469751 Chitin-Binding Proteins Induce Biomacromolecular Complexation: Inspirations from Insect Cuticle for Hierarchical Self-Assembly
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.
 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.