(228u) Chitin-Binding Proteins Induce Biomacromolecular Complexation: Inspirations from Insect Cuticle for Hierarchical Self-Assembly

Authors: 
Gehrke, S. H., University of Kansas
Dittmer, N. T., Kansas State University
Dhar, P., University of Kansas
Ghazvini, S., University of Kansas
Insect cuticle, the primary component of the exoskeleton, is one of the most widespread 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. 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-metal ion interactions. Here we examine the interactions between novel structural proteins that we have identified in the beetle Tribolium castaneum and their interactions with chitin, chitosan and metal ions. The goal of this work extends from understanding the in vivo roles of specific interactions to development of new biomaterial design motifs.1

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. In this work, we establish direct evidence of this protein-chitin binding by using an active microrheology technique, coupled with fluorescence and bright-field microscopy. While our results from active microrheology showed that addition of CPR27 to aqueous chitosan solutions caused a 2-fold decrease in viscosity, simultaneous visualization of the solution microstructure shows the formation of micron-sized particles. Together these results indicated that CPR27 complexed with chitosan as hypothesized to form micron-scale structures. Furthermore, by using fluorescently-labeled chitosan, our simultaneous fluorescence images confirm the presence of clusters of chitosan, suggesting that the protein CPR27 serves as a nucleation agent to induce the complexation of the polyelectrolyte. However, varying the concentration of the protein over several orders of magnitude does not alter the viscosity drop, suggesting that the RR interactions are the first step in inducing complexation of chitosan. In contrast, CP30, which does not contain the chitin-binding sequence, displayed no evidence of complexation. The role of quinone-crosslinking of both proteins with the catechol N-β-alanyldopamine (similar to that observed in mussel adhesion) was also examined. Microparticle formation was observed in solutions containing protein, catechol and the oxidative enzyme laccase. Understanding the interactions involving these 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.

REFERENCE

 [1] Lomakin, J.; Huber, P.A.; Eichler, C.; Arakane, Y.; Kramer, K.J.; Beeman, R.W.; Kanost, M.R.; Gehrke, S.H. Biomacromolecules (2011) 12, 321.