
liquid states. The liquid crystalline mesopases transition is influenced by
temperature and concentration, giving rise to different kinds of ordered
structures. Particularly, LCs play an important role in biology because of
their essential characteristic, the combination of order and mobility that is a
basic requirement for self-organization and structure formation in living
systems. Among biomacromolecules, many proteins and polypeptides can
form liquid crystalline structures both in vivo and in vitro. A detailed
systematic study of liquid crystalline behavior of proteins is required to better
understand the nature of protein liquid crystals in biological systems and for
implementing their utilization in medicine and material engineering.
Here we reviewed examples from biological liquid crystalline materials with a
focus on protein and polypeptides, both naturally occurring and synthetic.
Furthermore, we highlighted their potential as versatile materials for
applications in biotechnology and material science.
2
1.1
Liquid Crystalline mesophases
In nature matter exists in three different states: solid, liquid and gas. In each of
these states, atoms and molecules, the building blocks of matter, have a
different degree and type of order with respect to their neighbors. Two kinds
of order are distinguishable: i) positional order, where molecules are arranged
in any sort of ordered lattice, and ii) orientational order, where molecules are
mostly pointing in the same direction. In addition, the order can be either
short-range (i.e. only between molecules close to each other) or long-range
(i.e. extending to larger, sometimes macroscopic, dimensions). In
orientational order the direction of preferred alignment is usually described by
a unit vector, the director L, which is simply the symmetry axis of the
orientational distribution (Figure 1). Depending on the kind of order the
physical properties of matter are either uniform in all orientations and thus
implying identical properties in all directions, or directionally dependent.
These states of matter organization are isotropic and anisotropic, respectively1.
Crystalline solids have highly ordered structures, whilst gases do not show
any positional or orientational order and liquids possess only short-range but
no long-range ordering. The borders between the different states are not
always clear and there are a large number of other intermediate phases called
mesophases. In particular, intermediate mesophases - which exhibit longrange orientational order - exist between solid and liquid states. Those
mesophases are known as Liquid Crystals (LCs), since they reveal features
belonging to both solid and fluid states. This fourth state of matter generally
possesses orientational or weak positional order. Thus, it reveals several
physical properties of crystalline state and, at the same time, has the ability to
flow as a liquid.
Because of the orientational order, most physical properties of LCs are
anisoptropic. Additionally, there are new physical qualities, which do not
appear in simple liquids such as: magnetic susceptibility, optical birefringence,
elastic or frictional torques (rotational viscosity). All these properties are
extremely sensitive to external perturbations because of the liquid-like fluidity
of LCs. For example, the birefringence can be manipulated easily with the
help of rather weak external magnetic, electric or optical fields, leading to
magneto-optical, electro-optical and opto-optical effect. Therefore, the most
successful application of LCs is in display technology, which takes advantage
of the electro-optical effect.
Comprehensively, LCs can be partitioned into two classes: thermotropic LCs
3
and lyotropic LCs. The phase transitions of thermotropic LCs depend on
temperature, while those of lyotropic LCs depend on both temperature and
concentration.
FIGURE 1. THERMOTROPIC MESOPHASES. Schematic structures of various
thermotropic LCs phases as function of the temperature. i) Cristalline, ii) smectic and iii)
nematic are anisotropyic phases whereas iv) is the isotropic liquid state.
1.1.1
Thermotropic Liquid Crystals
LCs are defined as thermotropic if the transitions between the phases are due
to the action of heat alone. At high temperatures, the axes of the LC molecules
randomly orient resulting in the isotropic phase as schematically represented
in figure 1. One phase that forms with decreasing temperatures is the nematic
phase (Figure 1a). The nematic phase is the least ordered of the mesophases,
as the molecules possess only orientational but no long-range positional order.
If a nematic LC is made of chiral molecules a cholesteric LC phase is also
formed. The name cholesteric derives from the fact that the first molecules
4
found to display these properties were those related to cholesterol. In
cholesteric LC, the direction of preferred orientation forms a helical structure,
with the helical axis being perpendicular to the director L. The most
prominent characteristic of cholesteric LC is a set of equally spaced parallel
lines (bright and dark lines), somewhat reminiscent of a fingerprint when
observed by polarizing optical micrograph (POM) (Figure 4a). The distance
between the alternating bright and dark lines is called the periodicity S, which
is equal to half the pitch (p/2) of the torsion of cholesteric LC (Figure 1b). A
second mesophase having positional ordering is known as the smectic phase
(Figure 1a) and is due to layered molecular arrangement, characterized by
additional degrees of positional order. There are different kinds of smectic
phases, for example the smectic A, where the molecular orientation is
perpendicular to the layers, and the smectic C, where the director is tilted
(Figure 1b). Several other kinds of smectic phases can exist, depending on the
extent and nature of positional order within the layers. Below a certain
temperature, the crystalline state is eventually reached (Figure 1a). Such a
temperature dependence of the material phases is reversible and a specific
mesosphase can be equilibrated by temperature stabilization.
1.1.2
Lyotropic Liquid Crystals
In lyotropic LCs, phase transition between mesophases may also be affected
by the addition of appropriate solvents, such as surfactants, and their
concentration. The molecules that form lyotropic LCs are amphipatic. The
objects created by amphiphiles are usually spherical, as in the case of micelles,
but may also be disc-like, rod-like or biaxial. These anisotropic selfassembled nano-structures can order themselves forming large-scale versions
of all the thermotropic phases, in a similar way as thermotropic liquid crystals
do. A generic progression of phases, going from low to high amphiphile
concentration, is: i) discontinuous cubic phase (micellar cubic phase); ii)
hexagonal phase (hexagonal columnar phase); iii) lamellar phase; iv)
bicontinuous cubic phase; v) reverse hexagonal columnar phase; vi) and
inverse cubic phase (inverse micellar phase) (Figure 2).
A very simple model, which predicts lyotropic phase transitions, is the hardrod model proposed by Lars Onsager1,2. This model predicts that a solution of
rod-shaped objects, at sufficient concentration, will undergo a phase transition
into a nematic phase.
5
FIGURE 2. LYOTROPIC MESOPHASES. Some lyotropic LC phases as a function of
temperature and concentration of the amphiphilic molecules a) dissolved surfactant being in a
disordered state, b) micellar, c) cubic, d) cylindrical and e) lamellar phases.
1.2
Biological Liquid Crystals
The main characteristics of biological materials are hierarchical structures,
multi-functionality and self-assembly. The combination of order and mobility
in living systems is a basic requirement for self-organization and structure
formation in solution3,4, since solids do not easily respond to change and
isotropic liquids have less capacity to include functionality. As a result,
mesophases are present throughout nature5. All biological liquid crystals
involve the formation of lyotropic phases as they are formed by aggregates of
molecules in a solvent, mainly water.
In the past decades, the study of lyotropic liquid crystals from biomolecules
and bio molecular assemblies has attracted attention in diverse areas such as
tissue engineering6, neurodegenerative diseases7, gene therapy8, food science9,
and prebiotic chemistry 10. LC phases have been identified in many structural
and functional biological materials, here we summarize examples ranging
6
from phospholipid cell membranes, to some concentrated DNA and protein
solutions in the secretion of spiders used to generate silk (Table 1).
