Modular peptide binding interactions of sucker-ring teeth proteins

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.Doctor of Philosophy (MSE

Studies on the site-specific self cleavage of G-quadruplexes and the topological properties of DNA

In this thesis, the studies are classified under two main themes, namely the studies on G-quadruplexes and studies on the topological properties of DNA. Firstly, the effects of several factors that could affect the self-cleaving reactions of G-quadruplex were examined in this study. The optimum self-cleaving reaction condition observed was between pH 7.4 – 7.6 with potassium ion stabilizing the G-quadruplex structure in the central cavity. It was found that Mg2+ is the best metal ion to catalyze the hydrolysis self-cleavage process, whereas Ca2+ catalyzes the self-cleaving reactions best when the ligand, methionine was introduced. Secondly, structural polymorphism and the effect of the structure of the resulting of G-quadruplexes with varying loop length was studied by designing series of oligonucleotides with four repeat guanines sequences, d(G4Tn)3G4 (where n = 1-6). It was found that as the loop length increases, the possibility of forming a loop increases, and the formation of the types of loops ascends: no loop < lateral loop < diagonal loop < external loop. The conformation of G-quadruplexes can be predicted if we know the T-loop length. Lastly, the topological properties of DNA was explored. In order to examine if DNA curvature had any effect on human topoisomerase I, a series of DNA possessing intrinsic curvature was design, and it was found that hTopo I recognizes the curvature and binds preferably to the curved DNA with a higher degree of curvature. On the other hand, besides using EcTopo I, DNA gyrase was also able to help determine the superhelical density of circular DNA. Some very interesting findings were made with regards to the decatenation of kDNA by human Topoisomerase II alpha, whereby the enzyme might possibly be able to introduce supercoiling into circular DNA, when the self-crossing of the DNA backbone exist in the solution. Also, when the decatenation reaction was incubated past the completion of the decatenation, the enzyme seems to “re-catenate” the kDNA circles.​Master of Scienc

Examination of effect of thymine loop length on polymorphism of G-quadruplex.

Structural polymorphism is one of the important issue with regard to G-quadruplexes because the structural diversity may significantly affect their biological functions in vivo and also their physical properties in nano-material. A series of oligonucleotides with four repeat guanines sequences, d(G4Tn)3G4 (where n = 1-6) were designed for this study to investigate the effect of the length of loop on the polymorphism of G-quadruplex and the effect of the structure of the resulting G-quadruplex on its melting point. Via CD spectroscopy and comparison with the known CD spectrums of previously studied G-quadruplexes, and with the help of PAGE analysis, the structures of the G-quadruplexes could be predicted. A trend was also observed for these oligonucleotides while varying their T-loop length and this will be analyzed in this study.Bachelor of Science in Chemistry and Biological Chemistr

Controlling supramolecular chiral nanostructures by self-assembly of a biomimetic β-sheet-rich amyloidogenic peptide

Squid sucker ring teeth (SRT) have emerged as a promising protein-only, thermoplastic biopolymer with an increasing number of biomedical and engineering applications demonstrated in recent years. SRT is a supra-molecular network whereby a flexible, amorphous matrix is mechanically reinforced by nanoconfined β-sheets. The building blocks for the SRT network are a family of suckerin proteins that share a common block copolymer architecture consisting of amorphous domains intervened by smaller, β-sheet forming modules. Recent studies have identified the peptide A1H1 (peptide sequence AATAVSHTTHHA) as one of the most abundant β-sheet forming domains within the suckerin protein family. However, we still have little understanding of the assembly mechanisms by which the A1H1 peptide may assemble into its functional load-bearing domains. In this study, we conduct a detailed self-assembly study of A1H1 and show that the peptide undergoes β-strands-driven elongation into amyloid-like fibrils with a rich polymorphism. The nanostructure of the fibrils was elucidated by small and wide-angle X-ray scattering (SAXS and WAXS) and atomic force microscopy (AFM). The presence of His-rich and Ala-rich segments results in an amphiphilic behavior and drives its assembly into fibrillar supramolecular chiral aggregates with helical ribbon configuration in solution, with the His-rich region exposed to the solvent molecules. Upon increase in concentration, the fibrils undergo gel formation, while preserving the same mesoscopic features. This complex phase behavior suggests that the repeat peptide modules of suckerins may be manipulated beyond their native biological environment to produce a wider variety of self-assembled amyloid-like nanostructures

Barnacle cement protein as an efficient bioinspired corrosion inhibitor

Abstract To prevent corrosion damage in aggressive environments such as seawater, metallic surfaces are coated with corrosion inhibitors usually made of organic molecules. Unfortunately, these inhibitors often exhibit environmental toxicity and are hazardous to natural habitats. Thus, developing greener and effective corrosion inhibitors is desirable. Here, we present an alternative green inhibitor, the recombinant protein rMrCP20 derived from the adhesive cement of the barnacle Megabalanus rosa and show that it efficiently protects mild steel against corrosion under high salt conditions mimicking the marine environment. We reveal that these anti-corrosion properties are linked to the protein’s biophysical properties, namely its strong adsorption to surfaces combined with its interaction with Fe ions released by steel substrates, which forms a stable layer that increases the coating’s impedance and delays corrosion. Our findings highlight the synergistic action of rMrCP20 in preventing corrosion and provide molecular-level guidelines to develop alternative green corrosion inhibitor additives

