title: Unlocked concanavalin A forms amyloid-like fibrils from coagulation of long-lived "crinkled" intermediates.
abstract: Understanding the early events during amyloid aggregation processes is crucial to single out the involved molecular mechanisms and for designing ad hoc strategies to prevent and reverse amyloidogenic disorders. Here, we show that, in conditions in which the protein is positively charged and its conformational flexibility is enhanced, Concanavalin A leads to fibril formation via a non-conventional aggregation pathway. Using a combination of light scattering, circular dichroism, small angle X-ray scattering, intrinsic (Tryptophan) and extrinsic (ANS) fluorescence and confocal and 2-photon fluorescence microscopy we characterize the aggregation process as a function of the temperature. We highlight a multi-step pathway with the formation of an on-pathway long-lived intermediate and a subsequent coagulation of such "crinkled" precursors into amyloid-like fibrils. The process results in a temperature-dependent aggregation-coagulation pathway, with the late phase of coagulation determined by the interplay between hydrophobic and electrostatic forces. Our data provide evidence for the complex aggregation pathway for a protein with a highly flexible native conformation. We demonstrate the possibility to generate a long-lived intermediate whose proportion and occurrence are easily tunable by experimental parameters (i.e. temperature). As a consequence, in the case of aggregation processes developing through well-defined energy barriers, our results can open the way to new strategies to induce more stable in vitro on-pathway intermediate species through a minute change in the initial conformational flexibility of the protein. This will allow isolating and experimentally studying such transient species, often indicated as relevant in neurodegenerative diseases, both in terms of structural and cytotoxic properties.
authors.affiliation: Dipartimento di Fisica e Chimica, Università di Palermo, Palermo, Italy. valeria.vetri@unipa.it
authors.forename: Valeria
authors.initials: V
authors.lastname: Vetri
authors.forename: Maurizio
authors.initials: M
authors.lastname: Leone
authors.forename: Ludmilla A
authors.initials: LA
authors.lastname: Morozova-Roche
authors.forename: Bente
authors.initials: B
authors.lastname: Vestergaard
authors.forename: Vito
authors.initials: V
authors.lastname: Foderà
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D002942:Circular Dichroism; D003208:Concanavalin A / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D000682:Amyloid; D003208:Concanavalin A