TABLE 1. BIOLOGICAL LIQUID CRYSTALS. Compilation of liquid crystals from
biological molecules in vivo and in vitro.
BIOPOLYMER
FUNCTION
LC ORGANISATION
OCCURRENCE
Ref.
DNA
Chromosomal DNA
Efficient packing
Cholesteric
In vivo
13,19
Phage DNA
Efficient packing
Columnar
In vivo
12
Bacterial Plasmids
Efficient packing
Cholesteric
In vivo / In vitro
20
Structural
Helicoidal / Cholesteric
In vitro / In vivo
17
Starch
Reservoir of energy
Smectic
In vitro
9
Chitin
Structural
Helicoidal / Cholesteric
In vitro / In vivo
24,25
Structural/Functional
Layered lamellar
In vivo / In vitro
88
Structural
Nematic / Cholesteric
In vivo / In vitro
27,32
Spider Silk
Structural / Functional
Nematic
In vivo / In vitro
28
Silkworm Silk
Structural / Functional
Nematic
In vivo / In vitro
37
F-actin
Structural / Functional
Nematic / Smectic
In vitro
30
Microtubules
Structural / Functional
Nematic
In vitro
41
β-Lactoglobulin
Functional
Nematic
In vitro
45
Lisozyme
Functional
Nematic
In vitro
42
Insulin
Functional
Nematic
In vitro
44
Glycopolymers
Cellulose
Lipids
Phospholipids
Proteins
Collagen
7
The liquid crystalline state is important for different features found in nature:
i) efficient packing, as in concentrated DNA solutions11 in viruses12 and
sperm13; ii) surface directed self assembly, as in surface supported growth in
cellulose plywood14; iii) low viscosity, important in flow processing in silk
spinning15; iv) sensor actuator ability as in outer hair cells16; v) mechanical
strength, as in cellulose in plant cell walls, chitin in insect exocuticole and
collagen in human bones17 (Table 1).
The mesophase ordering in biological mesogens is usually chiral nematic, in
particular cholesteric. Biological LCs and fibrous biological materials are also
referred to as helicoidal plywoods18 because of their typical parallel fiber
arrangement (Figure 3). In the next sections, a brief overview is given on
naturally occurring liquid crystal phases according to molecular classes. The
emphasis is laid on natural and synthetic polypeptides and proteins since they
are a major subject of this thesis.
FIGURE 3. BIOLOGICAL PLYWOOD. (a–c) Schematics of the helicoidal plywood found
in animals and plants. Oblique sections (c) give the characteristic arc pattern observed in
extracellular fibrous composite biomaterials. (d and e) Side and normal view schematics of
the helix axis, defined by the helix orientation h (dashed line in d), the pitch p0 and the
rotation sense (counter-clockwise into the page in d) the layer distance between a 2π rotation
of the fibres is the pitch p0, whose magnitude is in the micrometer range.18
1.2.1
DNA
LC formation of DNA is observed where efficient packing is required,
examples are: the nuclei of cells such as dinoflagellates19, and sperm cells13,
the heads of bacteriophages12, or plasmid DNA within bacteria20. In
dinoflagellates, the twisted arrangement of chromosomes forms a cholesteric
liquid crystal phase19. In bacteriophages, DNA is packed in a columnar
fashion within concentric rings21. Plasmid DNA in E. coli cells forms dense
8
clusters with long-range order, and in vitro it shows birefringent liquid crystal
textures corresponding to a cholesteric phase at physiological concentrations20.
1.2.2
Glycopolymers/carbohydrates
Glycopolymers are also known to form LCs. Cellulose is one of the most
widely found biopolymers in nature and it is a component of the wall of many
types of plant cell. Cellulose micro-fibrils within plant cell walls exhibit a
helicoidal structure, which may be templated via a cholesteric liquid crystal
phase17,22. The cholesteric phase has also been observed in vitro in
concentrated solution of cellulose and derivatives of cellulose23. Starch has an
important energy storage function in plants. It consists of the linear glucan
amylose and the branched amylopectin. The amylopectin side chains form a
smectic structure and the packing depends on the hydration level9. Chitin has
a structural role in the exoskeleton of arthropods and is a long-chain polymer
of N-acetylglucosamine. α-Chitin can be processed by acid hydrolysis to
produce rod-like nanoparticles, which can form a chiral nematic phase24.
Iridescent colouring of certain beetle wings results from helicoidal stacking of
fibrous chitin layers, as for cellulose microfibrils25, and it is possible that LC
phases may act as precursors for these structures.
1.2.3
Lipids and Membranes
Lipids that constitute the cell membranes are perhaps the most important
example of a biological LC. All biological membranes are liquid-crystalline
structures and exhibit the typical layered lamellar structure26. Membranes
exert structural and functional roles such as separation of different fluid
compartments, selective transport and signal transduction. They consist of
phospholipid bilayers, with simple and conjugate proteins immersed in it.
Myelin, prominent in the transmission of electrical impulses by the nerve, is
also a LC and it consists of concentric cylindrical phospholipid bilayers.
1.2.4
Polypeptides and proteins
Polypeptides and proteins often self-assemble into fibrillar structures. In
(aqueous) solution, at sufficiently high concentration, these fibrillar structures
9
can orient and pack into lyotropic mesophases. As mentioned above, the
liquid crystalline state can play a pivotal role in the self-assembly process.
Many of the living world’s structural materials are based on self-assembled
protein fibers. Diverse examples include collagen27, silk28, tubulin29 and
actin30. Some of these materials, like collagen and silk, have recently been
described as a new class of matter: LC elastomers. LC elastomers can be
defined as lightly cross-linked main-chain or side-chain liquid crystalline
polymers showing elastomeric properties31. Interestingly, unlike conventional
rubbers, at rest, LC elastomers are ordered rather than disordered.
FIGURE 4. LIQUID CRYSTALLINITY IN COLLAGEN. a) Ordering of collagen
molecules in concentrated media observed in polarized light microscopy. Collagen fingerprint
patterns typical of a liquid crystalline cholesteric phase. bar =10 um. b) Structure of the
collagen fibril. Thin section of rat tail tendon observed in transmission electron microscopy
with uranyl acetate staining. bar = 100 nm.
