Competitive binding of fatty acids and the fluorescent probe 1-8-anilinonaphthalene sulfonate to bovine beta-lactoglobulin

Abstract

The use of spectroscopy in the study of fatty acids binding to bovine beta-lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty-acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS-BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid-binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.

Figure 1.

Chemical structure of the investigated ligands; fatty acids and ANS (1-8-anilinonaphthalene sulfonate).

Figure 2.

Fluorescence lifetimes of ANS bound to BLG versus fatty-acid concentration at pH 8.3. (□) Palmitic acid; (▵) lauric acid; (⋄) caprylic acid.

Figure 3.

Fractional intensities of ANS decay in the presence of BLG versus fatty-acid concentration at pH 8.3. Different symbols refer to different fractional intensities. (□) f₁, corresponding to the longer bound ANS lifetime; (○) f₂, corresponding to the shorter bound ANS lifetime; (▵) f₃, corresponding to the free ANS lifetime.

Figure 4.

Ratio f₁τ₂/f₂τ₁ = const’ × C_b1 versus fatty-acid concentration at the different investigated pH values: (▪) pH 8.3; (•) pH 7.2; (▴) pH 6.2. Broken lines refer to the fitting functions as defined in the text, Equations 4–6.

Figure 5.

Free energy difference for fatty-acid binding to BLG at pH 8.3 versus the number of methylene groups in the fatty-acid chain.

Figure 6.

Docking solutions obtained from GRID program for interaction of BLG with palmitic, lauric, caprylic acids, and ANS. (A) Superposition of predicted (blue) and experimentally observed (red) positions of palmitic acid within BLG. BLG backbone (PDB code 1bsy) is shown in gray in a ribbon representation. (B) Graphical representation of MIF in the region of palmitic acid binding site. Palmitic acid docking solution is shown in gray. The green and blue contours indicate regions of favorable interaction energies for hydrophobic (energy level = −0.5 Kcal/mole) and carboxyl oxygen (energy level = −6.2 Kcal/mole) probes, respectively. (C) Docking solutions obtained for lauric acid. (D) Docking solutions obtained for caprylic acid. Darker colors correspond to lower energy solutions. (E) Docking solution obtained for ANS. ANS molecule is colored by atom type. K60 and K69 side-chains are displayed in blue.