Modular peptide binding interactions of sucker-ring teeth proteins

• Main Author: Hiew, Shu Hui
• Other Authors: Ali Gilles Tchenguise Miserez
• Format: Theses and Dissertations
• Language: English
• Published: 2017
• Subjects: DRNTU::Engineering::Materials::Biomaterials
• Online Access: http://hdl.handle.net/10356/69615
• Institution: Nanyang Technological University

Biomimetics is highly integrated into our lives, inspiring us with ideas to advance technology and to design novel materials. The sucker ring teeth (SRT) of the Humboldt squid (Dosidicus gigas) is one such intriguing example, being a natural biotool that is both hard and strong, yet non-mineralized and fully proteinaceous (comprising of proteins named suckerins). With the aim of identifying the main building block of the SRT that endows the material with its mechanical strength, a bottom-up approach was undertaken to gain insight into the material’s design strategy and to obtain valuable molecular information. The first study attempts to dissect the suckerin-19 protein and examine the interactions between the highly repetitive modular suckerin peptide sequences found within, at the molecular level. Using a partial combinatorial chemistry approach, a library of identified modular peptides was screened for interactions with a peptide macro-array binding assay. Peptides that exhibit the highest “hits” indicate strong interactions, providing an insight to the propensity of high affinities between peptide sequences within the bio-system. Further examination of suckerin peptides using CD spectroscopy and FTIR spectroscopy found that they have high propensities to adopt -sheet secondary structures. His-rich suckerin peptides tend to adopt PPII and –sheet secondary structures and remain stable and soluble at high concentrations, while Ala-rich suckerin peptides self-assemble into anisotropic microfibers, with characteristics resembling those of amyloid -rich structures. MD simulations of Ala-rich fibers show the structures are highly stable while H/D exchange NMR experiments demonstrated their high stability even at elevated temperatures. These studies reveal that the assembly of SRT is most likely driven by -sheet seeding, similar to that of amyloid growth. The second study proceeds to explore the fabrication of new suckerin-peptide based material. Self-assembled Ala-rich microfibers were identified to have similar Young’s modulus to that of the native SRT by nanoindentation, indicating that the –sheet–forming sequence indeed contributes to the mechanical property of SRT. To increase the size of these fibers, a longer suckerin peptide sequence comprising of Ala-rich and His-rich segments was synthesized. With a solvent-driven method, an accelerated assembly of these peptides yielded larger and longer fibers with extended and organized hierarchical structures that were rich in –structures, as shown by WAXS and FTIR spectroscopy. By combining the two sequences that have –sheet–forming propensities, the solubility of the -sheet seeding Ala-rich suckerin peptide was drastically increased and longer fibers could be assembled at high peptide concentrations. These fibers maintained their mechanical robustness and possess a wide range of working conditions as they were highly tolerant of harsh environmental conditions; they resist degradation by strong denaturing chemical conditions and physical agitation, and have high melting point and thermal degradation temperature, as shown by DSC and TGA techniques respectively. The thesis highlights the importance of both amino acid sequence and molecular interactions in constructing protein/peptide-based structural materials. By first performing fundamental research, we can equip ourselves with the knowledge and understanding of the roles of different building blocks. This then allows us to select peptide sequences according to their intrinsic properties for constructing materials with their intended application-based properties. The studies presented here serve as a platform that equip us with a plethora of possibilities towards engineering new biomimetic materials; to intricately design and recreate proteinaceous and peptidic materials with tailored mechanical properties.