Modulation of mechanical properties of short bioinspired peptide materials by single amino-acid mutations

The occurrence of modular peptide repeats in load-bearing (structural) proteins is common in nature, with distinctive peptide sequences that often remain conserved across different phylogenetic lineages. These highly conserved peptide sequences endow specific mechanical properties to the material, such as toughness or elasticity. Here, using bioinformatic tools and phylogenetic analysis, we have identified the GX8 peptide with the sequence GLYGGYGX (where X can be any residue) in a wide range of organisms. By simple mutation of the X residue, we demonstrate that GX8 can be self-assembled into various supramolecular structures, exhibiting vastly different physicochemical and viscoelastic properties, from liquid-like coacervate microdroplets to hydrogels to stiff solid materials. A combination of spectroscopic, electron microscopy, mechanical, and molecular dynamics studies is employed to obtain insights into molecular scale interactions driving self-assembly of GX8 peptides, underscoring that π-π stacking and hydrophobic interactions are the drivers of peptide self-assembly, whereas the X residue determines the extent of hydrogen bonding that regulates the macroscopic mechanical response. This study highlights the ability of single amino-acid polymorphism to tune the supramolecular assembly and bulk material properties of GX8 peptides, enabling us to cover a broad range of potential biomedical applications such as hydrogels for tissue engineering or coacervates for drug delivery.Ministry of Education (MOE)Nanyang Technological UniversityThis research was funded by the Singapore Ministry of Education (MOE) through an Academic Research (AcRF) Tier 3 grant (grant No MOE 2019-T3-1-012).The authors also acknowledge financial support from the Strategic Initiativeon Biomimetic and Sustainable Materials(IBSM) at NTU. We thank the Facility for Analysis, Characterisation, Testing, and Simulation(FACTS) at NTU for the use of their electronmicro scopy facilities. M. Y. acknowledges support from MOE Tier 1 Grant RG27/21

A Short Peptide Hydrogel with High Stiffness Induced by 310‐Helices to β‐Sheet Transition in Water

Biological gels generally require polymeric chains that produce long‐lived physical entanglements. Low molecular weight colloids offer an alternative to macromolecular gels, but often require ad‐hoc synthetic procedures. Here, a short biomimetic peptide composed of eight amino acid residues derived from squid sucker ring teeth proteins is demonstrated to form hydrogel in water without any cross‐linking agent or chemical modification and exhibits a stiffness on par with the stiffest peptide hydrogels. Combining solution and solid‐state NMR, circular dichroism, infrared spectroscopy, and X‐ray scattering, the peptide is shown to form a supramolecular, semiflexible gel assembled from unusual right‐handed 3_10‐helices stabilized in solution by π–π stacking. During gelation, the 3_10‐helices undergo conformational transition into antiparallel β‐sheets with formation of new interpeptide hydrophobic interactions, and molecular dynamic simulations corroborate stabilization by cross β‐sheet oligomerization. The current study broadens the range of secondary structures available to create supramolecular hydrogels, and introduces 3_10‐helices as transient building blocks for gelation via a 3_10‐to‐β‐sheet conformational transition.ISSN:2198-384

Minimal reconstitution of membranous web induced by a vesicle–peptide sol–gel transition

Positive strand RNA viruses replicate in specialized niches called membranous web within the cytoplasm of host cells. These virus replication organelles sequester viral proteins, RNA, and a variety of host factors within a fluid, amorphous matrix of clusters of endoplasmic reticulum (ER) derived vesicles. They are thought to form by the actions of a nonstructural viral protein NS4B, which remodels the ER and produces dense lipid-protein condensates. Here, we used in vitro reconstitution to identify the minimal components and elucidate physical mechanisms driving the web formation. We found that the N-terminal amphipathic domain of NS4B (peptide 4BAH2) and phospholipid vesicles (∼100-200 nm in diameter) were sufficient to produce a gel-like, viscoelastic condensate. This condensate coexists with the surrounding aqueous phase and affords rapid exchange of molecules. Together, it recapitulates the essential properties of the virus-induced membranous web. Our data support a novel phase separation mechanism in which phospholipid vesicles provide a supramolecular template spatially organizing multiple self-associating peptides thereby generating programmable multivalency de novo and inducing macroscopic phase separation.Accepted versio

Interference of intrinsic curvature of DNA by DNA-intercalating agents

It has been demonstrated in our studies that the intrinsic curvature of DNA can be easily interrupted by low concentrations of chloroquine and ethidium bromide. In addition, the changes of DNA curvature caused by varying the concentration of these two DNA intercalators can be readily verified through using an atomic force microscope