Reversal of Protein Aggregation Provides Evidence for Multiple Aggregated States Martino Calamai 1 , Claudio Canale 2 , Annalisa Relini 2 , Massimo Stefani 3 Fabrizio Chiti 3 and Christopher M. Dobson 1 * 1 Department of Chemistry University of Cambridge Lensfield Road, Cambridge CB2 1EW, UK 2 Dipartimento di Fisica, Universita ` di Genova, Via Dodecaneso 33, 16146, Genova Italy 3 Dipartimento di Scienze Biochimiche, Universita ` degli Studi di Firenze, Viale Morgagni 50, 50134, Firenze Italy 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 acyl- phosphatase (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 (O5 mm) 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 w1s K1 . The 60–200 nm globular aggregates disaggregate with an apparent rate constant of w2.5!10 K3 s K1 while the 400–800 nm clusters disassembly more slowly with a rate constant of w3.1!10 K4 s K1 . The larger (O5 mm) 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. q 2004 Elsevier Ltd. All rights reserved. Keywords: amyloid; disaggregation kinetics; stability; refolding; size *Corresponding author Introduction One of the most exciting challenges in con- temporary cellular and structural biology is to develop a molecular description of the phenom- enon of protein misfolding and aggregation. 1 Recent studies have demonstrated that cells have evolved a complex system of defences against aggregation, centred on the ability of molecular chaperones to interact with incompletely folded and aggregation-prone proteins, thereby prevent- ing improper intermolecular interactions. 2,3 Such protective mechanisms are not infallible, however, and an increasing number of diseases is now recognized as being related directly to the aggrega- tion of misfolded proteins. 1,4,5 These pathological conditions include disorders such as Alzheimer’s disease and type II diabetes in which specific proteins are deposited within tissue as insoluble amyloid fibrils and plaques. 6–9 Despite major differences in the sequences and structures of the peptides and proteins involved, the fibrillar forms 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. Abbreviations used: AcP, human muscle acylphosphatase; TFE, 2,2,2 trifluoroethanol; ThT, thioflavin T; FTIR, Fourier transform infrared spectroscopy; DLS, dynamic light-scattering; AFM, atomic force microscopy; OM, optical microscopy; bzP, benzoyl phosphate; HypF-N, the N-terminal domain of E. coli HypF; a.u., arbitrary units. E-mail address of the corresponding author: cmd44@cam.ac.uk doi:10.1016/j.jmb.2004.11.067 J. Mol. Biol. (2005) 346, 603–616