(387d) Chitin-Protein Interactions That Control the Mechanical Properties of Beetle Elytral Cuticle, a Multicomponent Biomaterial

Authors: 
Dittmer, N. T., Kansas State University
Dhar, P., University of Kansas
Gehrke, S. H., University of Kansas

Insect cuticle, the primary component of the insect exoskeleton, is one of the most common materials in nature. It is a valuable model for the development of biomimetic materials because its moduli can vary by six orders of magnitude or more despite being comprised of little other than protein, chitin and catechol-derived pigments organized into a hierarchically ordered composite. Additionally, these components and their chemistry are broadly utilized in the animal kingdom. In a series of publications, we have used both molecular biological techniques(e.g., RNA interference) and mechanical test methods (e.g., dynamic mechanical analysis) of elytra (wing covers) from the model organisms Tribolium castaneum (red flour beetle) and Tenebrio molitor (yellow mealworm) to advance understanding of composition-structure-property relationships in cuticle. Our broad objective in this line of research has been to investigate molecular interactions among the components of elytral cuticle to understand their role in determining the in vivo properties. Here we examine the interactions between novel structural proteins that we have identified in T. castaneum and expressed in E. coli and their interactions with chitin, chitosan and multivalent metal ions. The goal of this in vitro work expands from understanding the in vivo roles of specific interactions to development of new biomaterial design motifs.

The exceptional properties of insect cuticle are hypothesized to arise from a combination of covalent and non-covalent interactions among proteins, catechols, chitosan and chitin nanofibers, including protein-catechol crosslinking, generation of catechol-derived microparticles and protein-Ni2+ interactions. Two abundant cuticle proteins in the elytra of T. castaneum that we named CPR27 and CP30 were the foci of this study. CPR27 has a conserved sequence of amino acids first hypothesized by Rebers and Riddiford to bind chitin. Our results from active microrheology showed that addition of CPR27 to fluorescently-labeled aqueous chitosan solutions caused a 2-fold decrease in viscosity. Since this technique allows for the simultaneous visualization of microstructure, the appearance of bright fluorescent spots was observed to parallel the drop in viscosity.  Together these results indicated that CPR27 complexed with chitosan as hypothesized to form micron-scale structures.  In contrast, CP30, which does not contain the conserved chitin-binding sequence, displayed no evidence of complexation.  The role of quinone-crosslinking of both proteins with the catechol N-b-alanyldopamine (similar to that observed in mussel adhesion) was examined using passive microrheology. Microparticle formation was observed in solutions containing protein and the oxidative enzyme laccase, catechol and laccase, and protein, catechol and laccase.  However, elasticity was observed only when the protein was present, consistent with the hypothesis developed in studies on whole elytra that quinone-crosslinking has a mechanical role in cuticle, as concluded by the studies on elytra. The very unusual sequence of highly repetitive short blocks of positive and then negative residues in CP30 suggested that it may self-assemble or complex with multivalent metal ions.  Indeed, CP30 was shown to interact with nickel ions to form microparticles that induced elasticity in the fluid.  Understanding the interactions involving these two cuticle proteins suggest new motifs that could be used in the design of new composite materials.  The rational design of recombinant proteins following these principles, with specific covalent and non-covalent interactions with polysaccharides or ions, inspired by insect cuticle, may lead to biomaterials with enhanced mechanical properties.