34
Collagen plays a vital structural role in tissues such as tendon, skin, bone and
cornea. It has a triple helix structure of peptide strands rich in glycine, in
particular in sequences of type Gly-Pro-Y or Gly-X-Hyp (where X and Y are
any amino acid other than glycine, proline or hydroxyproline). Many different
types of collagen are known. Type I collagen monomers can form nematic,
precholesteric and cholesteric phases in vitro, in dilute acid solution27. Their
liquid crystallinity and orientation was first observed, on the basis of
birefringence and magnetic field alignment in studies of collagen from rat-tail
tendon under acidic conditions32 (Figure 4). Pro-collagen fibrils can also form
nematic and precholesteric liquid crystal phases under physiological buffer
conditions33 (Figure 4). It has been proposed that collagen fibrils belong to the
10
class of biological LC elastomers, since they are weakly cross-linked
networks with elastic mechanical properties31. The arrangement of fibrils in
vivo may also be LC-like, for instance the ordering of twisted fibrils of bone
osteons resembles that of a cholesteric mesophase33. Moreover, liquid
crystallinity is observed in collagen in solution under conditions close to the
concentration in tissues (50–200 mg mL-1)34. Although direct evidences for
liquid crystallinity of collagen in vivo are lacking, LC ordering has been
proposed to play an important role in the determination of tissue form33. In
this context, the ordering of collagen in bones, fish scales, cornea and in chitin
fibers of arthropods has been suggested to rely on self-assembly within
mesophases and being the underlying principle for formation of these
extracellular matrices35. In another example, collagen (mainly type IV)
extracted from bovine lens capsules gives an X-ray diffraction pattern that can
be interpreted as nematic ordering of helical collagen chains36.
Spider (Nephila clavipes) dragline silk has also been proposed to be a type of
LC elastomer, due to its structural and mechanical properties31. Spider
dragline silk protein comprises β-crystallites, enriched in the polyalanine
repeats, within a matrix of the less ordered glycine-rich segments. The
polyalanine segments form transverse lamellae within oriented nanofibrils,
extruded from the duct31. These fibrils exhibit nematic ordering28,31. The
efficiency of silk spinning depends on the viscosity of the protein solution
dope, which is reduced by adopting well-aligned nematic ordering15. In
contrast to fibers extruded from silk glands, mechanically drawn fibers show a
banded texture, indicating a periodic variation in the orientation of the
director37.
Another type of silk, produced by silkworms (Bombyx mori), is consisting of
β-sheets and is rich in GAGAGS repeats. Efforts are underway to understand
the natural spinning process whereby an aqueous solution of silk fibroin is
extruded under ambient conditions to produce aligned and water-insoluble
fibrils. A nematic phase has been observed in aqueous solutions of silkworm
silk fibroin37, and it was suggested to be an intermediate state, orienting the
fibrils, in the in vivo processing route37,38.
F-actin is the polymerized form of actin adopting helically twisted
microfilaments. It has served as a model for a semiflexible biopolymer39. As
an important motor protein, it is involved in force transduction in muscles and
also plays a structural role in the cytoskeleton. Muscle cells are packed with
oriented arrays of these microfilaments, which arrange themselves in a way
reminiscent of nematic and smectic LC. Nematic-like ordering was observed
11
in concentrated solutions of actin30 and at the edge of evaporating droplets on
glass40.
Microtubules (neurofilaments) are the main component of the cytoskeleton in
axons of vertebrates. They comprise superstructures of coiled-coil dimers of
the central hydrophobic regions of the subunit proteins. The N- and Cterminal domains are unstructured and the latter adopt side arm structures
around the filament body. The neurofilaments, which are the main
components of neuronal cytoskeleton, can form nematic hydrogels under
appropriate conditions of protein and salt concentration41.
Besides in structural proteins, nematic phase formation was also observed for
functional proteins such as hen lysozyme42, insulin43,44 and β-lactoglobulin45,
all of which form amyloid fibrils under appropriate denaturing conditions
(generally acidic pH, and/or high temperature) (Table 1). It is worth to
mention that nematic order was also observed in supramolecular structures
that do not form fibrils, as in the tobacco mosaic virus in colloidal
solutions46,47 and the isolated gp8 coat protein of M13 bacteriophage48.
Interestingly, various types of mucus are essentially liquid crystalline
hydrogels, comprising an entangled network of chains with local rigidity due
to glycosylated side chains. For example, the proteoglycan mucin is a
polyelectrolyte with a bottlebrush structure characterized by highly charged
carbohydrate side chains attached to a semiflexible protein backbone. Smallangle neutron scattering was used to show that mucin from pig stomach forms
a nematic phase at high concentration49. Other proteoglycans such as those
found in slug slime trails also form nematic phases50, which exhibit
viscoelastic properties important to this mode of movement.
1.3
Liquid Crystals from synthetic polypeptides
Several examples of LC phases have been obtained with synthetic and semisynthetic peptides or polypeptides in vitro, in the presence of different kind of
solvents (Table 2). Polypeptides generally adopt α-helix conformation when
they are dissolved in organic solvents such as N,N-dimethylformamide,
chloroform or benzene. At high concentration, the side-by-side packing of
rigid polypeptide chains induces LC structures. Interestingly, the
characteristic ordering of polypeptides was first observed from LC structures51.
12
TABLE 2. SYNTHETIC POLYPEPTIDE LIQUID CRYSTALS. Liquid crystals based on
synthetic peptides and polypeptides reported in the literature.
POLYPEPTIDE
Poly(γ-benzyl Lglutamate) (PBLG)
LC
ORGANIZATION
Cholesteric
SOLVENT
CONDITIONS
Chloroform
(CHCl3 )
n.d.
PBLG
right/left-handed
cholesteric, nematic
m-cresol
PBLG
right/left-handed
cholesteric
Dioxanedichloroethan
e
poly(β-pchlorobenzyl Laspartate)
(PClBLA)
right/left-handed
cholesteric
poly(β-phenethyl Laspartate (PPLA)
right/left-handed
cholesteric, nematic
PBLG
(recombinant DNA)
Smectic
AcQQRFQWQFEQQNH2
NH2 -FF-COOH
nanowires
Nematic
Nematic
Trichloroethyl
ene (TCE)
Trichloroethyl
ene/dichloroa
cetic acid
CHCl3/trifluor
oacetic acid
(TFA)
Water
CS2
30–60°C,
>60°C
10-70°C
25-100°C
10-93°C
RT
NOTES
Ref.
54
30–60°C right-handed
cholesteric; 60°C
nematic; >60°C lefthanded cholesteric
from right-handed to lefthanded with increasing
volume fraction of
dichloroethane
left cholesteric (90°C),
nematic (97°C), right
cholesteric (102°C)
opposite to change in
screw sense of the
polypeptide backbone
The screw sense of the
polypeptide backbone
changed from lefthanded (T<25°C) to
right-handed
(25°C<T<89°C) and then
to left-handed (T>89°C)
(L–R–L) with
corresponding and
opposite change in
cholesteric LCs (R-L-R)
separate by nematic
phases
Smectic order only when
CHCl3 (97%) and TFA
(3%)
57
56
58
59
46
20°C
60
RT
61
13
AAKLVFF
βAβAKLVFF
Nematic
Methanol
RT
62
Nematic
Water
25°C
63
β-peptides
Nematic
Water
- RT
65
A6K
Nematic
Water
n.d.
64
Nematic, columnar
hexagonal
Water
RT
89
Nematic, cholesteric
Water
(RT)
Nematic when Xaa was
Val and cholesteric when
Xaa was Pro or Ala
72
n.d.
