Peptide-based materials and the assembly of peptide-based nanostructures
AffiliationChemical and Biomolecular Engineering
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2020-10-04.
© 2018 Dr. Mahshid Kalani
Amyloid fibrils are highly ordered protein aggregates associated with the disease that has been identified in deposits, including different organs and tissues of mammals, where they are known as corpora amylacea. κ-casein, a protein abundant in milk, has a high propensity to form amyloid fibrils, which could be associated with corpora amylacea, although this association has not been confirmed to date. Therefore, κ-casein is useful for fundamental studies of aggregation. The first objective of this thesis aimed to assess the kinetics of κ-casein fibril formation. κ -casein has two cysteine residues on its primary structure, which tend to aggregate to form oligomers. Therefore, in this study, these cysteine residues are reduced and capped to have a monomeric form of a κ-casein protein called reduced carboxymethylated κ-casein. In previous studies, two different mechanisms have been proposed for κ-casein fibril formation (i) either the micelle is the precursor structure or (ii) dissociation of the κ-casein oligomers leads to amyloidogenic species. In this study, reduced carboxymethylated κ-casein was able to form fibrils at a concentration below the critical micelle concentration (CMC), at which it is unlikely to have κ-casein micelle structure. Therefore, it is proposed in this research that the dissociation of κ-casein oligomeric form is the rate-limiting step of κ-casein fibrillation process, although further experiments are required to confirm this mechanism. Para κ-casein, the N-terminal fragment of κ-casein, is produced by the specific cleavage of κ-casein, at which occurs during both rennet cheese manufacturing processes and stomach digestion of κ-casein milk protein in some mammals. Para κ-casein is suspected to have a high propensity to form amyloid fibrils; the impact of such fibrils on cells is unknown and the aggregates may also have an impact on equipment fouling. The second objective of this research was to investigate the propensity of para κ-casein to form amyloid fibrils and to characterize these fibrils. Para κ-casein was successfully generated by specific cleavage of full length reduced carboxymethylated κ-casein and high-performance liquid chromatography was employed to purify para κ-casein. The para κ-casein fibrils were shorter and narrower in compared with the reduced carboxymethylated κ-casein fibrils. The kinetics of both RCM κ-casein and para κ-casein fibril formations was similar without any lag phase. Both X-ray diffraction patterns of RCM κ-casein and para κ-casein revealed the presence of spacing between adjacent hydrogen-bonded β-strands perpendicular to the fibril axis. Amyloid fibrils have also attracted attention in because of their high tensile strength and modulus, flexibility, resistance to degradation, stability, and ability to interact with non-biological solid surfaces. The third objective of this thesis was to form a hybrid biomaterial from nano-cellulose fibres reinforced with hen egg white lysozyme amyloid fibrils. The properties of the composite sheet of amyloid fibrils reinforced by micro-fibrillated cellulose are characterized. The fabrication of such composite sheets is relatively easy, and these sheets may have potential application in biomedical engineering or for use as a functionalized nano-scaffold template.
Keywordsamyloid fibril formation; protein aggregation; amyloidosis; kappa casein
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