PEG mediated destabilization of holo α-lactalbumin probed by in silico and in vitro studies: deviation from excluded volume effect

Abstract

Crowded and confined macromolecular milieus surround proteins, and both are stabilizing if the nature of the interaction between crowder and proteins are considered hard-core repulsive interactions. However, non-specific chemical interactions between a protein and its surroundings also play a significant role and the sum effect of both hard-core repulsion and soft interaction balances the overall effect of crowding/confinement. Previous studies showing the effect of polyethylene glycol (PEG) on protein and nucleic acid may be interpreted as either primarily excluded volume effect or, in some cases, chemical effect by changing solvent properties. In case of destabilizing interactions, charge-charge and hydrophobic contact have to gain more attention. For instance, in vitro and in vivo studies using protein as crowding agent revealed the destabilization of proteins induced by charge-charge interactions. To investigate the effect of PEG 10 kDa on holo α-lactalbumin (holo α-LA), structure and thermal stability of the protein were measured at different pH values using several techniques. Structural characterization by Trp-fluorescence, near-UV CD and far-UV measurements at different pH values clearly shows perturbation of tertiary and secondary structure of holo α-LA by PEG 10 kDa. Furthermore, the dynamic light scattering measurement shows that the protein is homogeneous under all experimental conditions. Analysis of the heat-induced denaturation profile in the presence of the crowder shows destabilization of the protein in terms of T m (midpoint of denaturation) and ΔG D 0 (Gibbs free energy change at 25 °C). To evaluate the interaction of PEG 10 kDa with holo α-LA and stability of PEG-α-LA complex, docking and molecular dynamic simulation were carried out for 100 ns. Communicated by Ramaswamy H. Sarma