Self-Assembly of Synthetic and Biological Polymeric Systems of ...

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Figure 4: Normalized maximum ThT fluorescence intensities of HSA samples incubated at different ethanol concentrations in the mixed
solvent at a) pH 7.4 and 65 ºC, b) pH 7.4 and 25 ºC, c) pH 2.0 and 65 ºC and d) pH 2.0 and 25 ºC. Normalization is made with respect to
the maximum ThT fluorescence observed at pH 7.4 and 65 ºC.
30 ethanol concentrations larger than 50% (v/v) the β-sheet content
increases, as denoted from the progressive shift of the minimum
in CD spectra to 215 nm, which is characteristic of β-strand
formation. In contrast, at acidic pH and 25 ºC (Figure 3d), the
structural content of protein samples at ethanol concentrations
35 lower than 60% (v/v) remains almost invariable. Since HSA
molecules are in a starting acid-denaturated state, alcohol
stabilizes the α-helix conformation of the acid-unfolded protein
monomers by minimizing the exposure of the peptide backbone.
In particular, at alcohol concentrations < 40% (v/v),
40 intermolecular interactions are disfavored by suppression of the
strong aggregate-stabilizing effect of negatively charged residues,
still resulting in an effective electrostatic repulsion between
positively charged chains. Hence, under these conditions alcohol
stabilizes the molten-globule state of HSA during incubation, and
only protein clusters can be formed. 63,64 45
The presence of these
clusters, confirmed by the presence of a small peak at relatively
low aggregate sizes (∼20-30 nm) (see Figures 2d), implies some
increase in light scattering from solution as shown previously
and, thus, possibly originates the slight decrease in ellipticity
50 observed in Figure 3d. Protein molecules experience additional
structural rearrangements at ethanol concentrations between 50-
90% (v/v) when the polarity of the medium is drastically
changed. This involves a decrease in α-helix structure and
formation of β-strands that, as observed from light scattering
55 data, are prone to aggregate. FT-IR measurements corroborate the
structural changes undergone by protein molecules depicted by
CD data (for additional comments see ESI).
Although reliable in detecting β-strands and probing hydrogen
bonding between them, CD and FT-IR spectroscopies fail to
60 discriminate between amorphous and ordered aggregates. Since
ThT dye strongly emits when bound to amyloid-like material
rather than to amorphous aggregates, we conducted ThT
fluorescence measurements of HSA samples under the different
solution conditions in order to determine the presence of ordered
65 aggregates (amyloid-like fibrils) in solution. Experimental data
were background corrected for solvent and monomeric protein
contributions and normalized to the largest value attained, i.e., at
pH 7.4 and 65 ºC at 70 % (v/v) ethanol. Figure 4 confirms that βsheet
rich structures (fibrils as shown by TEM pictures below) are
70 formed under all conditions except at acidic pH and 25 ºC at
alcohol concentrations below 60% (v/v). At pH 7.4 and 65 ºC
(Figure 4a), the amount of fibrils progressively increases up to an
ethanol content of 70% (v/v), and then decreases. This decrease
in ThT fluorescence can be associated with the presence of
75 mature fibril association/aggregation, as confirmed by TEM (see
below), which may reduce the protein exposed surface area and
the number of available ThT binding sites. 65 In contrast, at acidic
conditions (Figure 4c) the maximum level of fibril formation
takes place at the largest ethanol concentrations (70-90% v/v). On
80 the other hand, the temporal evolution of ThT binding confirms
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