Retinoic acid binding properties of the lipocalin member β-lactoglobulin studied by circular dichroism, electronic absorption spectroscopy and molecular modeling methods

Abstract

Interaction between the Vitamin A derivative all- trans retinoic acid and the lipocalin member bovine β-lactoglobulin (BLG) was studied by circular dichroism (CD) and electronic absorption spectroscopy at different pH values. In neutral and alkaline solutions achiral retinoic acid forms a non-covalent complex with the protein as indicated by the appearance of a negative Cotton effect around 347 nm associated to the narrowed and red shifted π–π ∗ absorption band of the ligand. The induced optical activity is attributed to the helical distortion of the conjugated chain caused by the chiral protein binding environment. As the disappearing CD activity showed in the course of CD–pH titration experiment, retinoic acid molecules dissociate from BLG upon acidification but this release is completely reversible as proved by the reconstitution of the CD and absorption spectra after setting the pH back to neutral. This unique behavior of the complex is explained by the conformational change of BLG (Tanford transition) which involves a movement of the EF loop at the entrance of the central cavity from open to closed conformation in the course of pH lowering. From these results it was inferred that retinoic acid binds within the hydrophobic calyx of the β-barrel.