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The 93-residue N-terminal fragment of apolipoprotein A-I (ApoA-I) is the major constituent of fibrils isolated from patients affected by the amyloidosis caused by ApoA-I mutations. We have prepared eight polypeptides corresponding to all the currently known amyloidogenic variants of the N-terminal region of ApoA-I, other than a truncation mutation, and investigated their aggregation kinetics and the associated structural modifications. All the variants adopted a monomeric highly disordered structure in solution at neutral pH, whereas acidification of the solution induced an unstable α-helical conformation and the subsequent aggregation into the cross-β structure aggregate. Two mutations (Δ70-72 and L90P) almost abrogated the lag phase of the aggregation process, three mutations (Δ60-71, L75P, and W50R) significantly accelerated the aggregation rate by 2- to 3-fold, while the remaining three variants (L64P, L60R, and G26R) were not significantly different from the wild type. Therefore, an increase in aggregation propensity cannot explain per se the mechanism of the disease for all the variants. Prediction of the protection factors for hydrogen exchange in the native state of full-length protein reveals, in almost all the variants, an expansion of the conformational fluctuations that could favour the proteolytic cleavage and the release of the amyloidogenic peptide. Such an event seems to be a necessary prerequisite for ApoA-I fibrillogenesis in vivo, but the observed increased aggregation propensity of certain variants can have a strong influence on the severity of the disease, such as an earlier onset and a faster progression. © 2011 Elsevier Ltd. All rights reserved.

Aggregation of the N-terminal domain of the Escherichia coli HypF (HypF-N) was investigated in mild denaturing conditions, generated by addition of 6-12% (v/v) trifluoroethanol (TFE). Atomic force microscopy indicates that under these conditions HypF-N converts into the same type of protofibrillar aggregates previously shown to be highly toxic to cultured cells. These convert subsequently, after some weeks, into well-defined fibrillar structures. The rate of protofibril formation, monitored by thioflavin T (ThT) fluorescence, depends strongly on the concentration of TFE. Prior to aggregation the protein has far-UV circular dichroism (CD) and intrinsic fluorescence spectra identical with those observed for the native protein in the absence of co-solvent; the quenching of the intrinsic tryptophan fluorescence by acrylamide and the ANS binding properties are also identical in the two cases. These findings indicate that HypF-N is capable of forming amyloid protofibrils and fibrils under conditions in which the protein is initially in a predominantly native-like conformation. The rate constants for folding and unfolding of HypF-N, determined in 10% TFE using the stopped-flow technique, indicate that a partially folded state is in rapid equilibrium with the native state and populated to ca 1%. A kinetic analysis reveals that aggregation results from molecules accessing such a partially folded state. The approach described here shows that it is possible to probe the mechanism of aggregation of a specific protein under conditions in which the protein is initially native and hence relevant to a physiological environment. In addition, the results indicate that toxic protofibrils can be formed from globular proteins under conditions that are only marginally destabilising and in which the large majority of molecules have the native fold. This conclusion emphasises the importance for cells to constantly combat the propensity for even the most stable of these proteins to aggregate.

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.

The conversion of specific proteins or protein fragments into insoluble, ordered fibrillar aggregates is a fundamental process in protein chemistry, biology, medicine and biotechnology. As this structural conversion seems to be a property shared by many proteins, understanding the mechanism of this process will be of extreme importance. Here we present a structural characterisation of a conformational state populated at low pH by the N-terminal domain of Escherichia coli HypF. Combining different biophysical and biochemical techniques, including near- and far-UV circular dichroism, intrinsic and 8-anilinonaphthalene-1-sulfonate-derived fluorescence, dynamic light scattering and limited proteolysis, we will show that this state is largely unfolded but contains significant secondary structure and hydrophobic clusters. It also appears to be more compact than a random coil-like state but less organised than a molten globule state. Increase of the total ionic strength of the solution induces aggregation of such a pre-molten globule state into amyloid-like protofibrils, as revealed by thioflavin T fluorescence and atomic force microscopy. These results show that a pre-molten globule state can be, among other possible conformational states, one of the precursor states of amyloid formation. In addition, the possibility of triggering aggregation by modulating the ionic strength of the solution provides one a unique opportunity to study both the initial precursor state and the aggregation process.