Modulation of protein conformation and stability by trifluoroethanol: effects on the IFABP abridged family.

CR ANGELANI, IS CABANAS, JJ CARAMELO, LM CURTO, JM DELFINO

La Plata

Congreso; Modulation of protein conformation and stability by trifluoroethanol: effects on the IFABP abridged family.; 2013

Δ98Δ and Δ78Δ are two functional all-β sheet 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. Although IFABP and Δ98Δ are monomers, Δ78Δ is dimeric. We have previously demonstrated that their intrinsic stability (IFABP≥Δ78Δ>Δ98Δ) do not bear a straightforward correlation with their aggregation propensity (Δ78Δ>IFABP≥Δ98Δ). As this finding appears at odds with the established notion that a perturbation of the native fold should necessarily favor the population of aggregation-prone species, we decided to explore the influence of sub-aggregating concentrations of TFE (≤ 10%v/v) on the conformation and stability of this protein family. Upon addition of TFE, the far UV circular dichroism (CD) spectrum of IFABP remains unchanged. Remarkably, the spectra of Δ78Δ and Δ98Δ are gradually modified, at 10% TFE being indistinguishable from IFABP. In this condition, near UV CD spectra of the three proteins remain native-like. This information points to the consolidation of the conformation of the abridged variants. Consistently, in the presence of TFE, Δ98Δ and Δ78Δ exhibit lower proteolytic susceptibility, as compared with buffer alone. Conversely, IFABP is equally -or even more extensively- digested. TFE causes a reduction in the thermal stability of all proteins. For IFABP, irreversible thermal aggregation occurs alongside with denaturation, giving rise to a greater reduction in the melting point temperature. In summary, exposure to sub-aggregating concentrations of TFE reveals the coalescence of all three proteins into conformations richer in β content and more akin in stability.