Smectic, hexagonal,
(twisted) cholesteric, and
other phases could be
ob- served depending on
the chemical composition
of the copolymer and the
history and processing of
the sample
90
CHEMICALLY
MODIFIED
FFKLVFF–PEG
triblock
copolymers: Glu5(Gly-Xaa-Hyp-GlyPro- Hyp)6- Glu5
Polystyrene-bPBLG
PS-b-poly(L-lysine)
dendron-helical
(DHP) PBLG
copolymers
hyperbranched
polylysine (HBPL)
Ferritin-(N,Ndimethyl-1,3propanediamine)240
Cholesteric,
hexagonal
Lamellar
Smectic
Columnar hexagonal
or lamellar
Smectic
1,4-Dioxane
THF, Water
CHCl3
Water, 1butanol/water
/ethanol
No solvent
n.d.
75
n.d.
66
90-115°C
Dilute solution, complex
with sodium alkyl
sulphate surfactants
71
30-37°C
Water free: Complex with
the anionic polymer
surfactant C9H19 -C6H4 (OCH2CH2)20 O(CH2 )3 SO3
(S)
78
The formation of polypeptide LCs requires two crucial characteristics: rigidity
of the polymer chain and ordering of the rod chains. Thus, the α-helical
polypeptides support the LC structures, while the helix-to-coil transition is
detrimental to LC ordering. In the presence of a denaturating acid, for
14
example, polypeptides tend to adopt a random coil state because the acid
interferes with the hydrogen bonds involved in the helical architecture52. In
that case an anisotropic–isotropic transition (known as reentrant transition)
occurs at low temperature since the random coil is unable to induce LC
ordering53.
LCs of polypeptides were first described in the 1950s by Elliott and Ambrose,
who observed a birefringent phase in a solution of poly(γ-benzyl Lglutamate)/ chloroform (PBLG/CHCl3) mixtures54. PBLG later became a
model system for studying LCs.
Polypeptides are, in general, able to form LCs with nematic, cholesteric and
smectic phases (Figure 5).
FIGURE 5. POLYPEPTIDE LIQUID CRYSTALS. Classification of polypeptide LCs: a)
smectic, b) nematic and c) cholesteric phases. S indicates the periodicity of cholesterol phase
1.3.1
Nematic and cholesteric phases
In the nematic phase (Figure 5b), polypeptide chains have no positional order
and self-align to have long-range directional order with their long axes
roughly parallel. The nematic cholesteric phase (Figure 5c) is the most
common LC structure observed in polypeptides. In the cholesteric phase, the
periodicity S and the chirality are found to be dependent on several
parameters such as the temperature, polymer concentration, solvent nature and
molecular weight55-57. For example, PBLG in the solvent m-cresol, forms
right-handed cholesteric LCs in the temperature range of 30–60 °C and the
15
periodicity S increases with increasing temperature57. At 60 °C, the
cholesteric character disappears and nematic LCs are formed. Above this
temperature, left-handed cholesteric structures appear and the periodicity S
decreases with increasing temperature. Regarding the dependence on the
nature of the solvent, results from the PBLG LCs formed in dioxanedichloroethane mixed solvent showed that the chirality of the LC transformed
from right-handed to left-handed with increasing volume fraction of
dichloroethane56. For such behavior, the dielectric constant of the solvents is
believed to be one of the crucial influencing factors. Generally, a solvent with
lower dielectric constant supports a right-handed cholesteric structure. The
chirality of polypeptide backbones was also found to have an influence on the
handedness of LC structures. For example, Abe and collaborators found that
the chirality of the LCs is opposite to the screw sense of the polypeptide
backbone in the LC structure of poly(β-p-chlorobenzyl Laspartate)/trichloroethylene (PClBLA/TCE) system58. At room temperature,
PClBLA shows a right-handed α-helical conformation, while the chirality of
the LC is left-handed. Between 80 and 100 °C the polypeptide α-helix
changes its conformation from right to left-handed. Accordingly, the LC
structures change from left-handed to right-handed cholesteric, separated by a
nematic
phase.
In
another
work
on
poly(β-phenethyl
Laspartate)/trichloroethylene/dichloroacetic acid (PPLA/TCE/DCA) systems,
the same authors found a more complex LC behavior59. With increasing
temperature of the solution, the screw sense of the polypeptide backbone
changed from left-handed to right-handed and then to left-handed (L–R–L).
Simultaneously with the helix–helix transition, the handedness of LCs
gradually changes from right to left and then to right (R–L–R) (Figure 6),
each phase separated by a nematic phase. The inversion of the cholesteric
sense is triggered by the reversal of the screw sense of the α-helical backbone.
Simple nematic ordering has also been studied for de novo designed short
peptides. For example, the peptide Ac-QQRFQWQFEQQ-NH2, designed to
form β-tapes, has been shown to form nematic fluid and nematic gel phases60.
Nematic liquid crystalline ordering of the aromatic dipeptide NH2-FF-COOH
nanowires in the solvent CS2 has recently been discussed61. The stable
dispersion of the peptide nanowires exhibited a LC phase for a broad
concentration range allowing the rapid alignment of nanowires under an
external electric field.
Other nanotube and fibril forming peptides, such as AAKLVFF62,
βAβAKLVFF (βA denotes the β2-alanine amino acid)63 and the model
16
amphiphilic peptide A6K64, form nematic phases in different solvents.
Hartgerink and co-workers reported the nematic phase formation of selfassembling synthetic β-peptides65.
FIGURE 6. PPLA BACKBONE AND LC CHIRALITY. Schematic representation of
PPLA conformation-induced transition of LC handedness.
1.3.2
91
Smectic phases
In the smectic phase (Figure 5a), as mentioned before, polypeptide chains are
positionally ordered along one direction, forming well-defined layers that can
slide over one another. Smectic ordering is common for low molecular weight
LC compounds, is mainly found for polypeptides with identical degrees of
polymerization and it is rarely observed in synthetic polypeptides due to the
polydisperse nature of the materials. To overcome this problem, Tirrell’s
group reported for the first time the formation of smectic LC structures from
monodisperse polypeptides derived from PBLG, which was synthesized via
17
recombinant DNA technology46,47 (Figure 7). The LCs of modified PBLG
were obtained in mixtures of CHCl3 (97%, v/v) and trifluoroacetic acid (TFA;
3%, v/v). The solution, observed under the polarized optical microscope
(Figure 7a), was iridescent with a fan-like texture indicative for smectic order.
The densitometer scan of the SAXD pattern of the film (Figure 7b, curve a)
showed a well-defined spacing. In contrast, the suggested smectic-like
ordering was not present in a solid film of polydisperse PBLG with
comparable molecular weight (it yielded no small-angle reflections in a
similar diffraction experiment) (Figure 7b, curve b). In figure 7c a illustration
for the smectic LC structures is shown.
The addition of TFA inhibits aggregation of PBLG in concentrated solutions
while the low concentration does not destroy the rigidity of polymer chains.
Another strategy is to cap the polypeptide ends with a compact and bulky
group. During self-assembly in concentrated solutions, such modified
polypeptides can self-assemble into a smectic LC phase66. For example,
Winnik and colaborators found that dendron-helical (DHP) PBLG copolymers
could form smectic phases in CHCl366. The dendritic block prevents the DHP
copolymers from aligning into a nematic phase.
