Modulation of protein conformation and aggregation of a β-barrel protein family.

ANGELANI, C.R.; PONCINO, M.; CURTO L.M.; DELFINO J.M.

Salto

Congreso; Latin American Crosstalk in Biophysics and Physiology; 2015

SAB - SBF.uy

Δ98Δ and Δ78Δ are two all-β variants of IFABP (intestinal fatty acid binding protein). These frameworks became useful to study the molecular determinants related to aggregation of β-barrel proteins. Albeit displaying increased conformational plasticity, these variants exhibit a native-like β-barrel topology and are able to support a cooperative folding behavior. Challenging these scaffolds with 25% v/v trifluoroethanol (TFE) readily triggers amyloid-like aggregation. We decided to assess the impact of low concentrations of TFE (≤ 15% v/v) on conformation and stability so as to understand if these concentrations -where the proteins are still soluble- would mimic the early events occurring at 25% v/v TFE. In this milieu, the structure of all three proteins coalesces and they also become more akin in stability. Evidence in support of this contention arises from several biophysical measurements, including the binding of oleic acid and ANS, the quenching of intrinsic fluorescence, and by thermal ramps and exposure to chemical denaturants. Additionally, we identified those fragments initially obtained after limited proteolysis with chymotrypsin either in the absence on in the presence of 10% v/v TFE. The new data led us to refine the structural models proposed for the abridged proteins. In this scenario, the organization of a disordered C-terminal stretch attached to a common well-packed core might underlie the structural compaction phenomenon observed upon TFE addition. Finally, those changes occurring immediately after the addition of 25% v/v TFE are compared with the structural information available at low TFE concentration. The cumulative evidence supports the hypothesis that the conformational changes observed would represent those leading to the aggregation-prone species.