Investigating the self-assembly and molecular chaperone ability of casein proteins and polyproline

Caseins are a group of intrinsically disordered proteins (IDPs) present in the milk of all mammals. A property common to many IDPs is their propensity to assemble into amyloid fibrils, which are associated with many age-related diseases. Based on the stability of their predicted 'steric zipper' structures, which form the backbone of amyloid fibrils, caseins from all species contain amyloidogenic sequences. Such sequences in caseins are mainly located in their proline/glutamine (P,Q)-rich regions which have a propensity to adopt polyproline-II helical structure and are proposed to play a significant role in casein association to form amorphous aggregates or to facilitate their molecular chaperone activity whereby they stabilize partly unfolded proteins. This thesis (i) examines the sequence and structures of caseins underlying their chaperone ability and fibril-forming propensity; and (ii) explores whether the fibril-forming propensity is a general feature of caseins. Firstly, the capacity of other caseins (bovine alphas1- and beta-casein, as well as caprine and camelid beta-caseins) to form amyloid fibrils under physiologically relevant conditions was demonstrated using thioflavin T binding, transmission electron microscopy and X-ray fibre diffraction and far-UV circular dichroism. Partial digestion of beta-casein fibrils and analysis by mass spectroscopy indicated a common amyloidogenic sequence in the P,Q rich, C-terminal region of the protein from all three species. Moreover, SAXS measurements showed that self-assembly of bovine alphas1- and beta-casein led to elongated and spherical oligomers, respectively (Chapter 2). The partial digestion of fibrils formed by intra- and intermolecularly disulfide-bonded alphas2-casein indicated that Ala81 Lys113 is incorporated into the fibril core, implying that this P,Q-rich region, which is predicted by several algorithms to be amyloidogenic, initiates fibril assembly of alphas2-casein. Comparable ANS binding and intrinsic fluorescence spectra for these two species suggested that differences exist in their oligomeric states rather than their conformational propensities or hydrophobic interactions (Chapter 3). Additionally, the chaperone ability and self-association of poly-L-proline (PLP) was investigated as a representative sequence for the polyproline-II helical motifs adopted by IDPs, e.g. caseins. PLP exhibited a selective chaperone ability to stabilize the molten globule state of alpha-lactalbumin and prevent its amyloid fibrillar and amorphous aggregation. At the supramolecular level, PLP self-associated via a nucleation-independent mechanism in a temperature-dependent, reversible process to form fibrillar aggregates that bind to amyloidophilic fluorescent dyes (Chapter 4). Finally, the effects of milk components (calcium, lactose, lipids, and heparan sulfate) and crowding agents were investigated on reduced and carboxymethylated (RCM) kappa-casein fibril formation. Longer-chain phosphatidylcholine lipids enhanced RCM kappa-casein fibril formation, whereas shorter chain phospholipids and triglycerides had little effect. Heparan sulfate, a component of the milk fat globule membrane and catalyst of amyloid deposition in extracellular tissue, had little effect on the kinetics of RCM kappa-casein fibril formation. Major nutritional components such as calcium and lactose also had no significant effect. Macromolecular crowding enhances protein-protein interactions. In contrast to other fibril-forming species, the extent of RCM kappa-casein fibril formation was reduced in the presence of crowding agents, consistent with the proposed mechanism of kappa-casein fibril formation involving dissociation from the oligomer (Chapter 5)