Biological function in a non-native partially folded state of a protein.

As structural flexibility is known to be required for enzyme catalysis and pattern recognition and a significant fraction of eukaryotic proteins appear to be unfolded or contain unstructured regions, biological activity of conformational states distinct from fully folded structures could be more common than previously thought. By applying a procedure that allows the recovery of enzymatic activity to be monitored in real time, we show that a non-native state populated transiently during folding of the acylphosphatase from Sulfolobus solfataricus is enzymatically active. The structural characterization of this partially folded state reveals that enzymatic activity is possible even if the catalytic site is structurally heterogeneous, whereas the remainder of the structure acts as a scaffold.

Protein aggregation starting from the native globular state.

Amyloid formation by globular proteins that normally adopt a compact folded structure is generally induced in vitro under harsh conditions involving low pH, high temperature, high pressure, or in the presence of organic solvents. Under these conditions, folded proteins are generally unfolded, at least partially. The approach described here shows a rationale and two detailed examples as to how the mechanism of aggregation of a globular protein can be probed under conditions in which it is initially in its folded conformation, and hence relevant to a physiological environment.

Stabilization of a native protein mediated by ligand binding inhibits amyloid formation independently of the aggregation pathway.

The acylphosphatases from Sulfolobus solfataricus and Drosophila melanogaster (Sso AcP and AcPDro2) were previously shown to form amyloid-like aggregates without the need to unfold initially. Inorganic phosphate (Pi), a competitive inhibitor binding specifically to the active site of these proteins, was found to stabilize, upon binding, the native state of AcPDro2 and to inhibit its conversion into amyloid-like fibrils. The inhibitory effect of Pi is suppressed only in a variant in which the Arg residue responsible for Pi binding is mutated.

Sequence and structural determinants of amyloid fibril formation.

Amyloid fibril formation is a process that represents an essential feature of the chemistry of proteins and plays a central role in human pathology and the biology of living organisms. In this Account, we shall describe some of the recent results on the sequence and structural determinants of protein aggregation. We shall describe the factors that govern aggregation of unfolded peptides and proteins.

Exploring the mechanism of formation of native-like and precursor amyloid oligomers for the native acylphosphatase from Sulfolobus solfataricus.

Over 40 human diseases are associated with the formation of well-defined proteinaceous fibrillar aggregates. Since the oligomers precursors to the fibrils are increasingly recognized to be the causative agents of such diseases, it is important to elucidate the mechanism of formation of these early species. The acylphosphatase from Sulfolobus solfataricus is an ideal system as it was found to form, under conditions in which it is initially native, two types of prefibrillar aggregates: (1) initial enzymatically active aggregates and (2) oligomers with characteristics reminiscent of amyloid protofibrils, with the latter originating from the structural reorganization of the initial assemblies.

Structure, conformational stability, and enzymatic properties of acylphosphatase from the hyperthermophile Sulfolobus solfataricus.

The structure of AcP from the hyperthermophilic archaeon Sulfolobus solfataricus has been determined by (1)H-NMR spectroscopy and X-ray crystallography. Solution and crystal structures (1.27 A resolution, R-factor 13.

Evidence for a mechanism of amyloid formation involving molecular reorganisation within native-like precursor aggregates.

The aggregation of the alpha/beta protein acylphosphatase from Sulfolobus solfataricus has been studied under conditions in which the protein maintains a native-like, although destabilised, conformation and that therefore bear resemblance to a physiological medium. Static and dynamic light-scattering measurements indicate that under these conditions the protein aggregates rapidly, within two minutes. The initial aggregates are enzymatically active and have a secondary structure that is not yet characterized by the high content of cross-beta structure typical of amyloid, as inferred from Fourier transform infra-red and circular dichroism measurements.

Amyloid formation from HypF-N under conditions in which the protein is initially in its native state.

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.

Monitoring the process of HypF fibrillization and liposome permeabilization by protofibrils.

Much information has appeared in the last few years on the low resolution structure of amyloid fibrils and on their non-fibrillar precursors formed by a number of proteins and peptides associated with amyloid diseases. The fine structure and the dynamics of the process leading misfolded molecules to aggregate into amyloid assemblies are far from being fully understood. Evidence has been provided in the last five years that protein aggregation and aggregate toxicity are rather generic processes, possibly affecting all polypeptide chains under suitable experimental conditions.

Aggregation of the Acylphosphatase from Sulfolobus solfataricus: the folded and partially unfolded states can both be precursors for amyloid formation.

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.