Preparation of Amyloidogenic Aggregates from EF-Hand ß-Parvalbumin and S100 Proteins.

Proteins containing EF-hand helix-loop-helix-binding motifs play essential roles in calcium homeostasis and signaling pathways. These proteins have considerable structural and functional diversity by virtue of their cation-binding properties, and occur as either Ca2+-bound or Ca2+-free states with distinct aggregation propensities. That is the case among ß-parvalbumins and S100 proteins, which under certain conditions undergo Ca2+-dependent self-assembly reactions with the formation of oligomers, amyloid-type aggregates and fibrils. These phenomena may be particularly relevant in human S100A6 protein and in fish Gad m 1 allergenic protein, which are implicated in human disease processes. Here, we describe detailed methods to generate and monitor the formation of amyloidogenic assemblies and aggregates of these two EF-hand proteins in vitro.