Molecular mechanism of barnacle adhesion : a structural approach and underlying biochemistry (2022)

Barnacles adhere themselves robustly and permanently to diverse underwater substrates through strong interactions of a multi-protein complex layer called the “cement”. However, the intermolecular interactions responsible for the strong adhesive properties of the barnacle cement remains poorly understood. A central hypothesis of this thesis is that underwater properties of the cement complex are intimately linked to the molecular characteristics of cement proteins (CPs) forming the cement complex. Previous studies have shown that the cement is made of amyloid-like nanofibrils that may contribute to adhesion. However, the protein responsible for the formation of these nanofibrils remain unknown. In this study, the nanoscale morphological features of recombinant cement proteins (CPs) from the barnacle Megabalanus rosa (MrCP19 and MrCP20, with the numbers indicating molecular weight of 19 kDa and 20 kDa respectively) were characterized by Circular Dichroism (CD) measurement, Thioflavin T (ThT) assay, Atomic Force Microscopy (AFM), and Transmission Electron Microscopy (TEM), suggesting the potential to form nano-fibrillar structures under certain conditions. Based on the proteins’ primary structure and surface morphology, mechanical, biochemical, and antimicrobial studies were conducted to understand the unique roles of these interfacial proteins on barnacle growth and surface attachment process, for instance biomineralization and biodegradation control. Measurements using Surface Force Apparatus (SFA) and Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) illustrated that electrostatic interactions play a key role in surface adsorption and adhesion of MrCP19 and MrCP20. In addition, the mutual influence of barnacle base plate growth (calcium carbonate mineralization) and the adjacent cement protein MrCP20 fibrillation was investigated using self-assembled monolayer (SAM) functionalized gold surfaces, Raman spectroscopy, QCM-D, X-ray photoelectron spectrometer (XPS), and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Concurrently, the influence of the external substrate adjacent cement protein MrCP19 on bacteria cells which are present in biofilm on underwater surfaces in marine environment was demonstrated using different microbiology tests including zone of inhibition test, Minimum inhibitory concentration (MIC) assay, TEM, fluorescence study, and so on. More interestingly, an intriguing hypothesis regarding amyloid fibrillation process and antimicrobial activity was suggested. Based on these preliminary examinations, the two interfacial CPs showed distinctive potential responsibilities on barnacle settlement not only with its adhesion but also with other functional roles at the interfaces. This work will improve our knowledge about individual contributions of MrCP19 and MrCP20 in cement complex and hence in overall underwater adhesion capacity of barnacles. In this regard, the thesis aims at providing molecular guidelines towards the development of CPs inspired polymeric (peptide or protein based) mimics from this bio-adhesive molecular system. Keywords: Barnacle cement proteins, Underwater adhesion, Protein conformation, Amyloid fiber, Fibril formation, Surface adsorption behavior, Surface adhesion energy, Calcium carbonate, Biomineralization, Antimicrobial activity