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Proteins exhibit a remarkable structural plasticity and may undergo conformational changes resulting in protein misfolding both in a biological context and upon perturbing physiopathological conditions. Such nonfunctional protein conformers, including misfolded states and aggregates, are often associated to protein folding diseases. Understanding the biology of protein folding diseases thus requires tools that allow the structural characterization of nonnative conformations of proteins and their interconversions. Here we present detailed procedures to monitor protein conformational changes and aggregation based on spectroscopic and biophysical methods that include circular dichroism, ATR-Fourier-transformed infrared spectroscopy, fluorescence spectroscopy and dynamic light scattering. To illustrate the application of these methods we report to our previous studies on misfolding, aggregation and amyloid fibril formation by superoxide dismutase 1 (SOD1), a protein whose toxic deposition is implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS).

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.