Aggregation of the Acylphosphatase from Sulfolobus solfataricus

Abstract

Protein aggregation is associated with a number of human pathologies including Alzheimer's and Creutzfeldt-Jakob diseases and the systemic amyloidoses. In this study, we used the acylphosphatase from the hyperthermophilic Archaea Sulfolobus solfataricus (Sso AcP) to investigate the mechanism of aggregation under conditions in which the protein maintains a folded structure. In the presence of 15-25% (v/v) trifluoroethanol, Sso AcP was found to form aggregates able to bind specific dyes such as thioflavine T, Congo red, and 1-anilino-8-naphthalenesulfonic acid. The presence of aggregates was confirmed by circular dichroism and dynamic light scattering. Electron microscopy revealed the presence of small aggregates generally referred to as amyloid protofibrils. The monomeric form adopted by Sso AcP prior to aggregation under these conditions retained enzymatic activity; in addition, folding was remarkably faster than unfolding. These observations indicate that Sso AcP adopts a folded, although possibly distorted, conformation prior to aggregation. Most important, aggregation appeared to be 100-fold faster than unfolding under these conditions. Although aggregation of Sso AcP was faster at higher trifluoroethanol concentrations, in which the protein adopted a partially unfolded conformation, these findings suggest that the early events of amyloid fibril formation may involve an aggregation process consisting of the assembly of protein molecules in their folded state. This conclusion has a biological relevance as globular proteins normally spend most of their lifetime in folded structures.

Polyglutamine Expansion in Ataxin-3 Does Not Affect Protein Stability

“…Disease-associated, aggregation-prone mutants of the prion protein have pre- viously been found to display very little or no difference in thermodynamic stability compared with the wild-type protein (43,44). Furthermore, a recent report also suggests that the map of fibrillization pathways may be more complex than previously thought (45). In their system, Plakoutsi et al (45) found that aggregation and fibril formation does not necessarily involve a passage through the unfolding pathways and partially unfolded forms but rather may also occur via native or nearly native conformations.…”

“…Furthermore, a recent report also suggests that the map of fibrillization pathways may be more complex than previously thought (45). In their system, Plakoutsi et al (45) found that aggregation and fibril formation does not necessarily involve a passage through the unfolding pathways and partially unfolded forms but rather may also occur via native or nearly native conformations. It would seem that a similar process applies to ataxin-3, whereby conformational changes caused by the expanded polyglutamine tract do not perturb the dynamics of un/folding but are sufficient to lower the activation barrier to the fibrillization pathway and thus increase the propensity of the protein to form fibrils from a native or nearly native conformation (Fig.…”

Structural and Dynamics Characteristics of Acylphosphatase from Sulfolobus solfataricus in the Monomeric State and in the Initial Native-like Aggregates

“…Under conditions in which protofibril formation occurs more rapidly (50 mM acetate buffer, 20% (v/v) TFE, pH 5.5, 25°C), it has been shown that Sso AcP adopts initially, before aggregation starts, an essentially native fold (8,10). Two sequential phases have been detected in the aggregation process (11).…”

“…However, by contrast with other acylphosphatases of known structure, Sso AcP contains an 11-residue, unstructured segment at the N terminus (7). In the presence of 15-25% (v/v) 2,2,2-trifluoroethanol (TFE), 50 mM acetate buffer, pH 5.5, 25°C, Sso AcP has been shown to aggregate within 30 min into structured spherical and chain-like protofibrils (8). Protofibril formation is also observed in the absence of TFE under the same conditions of pH and temperature, although the process requires weeks in this case (8).…”

“…In the presence of 15-25% (v/v) 2,2,2-trifluoroethanol (TFE), 50 mM acetate buffer, pH 5.5, 25°C, Sso AcP has been shown to aggregate within 30 min into structured spherical and chain-like protofibrils (8). Protofibril formation is also observed in the absence of TFE under the same conditions of pH and temperature, although the process requires weeks in this case (8). The addition of inorganic phosphate, a competitive inhibitor binding to the active site of the enzyme, stabilizes the native fold of the protein and inhibits protofibril formation (9).…”