Study of the structure, conformational stability, folding and aggregation processes of profilin-1 and its mutants associated with amyotrophic lateral sclerosis.

Human profiin-1 is a novel protein associated with a recently discovered form of familial amyotrophic lateral sclerosis. This urges the characterization of possible conformational states, different from the fully folded state, potentially able to initiate self-assembly. Under native conditions, profiin-1 is monomeric and possesses a well-defied secondary and tertiary structure. When incubated at low pH or with high urea concentrations, profiin-1 remains monomeric but populates unfolded states exhibiting larger hydrodynamic radius and disordered structure, as assessed by dynamic light scattering, far-UV circular dichroism and intrinsic florescence. Refolding from the urea unfolded state was studied at equilibrium and in real-time using a stopped-flow apparatus. The results obtained with intrinsic florescence and circular dichroism indicate a single phase without significant changes of the corresponding signals before the major refolding transition. However, such a transition is preceded by a burst phase with an observed increase of ANS florescence, which indicates the conversion into a transiently populated collapsed state possessing solvent exposed hydrophobic clusters. Kinetic analysis reveals that such state has a conformational stability comparable to that of the fully unfolded state. To our knowledge, profiin-1 is the first example of an amyloid-related protein where folding occurs in the absence of thermodynamically stable partially folded states.The PFN1 gene, coding for profilin-1, has recently been associated with familial amyotrophic lateral sclerosis (fALS), as five mutations, namely C71G, M114T, G118V, A20T and T109M have been found in patients with familial forms of the disease and others, E117G and Q139L, has been proposed to be a moderate risk factor for disease onset. In this second part of the thesis we have purified the four profilin-1 variants along with the wild type protein. The resulting aggregates appear to be fibrillar, to have a weak binding to ThT, and to possess a significant amount of intermolecular β-sheet structure. Using ThT fluorescence assays, far-UV circular dichroism, and dynamic light scattering, we found that all variants have an aggregation propensity higher than that of the wild-type counterpart. In particular, the C71G mutation was found to induce the most dramatic change in aggregation. Such a propensity was found not to strictly correlate with the conformational stability in this group of profilin-1 variants, determined using both urea-induced denaturation at equilibrium and folding/unfolding kinetics. However, it correlated with structural changes of the folded states, as monitored with far-UV circular dichroism, intrinsic fluorescence spectroscopy, ANS binding, acrylamide quenching, and dynamic light scattering. Overall, the results suggest that all mutations increase the tendency of profilin-1 to aggregate and that such aggregation behavior is largely determined by the mutation-induced structural changes occurring in the folded state of the protein.