Structural coalescence underlies the aggregation propensity of a β-barrel protein motif.

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

Size-exclusion chromatography.

<p>Size-exclusion chromatography of IFABP (solid line), Δ98Δ (dashed line) and Δ78Δ (dotted line). Proteins were sampled onto a Superdex-75 column and eluted at 10% v/v TFE in buffer PN8 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170607#sec002" target="_blank">Materials and methods</a>). Arrows indicate the elution volumes in the absence of TFE.</p

Fluorescence quenching.

<p>Quenching by acrylamide of the intrinsic fluorescence intensity of IFABP (■, □), Δ98Δ (●,○) and Δ78Δ (▲,△). Experiments were carried out in the absence (closed symbols) or in the presence of 10% v/v TFE (open symbols) in buffer PN8 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170607#sec002" target="_blank">Materials and methods</a>). The values of the Stern-Volmer constants are shown as an inset table.</p

SDS-PAGE separation.

<p>Separation by SDS-PAGE of the digestion mixture of proteins after treatment with chymotrypsin. Proteins were digested at a mass ratio of protein to protease of 200:1 for 30 min at 30°C (left panel) and 20:1, overnight at 30°C (right panel).</p

Ribbon structure of IFABP (PDB 2IFB).

<p>For Δ98Δ, the excised N- and C-terminal fragments are shown in black. The Δ78Δ variant -lacking also the 107–126 fragment (in purple)-adopts a dimeric structure. Residues belonging to the hydrophobic core (F47, F62, L64, F68, M84 and L89) are depicted with their side-chains in red polytube representation. W82, that also belongs to the hydrophobic core, and W6 are shown in green polytubes. The figure was generated with VMD [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170607#pone.0170607.ref004" target="_blank">4</a>] and rendered with POV-Ray.</p

Circular dichroism spectroscopy of IFABP (A, B), Δ98Δ (C, D) and Δ78Δ (E, F).

<p>Far (left panels) and near (right panels) UV CD spectra at increasing TFE concentrations (% v/v): 0 (solid thick line), 2.5 (dotted line), 5 (dashed line), 7.5 (dash-dot-dot-dashed line) and 10 (solid thin line). Fluorescence spectra corresponding to the same samples are shown as insets. The ordinate axes, indicating Fluorescence intensity arbitrary units are drawn in the same scale.</p

Thermally-induced unfolding transitions.

<p>Circular dichroism (CD) measurements recorded as temperature is increased for IFABP (A and B), Δ98Δ (C and D) and Δ78Δ (E and F). The transitions were monitored by the evolution of the ellipticity signal at 216 nm (left panels) at 0 (closed symbols) and 10% v/v TFE (open symbols). The value of the dynode voltage (V in Volts) at each condition is shown (right panels).</p

Urea-induced unfolding transitions.

<p>Evolution of the fluorescence emission (integrated intensity) as a function of urea concentration (Panel A) for IFABP (■, □), Δ98Δ (●,○) and Δ78Δ (▲,△). Experiments were carried out in the absence (closed symbols) or in the presence of 10% v/v TFE (open symbols). Fitted curves of the native molar fraction of IFABP (solid line), Δ98Δ (dashed line) or Δ78Δ (dotted line) are plotted at 0 (panel B) and 10% v/v TFE (panel C). Notice that for the dimeric construct Δ78Δ -that has been described to dissociate and unfold concomitantly- the free energies (ΔG° values) of the overall process (expressed in protomer equivalents, see reference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170607#pone.0170607.ref007" target="_blank">7</a>]) are tabulated.</p

Amino acid sequence and schematic representation of the secondary structure elements of IFABP.

<p>Amino acid residues belonging to the hydrophobic core are indicated with dotted lines and depicted with their side-chains in poly-tube representation on the ribbon structure of IFABP (PDB 2IFB). Linear depiction of the abridged variants and of a common early proteolytic fragment (below in light gray) obtained after cleavage by chymotrypsin. The latter is also painted in light gray in the 3D model.</p

ANS binding.

<p>Binding of ANS to IFABP (A), Δ98Δ (B) and Δ78Δ (C). The intensity of fluorescence emission of ANS was measured when bound to proteins at 0 (thick lines) or 10 (thin lines) % v/v TFE in buffer PN8 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170607#sec002" target="_blank">Materials and methods</a>). Spectra obtained in the presence of oleic acid at a 5:1 molar ratio with respect to protein are represented in dash-dot-dot-dash lines.</p