“ABSTRACT: Biological materials have mechanical
properties that have been highly tuned for specific functions. The properties
are governed by combinations of molecular interactions that are incorporated at
multiple length scales in hierarchical structures. Establishing the links
between molecular interactions and physical properties in biological materials
has the potential to improve the design of structural biomaterials by offering
effective design strategies. Insect cuticle, in particular, is an interesting
model for studying structure-property relationships because it can have a wide
range of mechanical properties despite a limited compositional makeup. The
elytron, or outer wing cover, of the beetle is a strong and tough biomaterial
made mostly of proteins, chitin, catechols and water. The objective of this
thesis was to investigate molecular interactions among the components of
elytral cuticle to both understand their role in governing in vivo properties
as well as their future potential as biomaterial design motifs. The objective
was addressed using two approaches. First, the intact elytron was studied to
identify material design concepts that contribute to the strong and tough
physical properties of the structure. Second, simplified systems of cuticular
components were examined in vitro to help understand the in vivo roles of
specific interactions with the future goal of providing new biomaterial design motifs.
Studies on the intact elytron from the yellow mealworm beetle, Tenebrio
molitor, determined that the cuticle is not compositionally uniform throughout
development. Instead, new material is continuously secreted from the epidermal
cells. The color change and the frequency dependence of the cuticle plateaued
after 48 hours, indicating that the initially secreted material matures within
48 hours of eclosion. The mature cuticle bears the tensile load while
additional material is subsequently deposited and matured over a week of
development. Important contributions of both covalent and non-covalent
interactions were identified in cuticle using dynamic mechanical analysis over
a broad range of frequency, temperature and by treatments with non-covalent
bond breaking-solvents. Indirect evidence pointed to the dominant role of
covalent interactions in stabilizing the cuticle shortly after secretion from
the epidermal cells. Dynamic mechanical analysis showed that the storage
modulus of fully tanned and dried elytra was less frequency dependent (n =
0.013 ± 0.004) than the modulus of untanned and dried elytra (n = 0.05 ±
0.024), even at frequencies as low as 0.01 rad/s. Additionally, increasing the
temperature from 25 to 150°C, up to the point of degradation, resulted in only
a 3.5 fold decrease in the storage modulus and modest rise in the tan
δ curve for fully tanned elytra. To further separate the role of
covalent and non-covalent interactions in elytral cuticle, solvents were used
to disrupt non-covalent interactions. More material could be extracted from
partially tanned elytra than from fully tanned elytra, most likely reflecting
the lower degree of covalent crosslinking in the immature layers of cuticle
within partially tanned elytra. Soaking fully tanned elytra in formic acid,
which disrupts hydrogen bonding, caused the static mechanical properties to
resemble those of partially tanned elytra, which is consistent with the view
that covalent crosslinks within the load bearing layers of cuticle are established
within 48 hours. The role of non-covalent interactions was also highlighted by
mechanical tests after treating with formic acid. The solvent caused a 5-fold
decrease in the Young's modulus of fully tanned elytra, suggesting that a
significant portion of the stiffness (which increases 20-fold with maturation)
is provided by non-covalent interactions. The added stiffness provided by
non-covalent interactions appears to have been provided without compromising
toughness; that is, apparent hydrogen bonding caused a proportional increase in
fracture stress and decrease in fracture strain, thus leaving toughness
unchanged. Despite uncovering important contributions from covalent and
non-covalent interactions within the network as a whole, the complexity of the
biological structure makes it difficult to identify the exact interactions
contributing to the physical properties. Thus, simplified systems were used to
study specific molecular interactions in vitro. The hypothesized interactions
that were studied in vitro, namely protein-chitin complexation,
protein-catechol and protein-protein crosslinks, and protein-Ni2+ complexation,
were studied using two abundant cuticle proteins in the elytra of the red flour
beetle, Tribolium castaneum, CPR27 and CP30. CPR27 has a conserved sequence of
amino acids that has been hypothesized to bind chitin. Results from active
microrheology showed that addition of CPR27 to fluorescently-labeled aqueous
chitosan solutions caused a 2-fold decrease in viscosity. Since this microrheology
technique allows for the simultaneous visualization of microstructure, the
appearance of bright fluorescent spots was observed along with the drop in
viscosity. Together the results indicated that CPR27 complexation with chitosan
caused aggregation that effectively removed material from the vicinity of the
microrheology probes. CP30, which does not contain the conserved chitin-binding
sequence, did not display similar evidence of complexation. The role of quinone
crosslinking of both proteins with the catechol, N-&beta-alanyldopamine,
was examined using passive microrheology. Microparticle formation was observed
in solutions containing protein and laccase, catechol and laccase, and protein,
catechol and laccase. However, elasticity was observed only when the protein
was present, consistent with the hypothesis that quinone crosslinking has a
mechanical role in cuticle, as concluded by the studies on elytra. However,
further tests are needed to reproduce the data and verify the origin of
elasticity. The highly unusual sequence of highly repetitive short blocks of
positive and negative residues in CP30 suggested that it may self-assemble or
interact with multivalent metal ions. The protein was found to aggregate or
adsorb to surfaces, preventing analysis, near the isoelectric point of 5.82.
However, spectroscopic studies suggested the protein had an extended
α-helical conformation at other pH conditions. CP30 was also shown to
interact with nickel ions to form microparticles that induced elasticity in the
fluid. The interactions involving these two cuticle proteins suggest new motifs
that could be used in the design of tunable biomaterials. The rational design
of proteins with specific covalent and non-covalent interactions with
polysaccharides or ions, inspired by insect cuticle, may lead to biomaterials
with enhanced mechanical properties.”
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