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Amyloid formation is basically featured by a protein-protein interaction in which the reacting regions are the segments assembling into cross β-sheets. To identify these segments both theoretical and experimental tools have been developed. Here, we focus on the use of peptide arrays to probe the binding of several amyloid-specific probes such as the OC and A11 anti-amyloid conformation-selective antibodies and of monomers and preformed fibrils. These arrays use libraries containing partly overlapping peptides derived from the sequence of Gad m 1, the major allergen from Atlantic cod, which forms amyloids under gastrointestinal relevant conditions.

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 Ca 2+ -bound or Ca 2+ -free states with distinct aggregation propensities. That is the case among β-parvalbumins and S100 proteins, which under certain conditions undergo Ca 2+ -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.

Atomic force microscopy (AFM) has become a conventional tool for elucidation of the molecular mechanisms of protein aggregation and, specifically, for analysis of assembly pathways, architecture, aggregation state, and heterogeneity of oligomeric intermediates or mature fibrils. AFM imaging provides useful information about particle dimensions, shape, and substructure with nanometer resolution. Conventional AFM methods have been very helpful in the analysis of polymorphic assemblies formed in vitro from homogeneous proteins or peptides. However, AFM imaging on its own provides limited insight into conformation or composition of assemblies produced in the complex environment of a cell, or prepared from a mixture of proteins as a result of cross-seeding. In these cases, its combination with fluorescence microscopy (AFFM) increases its resolution.