FIGURE 7. PBLG LIQUID CRYSTAL. a) POM image of PBLG in CHCl3/TFA. b)
Densitometer scans of the SAXD patterns of films prepared from solutions of monodisperse
PBLG (curve a) and polydisperse PBLG (curve b). c) Smectic structure of PBLG, showing
the origin of the 12.5 A and 114.5 A reflections. As can be seen, the helical rods arrange in
layers of thickness 114.5 A , and the distance between two PBLG rods is 12.5 A , which
corresponds to the diameter of PBLG rods.
91
LC phase formation was also observed for some peptide based pharmaceutical
active compounds - in particular, luteinizing-hormone releasing hormone
(LHRH) agonists leuprolide67, nafarelin68 and detirelix68,69, all of which form
β-sheet fibrils that form birefringent lyotropic gels in water, and in some cases
18
in organic solvents. For the latter two peptides, the liquid crystal phase in
aqueous solution was identified as nematic68. Certain compounds, such as the
cyclic peptide cyclosporine (natural occurring peptide), may also form
thermotropic liquid crystals70. The liquid crystal phase formation is actually a
problem observed in formulation that leads to unwanted turbidity,
precipitation and gelation69.
Hyperbranched polylysine (HBPL) complexed with various anionic, sodium
alkyl sulphate surfactants, were observed to form short-range liquid crystallike order, columnar hexagonal packing or lamellar assembly, depending on
the surfactant alkyl chain length71.
Synthetic ‘‘triblock’’ copolymers containing Glu5 end blocks and a central
collagen-like midblock, (Gly-Xaa-Hyp-Gly-Pro- Hyp)6, where Xaa was one
of the amino acids Ala, Pro, Ser or Val, were also shown to exhibit liquidcrystal like ordering72. Nematic ordering was observed when Xaa was Val,
and cholesteric-like ordering was obtained with Xaa being either Pro or Ala.
1.4
Liquid Crystals from polymer-peptide conjugate
Besides pristine synthetic polypeptides, polystyrene-b-PBLG diblock
copolymer may form cholesteric or hexagonal phases in organic solvents73.
Swelling of melt structures for the same class of diblock also leads to lamellar
phase formation in organic solvents74. In polystyrene-b-poly(L-lysine) diblock
copolymer studied by the same authors, deswelling of lamellar structures was
noted75. Diblocks of PBLG attached to short peptide blocks (e.g. leucine or
leucine/ valine oligomers) may form cholesteric phases, although structures
were only observed in cast (dried) films76. Hamley and collaborators have
reported on nematic and hexagonal columnar phase formation by the peptide–
PEG conjugate FFKLVFF–PEG in aqueous solution77. The peptide is based
on a fragment of the amyloid β-peptide, KLVFF, extended at the N-terminus
by two phenylalanine residues.
Besides peptides, also proteins were transformed into liquid crystals by
chemical modification. Recently, Mann and collaborators reported the first
example of a solvent-free liquid protein. The protein melt, based on ferritin
covalently coupled to N,N-dimethyl-1,3-propanediamine (DMPA) (240
groups per molecule) (Figure 8), exhibits thermotropic liquid-crystalline
phase behavior with smectic micro-structure, when complexed with the
anionic polymer surfactant (C9H19-C6H4-(OCH2CH2)20O(CH2)3SO3- (S))78.
19
FIGURE 8. SOLVENT-FREE FERRITIN LIQUIDS AND LIQUID CRYSTALS.
Proposed structure of the [C-Fn][S] melt at 32 °C showing a lamella liquid-crystalline phase
viewed side-on to the layer stacking direction (black arrow; C-Fn (Cationized Ferritin) red, S
(anionic polymer surfactant C9H19-C6H4-(OCH2CH2)20O(CH2)3SO3-) green), with a liquid-like
in-plane arrangement comprising short-range local ordering interactions between adjacent
polymer chains.
1.5
78
Applications and outlook
Besides their importance for display technology, several new applications for
LCs are emerging in recent years, in particular for LCs from
biomacromolecules.
Some reports have been published describing new liquid-crystal materials
with notable biomedical and biological implications. For example, templates
based on nematic and precholesteric (so-called ‘crimped’) structures of
collagen were used to control the growth and morphology of fibroblasts79. It
was suggested that the high concentration of collagen in the matrices could
improve biodegradation compared to dermal equivalents79. Moreover, type I
collagen showed to be a versatile template for the structuration of silica,
allowing the replication of the chiral nematic organisation in a biologically
inspired collagen-silica hybrid material80.
Devices and configurations based on liquid-crystal materials are being
developed for spectroscopy, imaging and microscopy, leading to new
techniques for optically probing biological systems81. A series of
20
investigations have reported biosensors, which use the interface of
thermotropic liquid crystals as a detection system82.
With advancing technology, the preparation of peptide-modified interfaces
has received a great deal of attention due to the potential utility of these
interfaces for monitoring enzymatic activities, controlling cellular behaviors
and manipulating peptide-protein interactions83,84. Lundgren and colleagues,
for example, developed a liquid crystal pixel array for a high number of signal
discrimination in array biosensors85.
Regarding advanced materials processing, as mentioned before, regeneration
of silk fibers via a wet spinning process from solutions containing silkworm
cocoons is of intense interest in order to understand the natural processing
route86,87, with the view that this might be used to process new genetically
engineered silk proteins.
The success in constructing highly ordered smectic phases in solution by
using modified polydisperse polypeptide homopolymers, rather than
monodisperse samples, is of significant value for the preparation of welldefined supramolecular structures. Highly ordered ultrathin PBLG films with
controlled layer spacing and thickness can be prepared from smectic PBLG
LCs. When the rods are oriented perpendicular to the substrate, they can be
used as nonlinear optical and piezoelectric materials47. When the rods in the
films are organized parallel to the substrate, the mono- or multilayer films are
useful in creating patterned arrays for use in sensor technology. In addition,
the LCs can be further functionalized through tethering bulky groups or
polymer chains.
In conclusion, the rapid growing synergism between materials science and
molecular biology has led to new sources of engineering materials and to new
applications for bioengineered proteins. Polypeptides can be produced that
either i) copy the principal consensus amino acid repeats that characterize a
natural protein; ii) contain the principal repeats from more than one protein,
for example hybrid molecules of silk and elastin; iii) contain functional
proteins in fusion; or, iv) even contain amino acids not found in nature.
Because subsequent hierarchical assembly steps can be conducted in vitro
with external control over the temperature, pressure and chemical
environment, there is the opportunity for obtaining products with properties
not found in nature. This holds especially true for obtaining novel peptidebased LCs with different mesophase for a vast range of potential applications.
21
1.6
Summary
Phases of the matter in between the liquid isotropic and the solid anisotropic
states are called Liquid Crystals and comprise different mesophases. These
mesophases vary depending on the temperature (thermotropic LCs) and
concentration of the mesogens (lyotropic LCs).
