Structural determination of toxic and nontoxic HypF-N oligomers by FRET.

The aggregation and accumulation of proteins into extracellular deposits of amyloid fibrils or into intracellular inclusions with amyloid-like properties is the hallmark of several human disorders, such as Alzheimer’s disease, Parkinson’s disease, type II diabetes and amyloidosis. It is nowadays accepted that oligomeric intermediates, rather than mature amyloid fibrils, are considered the primary pathogenic species in many protein deposition diseases. The ability of the N-terminal domain of the bacterial HypF protein from E. coli (HypF-N) to produce oligomers with different biological properties when incubated in different solution conditions allowed to focus the attention on the parameters that determine a higher or a lower capacity of the oligomers to cause cell dysfunction. Previous data revealed that toxic HypF-N oligomers present a high degree of solvent-exposure and flexibility of the hydrophobic cores, necessary to interact and permeabilize the cellular membrane and, subsequently, determine an increase of calcium influx, production of ROS, activation of caspase-3, and, finally, cellular death. This thesis work aims at contributing to elucidate the structural differences between toxic type A and nontoxic type B HypF-N oligomers with a level of detail higher than that obtained in previous studies. In a first set of experiments 12 mutational variants of HypF-N carrying a single cysteine residue located at different positions along the polypeptide chain were labelled with the fluorescent dye 1,5-IAEDANS and, then, were allowed to form oligomers under conditions A and B. The maximum fluorescence emission (max) of this dye is influenced by the dielectric constant of the medium in which it is located. Thus, it is indicative of the hydrophilic/hydrophobic environment around the labelled residue in the oligomer. The results show that the hydrophobic regions of the sequence are more solvent-exposed in type A oligomers than in type B oligomers, in agreement with the PM excimer ratio analysis previously performed. As a second set of experiments, a structural investigation of such toxic and nontoxic HypF-N oligomers was carried out using Förster Resonance Energy Transfer (FRET), using the 1,5-IAEDANS and 6-IAF dyes, that have the necessary prerequisites to be considered a suitable pair of donor/acceptor probes of FRET. Thus, all the single cysteine variants were labelled with 1,5-IAEDANS (donor) and 6-IAF (acceptor) and combined in a 1:1 molar ratio to form toxic and nontoxic oligomers. By calculating the FRET efficiency from the donor to the acceptor, it was possible to determine and compare the degrees of intermolecular interaction between various residues in the oligomers. The results show that FRET efficiency values are generally higher in the toxic oligomers than in the nontoxic oligomers. This means that the intermolecular distances between pairs of labelled residues in adjacent protein molecules in the oligomer are lower in the case of type A oligomers, indicating that these aggregates are more compact and structured than the oligomers B. However, most of the hydrophobic interactions appear to be less structured in the toxic type A oligomers, allowing the key solvent-exposed residues to be identified in the toxic assemblies.