Reversal of protein aggregation provides evidence for multiple aggregated states

Year: 2005

Authors: Calamai M., Canale C., Relini A., Stefani M., Chiti F., Dobson CM.

Autors Affiliation: Department of Chemistry University of Cambridge Lensfield Road, Cambridge CB2 1EW, UK;
Dipartimento di Fisica, Universita` di Genova, Via Dodecaneso 33, 16146, Genova Italy;
Dipartimento di Scienze Biochimiche, Universita` degli Studi di Firenze, Viale Morgagni 50, 50134, Firenze Italy

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 mum) 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 similar to 1 s(-1). The 60-200 nm globular aggregates disaggregate with an apparent rate constant of similar to 2.5 x 10(-3) s(-1) while the 400-800 run clusters disassembly more slowly with a rate constant of similar to 3.1 x 10(-4) s(-1). The larger (> 5 mum) 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. (C) 2004 Elsevier Ltd. All rights reserved.

Journal/Review: JOURNAL OF MOLECULAR BIOLOGY

Volume: 346 (2)      Pages from: 603  to: 616

More Information: We thank Mark Krebs and Leopold L. Ilag for assistance with certain experiments, and Niccolo Taddei, Alessandra Gliozzi, Ranieri Rolandi, Damien Hall, Jesus Zurdo, Natalia Carulla and Reto Bader for valuable discussions. This work was supported by grants from the Wellcome Trust, from the European Commission (Research Directorates, project “Protein (mis)folding”, proposal no RTN2-2001-00364) and from the Italian MIUR (FIRB 2001 RBNE01S29H, PRIN 2002058218). Appendix
KeyWords: acylphosphatase; amyloid, article; atomic force microscopy; bright field microscopy; light scattering; particle size; priority journal; protein aggregation; protein assembly; protein degradation; protein folding; protein stability; protein structure; structure analysis, Acid Anhydride Hydrolases; Amyloid; Dimerization; Humans; Kinetics; Microscopy, Atomic Force; Muscles; Particle Size; Polytetrafluoroethylene; Protein Denaturation; Protein Renaturation; Solubility
DOI: 10.1016/j.jmb.2004.11.067

Citations: 75
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