Research Article: Structural coalescence underlies the aggregation propensity of a β-barrel protein motif

Date Published: February 10, 2017

Publisher: Public Library of Science

Author(s): Carla R. Angelani, Julio J. Caramelo, Lucrecia M. Curto, José M. Delfino, Reza Khodarahmi.


A clear understanding of the structural foundations underlying protein aggregation is an elusive goal of central biomedical importance. A step toward this aim is exemplified by the β-barrel motif represented by the intestinal fatty acid binding protein (IFABP) and two abridged all-β sheet forms (Δ98Δ and Δ78Δ). At odds with the established notion that a perturbation of the native fold should necessarily favor a buildup of intermediate forms with an enhanced tendency to aggregate, the intrinsic stability (ΔG°H2O) of these proteins does not bear a straightforward correlation with their trifluoroethanol (TFE)-induced aggregation propensity. In view of this fact, we found it more insightful to delve into the connection between structure and stability under sub-aggregating conditions (10% TFE). In the absence of the co-solvent, the abridged variants display a common native-like region decorated with a disordered C-terminal stretch. Upon TFE addition, an increase in secondary structure content is observed, assimilating them to the parent protein. In this sense, TFE perturbs a common native like region while exerting a global compaction effect. Importantly, in all cases, fatty acid binding function is preserved. Interestingly, energetic as well as structural diversity in aqueous solution evolves into a common conformational ensemble more akin in stability. These facts reconcile apparent paradoxical findings related to stability and rates of aggregation. This scenario likely mimics the accrual of aggregation-prone species in the population, an early critical event for the development of fibrillation.

Partial Text

Achieving full understanding of the mechanism of protein aggregation will represent a breakthrough in the context of both physiological and pathological phenomena occurring in nature. Such information will likely be of great use to shed light on normal processes or-when the outcome goes astray- on the origin of pathologies. Undoubtedly, this new comprehension will be of fundamental value in establishing modes of intervention on aggregation diseases with effector molecules, hopefully leading to the development of new therapies [1,2].

The co-solvent TFE is generally employed to induce helical structures on peptides, but it can also perturb the native state of proteins [15]. Therefore, the effect of low concentration of TFE on the conformation of IFABP and the abridged variants Δ98Δ and Δ78Δ was assayed. For IFABP, no change is observed -either in the far or in the near UV CD spectra- a fact indicative of lack of any significant conformational change (Fig 2A and 2B). However, a different behavior is observed for the abridged variants (Fig 2C–2E). Even at the lowest concentration assayed (2.5%), each abbreviated protein suffers a dramatic change in the shape of the far UV CD spectrum. The minimum is red-shifted (4 nm), giving rise to a spectrum coincident with that observed for the parent protein: similar molar ellipticity and minimum at ~ 216 nm. Additionally, a strong positive band centered at ~ 200 nm appears. Above that concentration, the spectra do not further change in shape, but the latter signal progressively increases in magnitude. Remarkably, at 10% TFE the far UV CD spectrum of all three proteins is almost identical. In the near UV CD range, minor spectral changes take place by the addition of 10% TFE, indicating a full conservation of the fine structure.

Previous work from of our laboratory [8,9] showed that the aggregation of IFABP, Δ98Δ and Δ78Δ triggered by 25% TFE share a common nucleation-elongation mechanism. The first event is a fast equilibrium step, whereby TFE induces a conformational rearrangement of the native protein (P), giving rise to an aggregation-prone state (P*). In all cases, the ensuing process includes the formation of a dimeric nucleus followed by the growth of the fibrillar aggregates by the irreversible apposition of more monomer [8,20]. Contrary to common expectation, the intrinsic stability of these proteins in water does not bear a straightforward correlation with their aggregation propensity. This observation is at odds with the established notion that a perturbation of the native fold would necessarily favor the population of aggregation-prone species [21]. In this sense, it might be more insightful to correlate aggregation propensity with conformational stability measured in the presence of up to 10% TFE, the maximal concentration at which all proteins remain soluble.




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