AMYLOIDINTERMEDIATE · The structural and dynamical ensemble of an amyloidogenic intermediate

The structural and dynamical ensemble of an amyloidogenic intermediate

The majority of proteins have evolved to adopt distinctive and well-defined functional states under physiological conditions, either as monomers or as complexes. The structures corresponding to these states are encoded in the sequence as is the crucial ability of the molecules to remain soluble within the crowded cellular environment. It is increasingly evident, however, that even under physiological conditions the aggregated states of proteins, such as the highly ordered amyloid form, can be thermodynamically more stable than native states, indicating that kinetic factors are of key importance in enabling protein homeostasis to be maintained. Proteins in vivo only rarely convert into aberrant aggregated states, such as those associated with pathological conditions such as Alzheimer's disease and type II diabetes, despite their inherent tendency to do so in vitro. In the present research, we used a combination of NMR experiments and molecular dynamics simulations to identify the characteristic features of the free energy landscapes that enable the majority of the proteins to avoid aggregation under physiological conditions. We chose for this scope the acylphosphatase from Drosophila melanogaster (AcPDro2) as this is a particularly well-suited system for investigating the molecular strategies used by living systems for the maintenance of protein solubility. AcPDro2 in its native state is a globular and monomeric protein with a structure consisting of five ß-strands (S1-S5), which form a single ß-sheet, and two a-helices (H1 and H2) that lie adjacent to this ß-sheet. The importance that subtle intrinsic factors play in enabling this protein to remain soluble is clearly shown by the fact that a very low concentration (5 % v/v) of trifluoroethanol (TFE) is sufficient to induce rapid formation of amyloid fibrils although the protein still populates a highly native-like conformational ensemble before aggregation occurs. Indeed, under these conditions, the hydrodynamic radius, intrinsic fluorescence, secondary structure content and enzymatic activity of AcPDro2 in its monomeric state are indistinguishable those of the protein in the absence of TFE, where the propensity of AcPDro2 to aggregate is extremely low. Moreover, within the experimental error, AcPDro2 has the same thermodynamic stability (i.e. the same free energy of unfolding, ?GU-F) in the presence and absence of 5 % v/v TFE. By contrast to AcPDro2, most folded proteins aggregate in the presence of much higher concentrations of TFE (15-30 %) where a significant portion of the molecules are unfolded or strongly destabilised. We could determine a series of energy landscapes of AcPDro2 thus analysing the differences in the structures, dynamics and energy surfaces of the protein in its soluble state or in situations where it aggregates. The study identifies the nature of the energy barriers that under normal physiological conditions prevent the protein ensemble from populating dangerous aggregation-prone states. We found that such states, although similar to the native conformation, have altered surface charge distribution, alternative topologies of the ß-sheet region and modified solvent exposure of hydrophobic surfaces and aggregation prone regions of the sequence. The identified barriers allow the protein to undergo functional dynamics while remaining soluble and without a significant risk of misfolding and aggregation into non-functional and potentially toxic species.

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