TRUNCATION OF A β-BARREL SCAFFOLD DISSOCIATES INTRINSIC STABILITY FROM ITS PROPENSITO TO AGGREGATION

CABANAS, I.S; CURTO L.M.; ANGELANI, C.R.; CARAMELO, J.J.; DELFINO J.M.

San Diego

Congreso; The 26th Annual Symposium of The Protein Society; 2012

The Protein Society

Δ98Δ and Δ78Δ are two functional all-β sheet variants of IFABP (intestinal fatty acid binding protein) that were generated by controlled proteolysis. 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. Remarkably, although full length IFABP and Δ98Δ are monomers in solution 1,2, Δ78Δ adopts a stable dimeric structure 3. Despite the fact that naturally occurring β-sheet proteins avoid edge-to-edge aggregation, the addition of the structure-promoting co-solvent 2,2,2-trifluoroethanol (TFE) can induce conformational changes that might trigger the onset of aggregation. With the aim of identifying the structural features involved in promoting or delaying aggregation, we compare here the effects of TFE on the structure and the propensity to aggregate of IFABP, Δ98Δ and Δ78Δ. In all cases, turbidity measurements at 350 nm revealed a maximum value at around 25% v/v. Furthermore, Congo Red and Thioflavin-T binding to the aggregates suggest an amyloid-like character, a fact consistent with data from transmission electron microscopy (TEM) that do not exclude their fibrillar morphology. In addition, substantial effects of TFE at various concentrations on the overall protein conformation were assessed by fluorescence and circular dichroism spectroscopies. The constructs share a common nucleation-elongation mechanism, whereby the population of stable dimeric nuclei occurs prior to the association of protein to the growing aggregates. Unexpectedly, the intrinsic conformational stability of IFABP and its truncated forms do not correlate with their tendency to aggregate. These findings appear at odds with the established notion that a perturbation of the native compact fold should necessarily favor the population of aggregation-prone species.