Amyloid Fibrils Formation of Concanavalin A at Basic pH

Mechanisms of partial unfolding and aggregation of proteins areof extreme interest in view of the fact that several human pathologies arecharacterized by the formation and deposition of protein-insoluble material,mainly composed of amyloid fibrils.Here we report on an experimental study onthe heat-induced aggregation mechanisms, at basic pH, of concanavalin A(ConA), used as a model system. Thioflavin T (ThT) fluorescence andmultiangle light scattering allowed us to detect different intertwined steps inthe formation of ConA aggregates. In particular, the ThT fluorescence increase,observed in the first phase of aggregation, reveals the formation of intermolecularβ-sheet structure which constitutes a rate-limiting step of the process. Theintertwining between the formation of β-aggregate structures and the wholeaggregation process is discussed as a function of protein concentration: acoagulation process produces the same kind of aggregates at the differentconcentrations studied. Multiangle light-scattering data highlight the onset ofthe condensation process which gives rise to formation of compact fractalaggregates. AFM microscopy supports this conclusion showing thin fibrils of ConA, formed in the early stage of aggregation, whichfurther interact to form larger structures with a netlike spatial organization.