Coacervates derived from mussel foot proteins

• Main Author: Guo, Qi
• Other Authors: Yu Jing
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2022
• Subjects: Science::Biological sciences::Biochemistry; Science::Biological sciences::Molecular biology
• Online Access: https://hdl.handle.net/10356/158713
• Institution: Nanyang Technological University

Biomimetics has inspired the development of various advanced technologies and novel materials, in which mussel adhesion has been regarded as a practical strategy to investigate the underwater adhesives with robust wet adhesion capacities attributed to the abundant post-translationally modified amino acid l-3,4-dihydroxyphenylalanine (Dopa) in mussel foot proteins (Mfps). Moreover, recent studies also reveal that the secretion of Mfps is conducted with a concentrated fluidic phase regulated by a liquid-liquid phase separation (LLPS) process. With the aim of exploring the phase behaviors of mussel plaque proteins and their Dopa-involved mechanisms, two mussel plaque proteins of different mussel species were chosen to gain insight into the natural secretion process and to have a deep understanding of the interaction information to the molecular level. The first study focuses on the mussel adhesive protein Pvfp-5 of the Asian green mussels with the attempt to examine the LLPS behaviors of mussel plaque proteins and figure out the decisive interactions within proteins at the molecular level. Based on the fact that only parts of Tyr residues are post-modified into Dopa residues within native Pvfp-5β, two Pvfp-5 variants, Pvfp-5-Tyr and Pvfp-5-Dopa, were expressed to investigate the exact roles of Dopa residues. Both Pvfp-5 variants exhibited comparable adhesion forces, which indicated the contribution of Dopa residues to form interfacial hydrogen bonds was negligible. Nonetheless, Pvfp-5-Dopa showed different phase behaviors compared to Pvfp-5-Tyr. Pvfp-5-Dopa could form coacervate microdroplets under seawater conditions, while Pvfp-5-Tyr could only form gel-like precipitates. Further characterization results using periodate oxidation, NMR and QC calculation found that Dopa residues could form a transient triangular hydrogen bonding network which is specific for Pvfp-5-Dopa. Pvfp-5-Dopa tended to form microdroplets through transient hydrogen bonds and π-π stacking, while Pvfp-5-Tyr preferred to form insoluble precipitates by the strong cation-π interactions. The second study investigated the GK-16 peptide derived from mussel adhesive protein Mfp-5 of the California blue mussels to identify the LLPS behaviors and to understand the molecular interactions involved in the phase separation process. Repeated three times within the Mfp-5 sequence, enzymatically modified GK-16* could undergo LLPS triggered by certain ionic strength at pH 3, in which the mechanical properties of coacervate droplets would be adjusted by salt concentration and urea concentration. CD and FTIR spectra also characterized the formation of localized β-sheet structures with the addition of salts. To further understand the specific roles of amino acids to the molecular level, several GK-16 variants were designed with a series of amino acid substituents. Observed from the phase diagrams, CD spectra and MD simulations, Dopa involved hydrogen bonds formed with a triangle configuration dominate within the LLPS process, followed by the side chain-main chain hydrogen bonds among Dopa residues and Gly residues. The thesis highlights the significance of Dopa residues, which could not only promote the wet adhesion by forming interfacial hydrogen bonds, but also facilitate the phase separation of mussel plaque proteins through intermolecular hydrogen bonds. By performing such fundamental studies, the natural mussel secretion procedure could be further decrypted and the detailed molecular interactions taking function could be acknowledged. Such results could provide sequence models according to their intrinsic properties to further design mussel-inspired materials with tailored mechanical properties. Especially for wet adhesives, the studies emphasize the multifunctional Dopa residues, which could construct the coacervate-based platform to promote the adhesion efficiency of Dopa residues.