LC is, thus, a “delicate phase of matter” and it is characteristic of many
biological materials from DNA to membranes. The perfect balance between
order and fluidity appear to be a necessary prerequisite for life and the selfassembly driven by the formation of lyotropic liquid crystal phases takes part
in several biological processes. Several protein and polypeptide LC mesogens
are present in nature and they take over important structural and functional,
cellular and extracellular processes. Moreover, synthetic peptides and
polypeptides that show liquid crystalline behavior have been produced thanks
to advances of chemical synthesis and genetic engineering.
There are many challenges arising for LCs based on proteins and polypeptides.
They range from the design of naturally inspired new materials, with extreme
control over the synthesis and introduction of functionalities, to a better
understanding of fundamental processes in biology.
1.7
Motivation and thesis overview
The overall goal of the work described in this thesis was to explore the
physical properties of a novel anhydrous thermotropic liquid crystalline
material based on protein polyelectrolytes.
In Chapter 2 we describe the construction of a genetically engineered elastinlike polypeptide (ELP) carrying several negative charges. The protein
electrolyte was monodisperse and had a precisely defined amino acid
composition. We further explored the production of thermotropic liquid
crystals in the absence of water. To perform this we used a generic method
based on the formation of a complex with cationic surfactants containing
flexible alkyl tails followed by dehydration. In the second chapter, the phase
behavior of these polypeptide-surfactant complexes was investigated
employing different techniques such as differential scanning calorimetry
(DSC), polarized optical microscopy (POM), small-angle x-ray scattering
(SAXS) and wide-angle x-ray scattering (WAXS).
After thoroughly characterizing the liquid crystal properties of the pristine
22
complexes, we tested the possibility of incorporating a functional protein
within the anhydrous thermotropic LCs, as described in Chapter 3. We first
fused the ELP proteins to a Green Fluorescent Protein (GFP) and then we
studied the formation of LC complexes. The ELP-GFP fusion proteins were
still able to form LCs structures without hampering the fluorescence of GFP
even in the anhydrous mesophase. Additionally, we analysed the effect of the
length of the ELP segment and of the alkyl chain of the surfactant on the
liquid-crystalline behavior.
In Chapter 4 we investigate the elastic properties and rheological studies of
the ELP-GFP surfactant complexes. They showed elastic behavior similar to
native elastin. The elastic values were comparable to those of liquid crystal
elastomers in which the mesogens usually need to be cross-linked by
polymerization to achieve elastic behavior. Moreover, it was shown how the
elasticity of the material can be controlled by varying the lengths of alkyl
chains of the surfactants or the molecular weight of the ELP segment.
In Chapter 5 we tested the properties of the pristine ELP-GFP material in the
absence of surfactant. In particular, we investigated the cytotoxicity and the
ability of the ELP-GFP to be internalized by mammalian cells. Thus, we
generated positively charged variant of ELP-GFP fusion proteins and tested
their cytotoxicity and uptake in mammalian cells. The ELP-GFP fusion
proteins showed low cytotoxicity and six-fold enhanced cellular uptake
compared to the GFP alone. Finally, the mechanism of cellular uptake was
investigated.
23
1.8
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
24
Bibliography
Sengupta, A. Liquid Crystal Theory. 7–36 (2013).
Onsager, L. The effects of shape on the interaction of colloidal
particles. Ann. N. Y. Acad. Sci. 51, 627–659 (1949).
Hamley, I. W. Liquid crystal phase formation by biopolymers. Soft
Matter 6, 1863–1871 (2010).
Hamley, I. W. & Castelletto, V. Biological Soft Materials. Angew.
Chem. Int. Ed. 46, 4442–4455 (2007).
Rey, A. D., Herrera-Valencia, E. E. & Murugesan, Y. K. Structure and
dynamics of biological liquid crystals. Liq. Cryst. 41, 430–451 (2014).
Chung, W. J. et al. Biomimetic self-templating supramolecular
structures. Nature 478, 364–368 (2011).
Conway, K. A. et al. Acceleration of oligomerization, not fibrillization,
is a shared property of both alpha-synuclein mutations linked to earlyonset Parkinson's disease: Implications for pathogenesis and therapy.
Proc. Natl. Acad. Sci. 97, 571–576 (2000).
Koltover, I., Salditt, T., Rädler, J. O. & Safinya, C. R. An inverted
hexagonal phase of cationic liposome-DNA complexes related to DNA
release and delivery. Science 281, 78–81 (1998).
Waigh, T. A., Perry, P., Riekel, C., Gidley, M. J. & Donald, A. M.
Chiral side-chain liquid-crystalline polymeric properties of starch.
Macromolecules 31, 7980–7984 (1998).
Nakata, M. et al. End-to-End Stacking and Liquid Crystal
Condensation of 6- to 20-Base Pair DNA Duplexes. Science 318,
1276–1279 (2007).
Livolant, F. & Bouligand, Y. Liquid crystalline phases given by helical
biological polymers (DNA, PBLG and xanthan). Columnar textures. J.
Phys. France 47, 1813–1827 (1986).
Earnshaw, W. C. & Casjens, S. R. DNA packaging by the doublestranded DNA bacteriophages. Cell 21, 319–331 (1980).
Livolant, F. Ordered phases of DNA in vivo and in vitro. Physica A
176, 117–137 (1991).
Giraud-Guille, M. M. Plywood structures in nature. Curr. Opin. Solid
St. M. 3, 221–227 (1998).
Vollrath, F. & Knight, D. P. Liquid crystalline spinning of spider silk.
Nature 410, 541–548 (2001).
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
Bouligand, Y. Liquid crystals and biological morphogenesis: Ancient
and new questions. Comptes Rendus Chimie 11, 281–296 (2008).
Neville, A. C. A Pipe-Cleaner Molecular-Model for Morphogenesis of
Helicoidal Plant-Cell Walls Based on Hemicellulose Complexity. J.
Theor. Biol. 131, 243–254 (1988).
Rey, A. D. Liquid crystal models of biological materials and processes.
Soft Matter 6, 3402 (2010).
Livolant, F. & Bouligand, Y. New observations on the twisted
arrangement of dinoflagellate chromosomes. Chromosoma 68, 21–44
(1978).
Reich, Z., Wachtel, E. J. & Minsky, A. Liquid-crystalline mesophases
of plasmid DNA in bacteria. Science 264, 1460–1463 (1994).
Cerritelli, M. Encapsidated Conformation of Bacteriophage T7 DNA.
Cell 91, 271–280 (1997).
Roland, J. C., Vian, B. & Reis, D. Further Observations on Cell-Wall
Morphogenesis and Polysaccharide Arrangement During Plant-Growth.
Protoplasma 91, 125–141 (1977).
Gilbert, R.-D. & Patton, P. A. Liquid crystal formation in cellulose and
cellulose derivatives. Prog. Polym. Sci. 9, 115–131 (1983).
Belamie, E., Davidson, P. & Giraud-Guille, M. M. Structure and
chirality of the nematic phase in α-chitin suspensions. J. Phys. Chem.
B 108, 14991–15000 (2004).
Jewell, S. A., Vukusic, P. & Roberts, N. W. Circularly polarized
colour reflection from helicoidal structures in the beetle Plusiotis
boucardi. New J. Phys. 9, 99–99 (2007).
