Self-Assembly of a Barnacle Cement Protein (MrCP20) into Adhesive Nanofibrils with Concomitant Regulation of CaCO₃ Polymorphism

Barnacles are convenient model organisms to study both biomineralization and bioadhesion phenomena. They secrete a proteinaceous adhesive from cement proteins (CPs) to strongly attach to solid immersed substrates. More recently, it has been suggested that some CPs also play a key role in regulating the calcification of barnacles’ protective shells. In this study, combining both solution and solid-state NMR spectroscopy, Raman and infrared spectroscopy studies, and atomic force microscopy (AFM) and transmission electron microscopy (TEM) imaging, we have explored the CaCO₃ mineralization pathway regulated by Megabalanus rosa CP 20 (MrCP20). Our data show that MrCP20 can sequestrate inorganic calcium and carbonate ions from the solution state, which quickly coarsen into liquid-like microdroplets and subsequently form protovaterite amorphous CaCO₃ (ACC) particles. This pathway leads to the stabilization of the metastable vaterite polymorphism of CaCO₃. Simultaneously, AFM and TEM investigations show that MrCP20 undergoes fibrillization triggered by a pH drift arising during CaCO₃ mineralization, leading to amyloid-like nanofibrils. Based on protein NMR, this mechanism appears to be stabilized by the reduction of intramolecular disulfide bonds. Collectively, our results demonstrate that MrCP20 plays a synergistic role of regulating CaCO₃ biomineralization while concomitantly self-assembling into adhesive nanofibrils.