title: Reversal of protein aggregation provides evidence for multiple aggregated States.
abstract: Observations that prefibrillar aggregates from different amyloidogenic proteins can be solubilised under some conditions have raised questions as to the generality of this phenomenon and the nature of the factors that influence it. By studying aggregates formed from human muscle acylphosphatase (AcP) under mild denaturing conditions, and by using a battery of techniques, we demonstrate that disaggregation is possible under conditions close to physiological where the protein is stable in its native state. In the presence of 25% (v/v) trifluoroethanol (TFE) AcP undergoes partial unfolding and globular aggregates (60-200 nm in diameter) that can assemble further into clusters (400-800 nm in diameter) develop progressively. Yet larger superstructures (>5 microm) are formed when the concentration of the globular aggregates exceeds a critical concentration. After diluting the sample to give a solution containing 5% TFE, the fraction of partially unfolded monomeric protein refolds very rapidly, with a rate constant of approximately 1s(-1). The 60-200 nm globular aggregates disaggregate with an apparent rate constant of approximately 2.5 x 10(-3)s(-1) while the 400-800 nm clusters disassembly more slowly with a rate constant of approximately 3.1 x 10(-4)s(-1). The larger (>5 microm) superstructures are not disrupted under the conditions used here. These results suggest that amyloid formation occurs in discrete steps whose reversibility is increasingly difficult, and dependent on the size of the aggregates, and that disaggregation experiments can provide a powerful method of detecting different types of species within the complex process of aggregation. In addition, our work suggests that destabilization of amyloid aggregates resulting in the conversion of misfolded proteins back to their native states could be an important factor in both the onset and treatment of diseases associated with protein aggregation.
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
authors.forename: Martino
authors.initials: M
authors.lastname: Calamai
authors.forename: Claudio
authors.initials: C
authors.lastname: Canale
authors.forename: Annalisa
authors.initials: A
authors.lastname: Relini
authors.forename: Massimo
authors.initials: M
authors.lastname: Stefani
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Chiti
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dobson
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000737:chemistry*; D000682:Amyloid / Q000737:chemistry*; D019281:Dimerization; D006801:Humans; D007700:Kinetics; D018625:Microscopy, Atomic Force; D009132:Muscles / Q000201:enzymology; D010316:Particle Size; D011138:Polytetrafluoroethylene; D011489:Protein Denaturation; D020673:Protein Renaturation; D012995:Solubility; D000097024:Acylphosphatase
chemical_list: D017766:Acid Anhydride Hydrolases; D000682:Amyloid; D011138:Polytetrafluoroethylene; D000097024:Acylphosphatase