Folding Steps in the Fibrillation of Functional Amyloid: Denaturant Sensitivity Reveals Common Features in Nucleation and Elongation.

Functional bacterial amyloids (FuBA) are intrinsically disordered proteins (IDPs) which rapidly and efficiently aggregate, forming extremely stable fibrils. The conversion from IDP to amyloid is evolutionarily optimized and likely couples folding to association. Many FuBA contain several imperfect repeat sequences which contribute to the stability of mature FuBA fibrils. Aggregation can be considered an intermolecular extension of the process of intramolecular protein folding which has traditionally been studied using chemical denaturants. Here we employ denaturants to investigate folding steps during fibrillation of CsgA and FapC. We quantify protein compactification (i.e. the extent of burial of otherwise exposed surface area upon association of proteins) during different stages of fibrillation based on the dependence of fibrillation rate constants on the denaturant concentration (m-values) determined from fibrillation curves. For both proteins, urea mainly affects nucleation and elongation (not fragmentation), consistent with the fact that these steps involve both intra- and intermolecular association. The two steps have similar m-values, indicating that activation steps in nucleation and elongation involve the same level of folding. Surprisingly, deletion of two or three repeats from FapC leads to larger m-values (i.e. higher compactification) during the activation step of fibril growth. This observation is extended by SAXS analysis of the fibrils which indicates that weakening of the amyloidogenic core caused by repeat deletions causes a larger portion of normally unstructured regions of the protein to be included into the amyloid backbone. We conclude that the sensitivity of fibrillation to denaturants can provide useful insight into molecular mechanisms of aggregation.

Dynamic content exchange between liprotides.

Liprotides are complexes composed of partially denatured proteins and fatty acids in which the fatty acids form a micelle-like core surrounded by a shell of proteins. Liprotides, composed of α-lactalbumin (aLA) and oleic acid (OA), are similar in components and cytotoxicity to the original HAMLET protein-fatty acid complex. Liprotides composed of aLA and OA kill tumor cells by transferring the OA component to, and thus destabilizing, the cell membrane. Here we investigate liprotides' dynamics of transfer of contents between themselves and membranes using the hydrophobic fluorescent probe pyrene. We find that pyrene incorporated into liprotides is exchanged between liprotides within the dead time of a stopped-flow instrument, while the transfer to membranes occurs within 20s. Transfer kinetics was not affected by the presence of the membrane stabilizing lipid cholesterol. Thus, transfer is a remarkably rapid process which illustrates liprotides' efficacy as transporters of hydrophobic compounds.