TFE unveils the existence of intermediate species in intestinal fatty acid binding protein (IFABP), a classically recognized two state protein model

CURTO L.M.; NICOLEYSON, A; ANGELANI, C.R.; DELFINO J.M.

CABA

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

IFABP is a 15 kDa protein exhibiting a β-barrel fold that resembles a clamshell consisting of two perpendicular five-stranded β-sheets with an intervening helix-turn-helix motif between strands A and B. Despite the fact that naturally occurring β-sheet proteins avoid aggregation through a variety of strategies, the addition of high concentrations of a structure-promoting cosolvent such as 2,2,2-trifluoroethanol (~ 25% v/v TFE) induces conformational rearrangements that trigger the onset of amyloid-like aggregation. Previous work from our group revealed that, in equilibrium, low concentrations of TFE (up to 15 % v/v) foster conformational changes akin to those leading to aggregation-prone species. Most significantly, the protein remains functional as attested by its capacity to bind fatty acids. As retaining function is a signature of the native state, it can be concluded that this level of co-solvent favors the population of alternative conformations scarcely explored in water. In view of this fact, we decided to evaluate which were the most relevant conformational rearrangements. Hardly any structural change in IFABP is observed by the full preservation of secondary structure and the minimal perturbations in tertiary structure, evidenced by the circular dichroism (CD) spectra and the unaffected λmax of fluorescence emission. Strikingly, even TFE concentrations as low as 2.5% turn the protein susceptible to thermal irreversible unfolding. Interestingly, data derived from guanidinium chloride-induced equilibrium unfolding transitions reveal the population of an intermediate species. This result demonstrates for the first time that IFABP does not follow a simple two-step folding behavior in equilibrium. Full characterization of this hidden intermediate might shed light on the genesis of aggregation-prone species.