| Summary: | The formation of nucleated clusters of β-Lactoglobulin (β-Lg) under alcohol-induced denaturation has been studied by applying spectroscopic and scattering techniques. The concentration dependence of the cluster formation process of β-Lg in 50% (v/v) buffer-ethanol solutions was study at pH 7 and 22°C. Near UV circular dichroism results indicated that the tertiary structure of the native protein was completely lost upon addition of ethanol. Changes in the secondary structure were characterised by a drastic increase of the α-helical content (and decrease of β-sheets content) immediately after ethanol addition. The percentages of these two types of secondary structure evolved further during the 10 days of incubation at room temperature attained an equilibrium state which differs from the secondary structure found in the native protein: while dimeric β-Lg in its native state has a majority content of β-sheets, clusters presented a majority content of α-helix. Similar results on the evolution of the secondary structure of the protein upon ethanol addition were found with attenuated total reflection infrared spectroscopy (ATR-IR). ThT-binding fluorescence spectroscopy revealed that the kinetics of structural rearrangement follows a nucleation dependent growth mechanism for the lowest protein concentration studied here (0.5 mg/ml and 1 mg/ml) where a lag phase, an exponential growth, and a plateau phase could be clearly identified and quantified. For higher protein concentrations (from 4 to 40 mg/ml) the structural transition kinetics of the protein was much faster and no lag phase was observed. CD and fluorescence results suggest that the high amount of β-helix structure formed immediately after ethanol addition for the lowest concentrated samples acts as an activation barrier and slows down the nucleation process leading to a distinct lag phage. Kinetic of cluster growth was monitored by dynamic light scattering, indicating that clusters grew rapidly after ethanol addition reaching a constant hydrodynamic radius shortly after the organic solvent was present in solution. Correlation with spectroscopic results revealed that structural changes were still taking place even after the cluster size attained its equilibrium value for high concentrations of protein (10 and 40 mg/ml). DLS results were in good agreement ii with those obtained by small angle X-ray scattering (SAXS) and were consistent with compact sphere-like structure of clusters. Our results indicate that clusters formed in the presence of 50% (v/v) of ethanol corresponded to a thermodynamically controlled intermediate on the β-Lg aggregation pathway. However, the internal structure of these clusters must contain suitable β-sheet arrangements with enhanced binding of the ThT dye to explain the fluorescence intensity increase found when the clusters were formed.
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