Searching for a hidden intermediate. Thermal Denaturation and Aggregation propensity of intestinal fatty acid binding protein (IFABP)

CURTO L.M.; BALLATORE, M; DELFINO J.M.

San Luis

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

IFABP is a 15 kDa protein characterized by a β-barrel fold resembling a clamshell. It consists of two perpendicular five-stranded β-sheets (A-E and F-J) decorated with an intervening helix-turn-helix motif between strands A and B. The addition of TFE (2,2,2- trifluoroethanol) -a structure-promoting co-solvent- at high concentration (~25% v/v TFE) triggers the onset of amyloid-like aggregation. Previous work from our group revealed that, under equilibrium conditions, a low concentration of TFE (up to 15 % v/v) fosters 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 was concluded that this level of co-solvent favors the population of alternative native-like conformations scarcely explored in water. Interestingly, equilibrium unfolding transitions induced by guanidinium chloride (GdmCl) reveal that, in the presence of TFE, IFABP does not follow a simple two-step folding behavior. Indeed, TFE unveils the existence of intermediate species in IFABP (populated at ~ 1.25 M GdmCl), a classically recognized two-state protein model. As attested by far UV circular dichroism (CD), this hidden intermediate is rich in β structure, although different from that prevalent in native IFABP. Despite the virtual absence of any structural change evident by far UV CD upon the addition of TFE at low concentration (5 % v/v at 25 °C), the protein becomes susceptible to irreversible thermal unfolding. In view of these facts, we propose that an increase in TFE concentration leads to the accretion in the population of the β-rich intermediate, thus leading to a decreased thermal stability. The main gist of this work points to discovering the connection between the conformational rearrangements exerted by TFE, leading to an imbalance in the population of intermediate species, and their relevance for the thermally induced aggregation propensity of the protein.