Goodby, B. J. Liquid crystals and life. Liq. Cryst. 24, 25–38 (1998).
Giraud-Guille, M. M. Liquid Crystallinity in Condensed Type-I
Collagen Solutions - a Clue to the Packing of Collagen in Extracellular
Matrices. J. Mol. Biol. 224, 861–873 (1992).
Knight, D. P. & Vollrath, F. Liquid crystals and flow elongation in a
spider's silk production line. P. Roy. Soc. B-Biol. Sci. 266, 519–523
(1999).
Janmey, P. A. Mechanical properties of cytoskeletal polymers. Curr.
Opin. Cell Biol. 3, 4–11 (1991).
Coppin, C. M. & Leavis, P. C. Quantitation of liquid-crystalline
ordering in F-actin solutions. Biophys. J. 63, 794–807 (1992).
Knight, D. P. & Vollrath, F. Biological liquid crystal elastomers. Phil.
Tr. Roy. Soc. B 357, 155–163 (2002).
25
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
26
Murthy, N. S. Liquid Crystallinity in Collagen Solutions and Magnetic
Orientation of Collagen Fibrils. Biopolymers 23, 1261–1267 (1984).
Martin, R. et al. Liquid crystalline ordering of procollagen as a
determinant of three-dimensional extracellular matrix architecture. J.
Mol. Biol. 301, 11–17 (2000).
Giraud-Guille, M.-M., Mosser, G. & Belamie, E. Liquid crystallinity
in collagen systems in vitro and in vivo. Curr. Opin. Colloid In. 13,
303–313 (2008).
Belamie, E., Mosser, G., Gobeaux, F. & Giraud-Guille, M.-M.
Possible transient liquid crystal phase during the laying out of
connective tissues: α-chitin and collagen as models. J. Phys-Condens.
Mat. 18, 115-129 (2006).
Barnard, K. & Gathercole, L. J. Short and long range order in
basement membrane type IV collagen revealed by enzymic and
chemical extraction. Int. J. Biol. Macromol. 13, 359–365 (1991).
Kerkam, K., Viney, C., Kaplan, D. & Lombardi, S. Liquid
Crystallinity of Natural Silk Secretions. Nature 349, 596–598 (1991).
Viney, C. & Bell, F. I. Inspiration versus duplication with
biomolecular fibrous materials: learning nature‘s lessons without
copying nature’s limitations. Curr. Opin. Solid St. M. 8, 165–171
(2004).
Käs, J. et al. F-actin, a model polymer for semiflexible chains in dilute,
semidilute, and liquid crystalline solutions. Biophys. J. 70, 609–625
(1996).
Vonna, L., Limozin, L., Roth, A. & Sackmann, E. Single-filament
dynamics and long-range ordering of semiflexible biopolymers under
flow and confinement. Langmuir 21, 9635–9643 (2005).
Jones, J. B. & Safinya, C. R. Interplay between liquid crystalline and
isotropic gels in self-assembled neurofilament networks. Biophys. J. 95,
823–835 (2008).
Corrigan, A. M., Müller, C. & Krebs, M. R. H. The Formation of
Nematic Liquid Crystal Phases by Hen Lysozyme Amyloid Fibrils. J.
Am. Chem. Soc. 128, 14740–14741 (2006).
Krebs, M. R. H., Bromley, E. H. C., Rogers, S. S. & Donald, A. M.
The Mechanism of Amyloid Spherulite Formation by Bovine Insulin.
Biophys. J. 88, 2013–2021 (2005).
Krebs, M. R. H. et al. The formation of spherulites by amyloid fibrils
of bovine insulin. Proc. Natl. Acad. Sci. 101, 14420–14424 (2004).
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
Sagis, L. M. C., Veerman, C. & van der Linden, E. Mesoscopic
Properties of Semiflexible Amyloid Fibrils. Langmuir 20, 924–927
(2004).
Yu, S., Soto, C. M. & Tirrell, D. A. Nanometer-scale smectic ordering
of genetically engineered rodlike polymers: Synthesis and
characterization of monodisperse derivatives of poly(gamma-benzyl
alpha,L-glutamate). J. Am. Chem. Soc. 122, 6552–6559 (2000).
Tirrell, D. A. et al. Smectic ordering in solutions and films of a rodlike polymer owing to monodispersity of chain length : Article :
Nature. Nature 389, 167–170 (1997).
Fiester, S. E., Jákli, A. & Woolverton, C. J. Liquid crystal properties of
a self-assembling viral coat protein. Liq. Cryst. 38, 1153–1157 (2011).
Waigh, T. A. et al. Entanglement Coupling in Porcine Stomach Mucin.
Langmuir 18, 7188–7195 (2002).
Viney, C., Huber, A. E. & Verdugo, P. Liquid-Crystalline Order in
Mucus. Macromolecules 26, 852–855 (1993).
Robinson, C. Liquid-Crystalline Structures in Solutions of a
Polypeptide. T. Faraday Soc. 52, 571–592 (1956).
Lin, J. P. Re-entrant isotropic transition of polypeptide liquid crystal.
Polymer 38, 4837–4841 (1997).
Lin, J. P. Reentrant isotropic transition of polypeptide liquid crystal:
effect of steric and orientation-dependent interactions. Polymer 39,
5495–5500 (1998).
Elliott, A. & Ambrose, E. J. Evidence of Chain Folding in
Polypeptides and Proteins. Discuss. Faraday Soc. 246–251 (1950).
Watanabe, J. & Nagase, T. Thermotropic Polypeptides .5.
Temperature-Dependence of Cholesteric Pitches Exhibiting a
Cholesteric Sense Inversion. Macromolecules 21, 171–175 (1988).
Toriumi, H., Minakuchi, S., Uematsu, Y. & Uematsu, I. Helical
Twisting Power of Poly(Gamma-Benzyl L-Glutamate) Liquid-Crystals
in Mixed-Solvents. Polym. J. 12, 431–437 (1980).
Toriumi, H., Kusumi, Y., Uematsu, I. & Uematsu, Y. Thermally
Induced Inversion of the Cholesteric Sense in Lyotropic Polypeptide
Liquid-Crystals. Polym. J. 11, 863–869 (1979).
Abe, A., Hiraga, K., Imada, Y., Hiejima, T. & Furuya, H. Screw‐sense
inversion characteristic of α‐helical poly (β‐p‐chlorobenzyl L‐aspartate)
and comparison with other related polyaspartates. Pept. Sci. 80, 249–
257 (2005).
27
59.
60.
61.
62.
63.
Abe, A., Furuya, H. & Okamoto, S. Spatial configurations,
transformation, and reorganization of mesophase structures of
polyaspartates - A highly intelligent molecular system. Biopolymers 43,
405–412 (1997).
Aggeli, A. et al. Hierarchical self-assembly of chiral rod-like
molecules as a model for peptide beta-sheet tapes, ribbons, fibrils, and
fibers. Proc. Natl. Acad. Sci. 98, 11857–11862 (2001).
