Resveratrol Effect on α-Lactalbumin Thermal Stability

Abstract

The effect of resveratrol (RESV) on α-lactalbumin (α-LA) thermal stability was evaluated using differential scanning calorimetry (DSC), circular dichroism (CD) and dynamic light scattering (DLS) measurements. Complementary information offered by molecular docking served to identify the binding site of the ligand on the native structure of protein and the type of interacting forces. DSC thermograms revealed a double-endotherm pattern with partial overlapping of the two components. The most relevant effect of RESV is manifested in the narrowing of the protein thermal fingerprint: the first process (peak temperature T₁) is shifted to higher temperatures while the second one (peak temperature T₂) to lower values. The CD data indicated partial conformational changes in the protein non-α-helix domain at T₁, resulting in a β-sheet richer intermediate (BSRI) with an unaffected, native-like α-helix backbone. The RESV influence on this process may be defined as slightly demoting, at least within DSC conditions (linear heating rate of 1 K min^-1). On further heating, unfolding of the α-helix domain takes place at T₂, with RESV acting as a promoter of the process. Long time incubation at 333 K produced the same type of BSRI: no significant effect of RESV on the secondary structure content was detected by CD spectroscopy. Nevertheless, the size distribution of the protein population obtained from DLS measurements revealed the free (non-bound) RESV action manifested in the developing of larger size aggregates.

Keywords: CD; DSC; aggregates; lactalbumin; resveratrol; unfolding.

Figure 1

DSC scans of α-LA denaturation in the absence and presence of RESV at different protein:ligand molar ratios in 30 mM phosphate buffer, pH 7.2.

Figure 2

The CD spectra of α-LA after incubation at (A) 24 h at 277 K and (B) for different time intervals at 333 K, in the absence and presence of different concentration of RESV.

Figure 3

The secondary structure content of α-LA at different incubation time intervals and temperatures, in the absence and presence of RESV.

Figure 4

CD thermal denaturation curves of α-LA, pH 7.2, in the absence and presence of RESV. Samples were heated at the scan rate of 1 K min⁻¹, and CD values (±standard deviation) were monitored at 222 nm.

Figure 5

Hydrodynamic diameter and polydispersity index for different RESV: α-LA molar ratios after 24 h’ incubation at 277 K.

Figure 6

Molecular docking of RESV and α-LA; (A) RESV is presented with a stick-and-balls model and the protein as solid ribbon. (B) The 2D representation of RESV–α-LA complex; the close amino acid residues are presented in green, dashed lines represent intermolecular interactions of different origin (alkyl–alkyl—pink lines; hydrogen bond—green line; van der Waals interactions—light green lines).