Han, T. H. et al. Liquid Crystalline Peptide Nanowires. Adv. Mater. 19,
3924–3927 (2007).
Krysmann, M. J. et al. Self-assembly of Peptide nanotubes in an
organic solvent. Langmuir 24, 8158–8162 (2008).
Castelletto, V., Hamley, I. W., Hule, R. A. & Pochan, D. Helical‐
Ribbon Formation by a β‐Amino Acid Modified Amyloid β‐Peptide
64.
65.
66.
67.
68.
69.
70.
28
Fragment. Angew. Chem. Int. Ed. Engl. 48, 2317–2320 (2009).
Bucak, S., Cenker, C., Nasir, I., Olsson, U. & Zackrisson, M. Peptide
Nanotube Nematic Phase. Langmuir 25, 4262–4265 (2009).
Pomerantz, W. C. et al. Nanofibers and Lyotropic Liquid Crystals
from a Class of Self‐Assembling β‐Peptides. Angew. Chem. 120, 1261–
1264 (2008).
Kim, K. T., Park, C., Kim, C., Winnik, M. A. & Manners, I. Selfassembly of dendron-helical polypeptide copolymers: organogels and
lyotropic liquid crystals. Chem. Commun. 13, 1372–1374 (2006).
Tan, M. M., Corley, C. A. & Stevenson, C. L. Effect of gelation on the
chemical stability and conformation of leuprolide. Pharm. Res. 15,
1442–1448 (1998).
Powell, M. F., Sanders, L. M., Rogerson, A. & Si, V. Parenteral
Peptide Formulations: Chemical and Physical Properties of Native
Luteinizing Hormone-Releasing Hormone (LHRH) and Hydrophobic
Analogues in Aqueous Solution. Pharm. Res. 08, 1258–1263 (1991).
Powell, M. F., Fleitman, J., Sanders, L. M. & Si, V. C. Peptide Liquid
Crystals: Inverse Correlation of Kinetic Formation and
Thermodynamic Stability in Aqueous Solution. Pharm. Res. 11, 1352–
1354 (1994).
Lechuga Ballesteros, D., Abdul Fattah, A., Stevenson, C. L. & Bennett,
D. B. Properties and stability of a liquid crystal form of cyclosporine—
the first reported naturally occurring peptide that exists as a
thermotropic liquid crystal. J. Pharm. Sci. 92, 1821–1831 (2003).
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
Canilho, N., Scholl, M., Klok, H.-A. & Mezzenga, R. Thermotropic
Ionic Liquid Crystals via Self-Assembly of Cationic Hyperbranched
Polypeptides and Anionic Surfactants. Macromolecules 40, 8374–8383
(2007).
Martin, R., Waldmann, L. & Kaplan, D. L. Supramolecular assembly
of collagen triblock peptides. Biopolymers 70, 435–444 (2003).
Schlaad, H. Solution properties of polypeptide-based copolymers. 53–
73 (2006).
Douy, A. & Gallot, B. Block copolymers with a polyvinyl and a
polypeptide block: factors governing the folding of the polypeptide
chains. Polymer 23, 1039–1044 (1982).
Billot, J.-P., Douy, A. & Gallot, B. Synthesis and structural study of
block copolymers with a hydrophobic polyvinyl block and a
hydrophilic polypeptide block: Copolymers polystyrene/poly(L-lysine)
and polybutadiene/poly(L-lysine) - Billot - 2003 - Die
Makromolekulare Chemie - Wiley Online Library. Makromol. Chem.
177, 1889–1893 (1976).
Minich, E. A., Nowak, A. P., Deming, T. J. & Pochan, D. J. Rod–rod
and rod–coil self-assembly and phase behavior of polypeptide diblock
copolymers. Polymer 45, 1951–1957 (2004).
Hamley, I. W., Krysmann, M. J., Castelletto, V. & Noirez, L. Multiple
Lyotropic Polymorphism of a Poly(ethylene glycol)-Peptide Conjugate
in Aqueous Solution. Adv. Mater. 20, 4394–4397 (2008).
Perriman, A. W., Cölfen, H., Hughes, R. W., Barrie, C. L. & Mann, S.
Solvent-free protein liquids and liquid crystals. Angew. Chem. Int. Ed.
Engl. 48, 6242–6246 (2009).
Besseau, L., Coulomb, B., Lebreton-Decoster, C. & Giraud-Guille, M.
M. Production of ordered collagen matrices for three-dimensional cell
culture. Biomaterials 23, 27–36 (2002).
Eglin, D., Mosser, G., Giraud-Guille, M.-M., Livage, J. & Coradin, T.
Type I collagen, a versatile liquid crystal biological template for silica
structuration from nano- to microscopic scales. Soft Matter 1, 129–131
(2005).
Woltman, S. J., Jay, G. D. & Crawford, G. P. Liquid-crystal materials
find a new order in biomedical applications. Nat. Mater. 6, 929–938
(2007).
Lowe, A. M. & Abbott, N. L. Liquid Crystalline Materials for
Biological Applications. Chem. Mater. 24, 746–758 (2012).
29
83.
84.
85.
86.
87.
88.
89.
90.
91.
30
Park, J.-S. & Abbott, N. L. Ordering transitions in thermotropic liquid
crystals induced by the interfacial assembly and enzymatic processing
of oligopeptide amphiphiles. Adv. Mater. 20, 1185–1190 (2008).
Park, J.-S. et al. Formation of Oligopeptide-Based Polymeric
Membranes at Interfaces between Aqueous Phases and Thermotropic
Liquid Crystals. Chem. Mater. 18, 6147–6151 (2006).
Lundgren, J. S., Watkins, A. N., Racz, D. & Ligler, F. S. A liquid
crystal pixel array for signal discrimination in array biosensors.
Biosens. Bioelectron. 15, 417–421 (2000).
Ha, S.-W., Tonelli, A. E. & Hudson, S. M. Structural Studies of
BombyxmoriSilk Fibroin during Regeneration from Solutions and Wet
Fiber Spinning. Biomacromolecules 6, 1722–1731 (2005).
Liivak, O., Blye, A., Shah, N. & Jelinski, L. W. A microfabricated
wet-spinning apparatus to spin fibers of silk proteins. Structureproperty correlations. Macromolecules 31, 2947–2951 (1998).
Luzzati, V. & Husson, F. The structure of the liquid-crystalline phasis
of lipid-water systems. J. Cell Biol. 12, 207–219 (1962).
Hamley, I. W. et al. Nematic and Columnar Ordering of a PEGPeptide Conjugate in Aqueous Solution. Chem. Eur. J. 14, 11369–
11375 (2008).
Gallot, B. Comb-like and block liquid crystalline polymers for
biological applications. Prog. Polym. Sci. 21, 1035–1088 (1996).
Cai, C., Lin, J., Zhuang, Z. & Zhu, W. Ordering of Polypeptides in
Liquid Crystals, Gels and Micelles. Controlled Polymerization and
Polymeric Structures: Flow Microreactor Polymerization, Micelles
Kinetics, Polypeptide Ordering, Light Emitting Nanostructures 259,
159–199 (2013).