Polyethylene glycol induced structural modulation of holo alpha-lactalbumin: In vitro and in vivo approach towards protein stability

Macromolecular crowding is driven by hard-core repulsions and soft interactions, may have both the stabilizing and destabilizing effects on protein folding and stability, which has gradually gained attention in recent years. Here, we investigated the impact of polyethylene glycol (PEG), a polymer of ethylene glycol and a constituent of many drugs and eye drops on the structure and stability of holo alpha-lactalbumin (alpha-LA). Results showed that there was a substantial alteration in the secondary structure of holo alpha-LA. Further, it was found that PEG dis-rupted the tertiary structure. In holo alpha-LA, PEG has been found to create a molten globule state, where the intermediate state contains hydrophobic patches and has a greater hydrodynamic volume than the native pro-tein. Additionally, at the physiological pH, isothermal titration calorimetry revealed a significant binding be-tween holo alpha-LA and PEG 20,000 Da. We observed that while researching macromolecular crowding, it is extremely important to consider protein binding with crowder and other soft interactions carefully. In this scenario, destabilizing protein-crowder interactions dominated the stabilizing exclusion volume effect.

Automated summary

Macromolecular crowding, driven by hard-core repulsions and soft interactions, has both stabilizing and destabilizing effects on protein folding and stability, which has recently gained attention. This study investigated the impact of polyethylene glycol (PEG) on the structure and stability of holo alpha-lactalbumin (alpha-LA), and found significant alterations in secondary and tertiary structure. PEG created a molten globule state in alpha-LA, with hydrophobic patches and a larger hydrodynamic volume. Isothermal titration calorimetry revealed significant binding between holo alpha-LA and PEG 20,000 Da at physiological pH. Protein-crowder interactions should be carefully considered in macromolecular crowding studies, as destabilizing interactions dominated stabilizing exclusion volume effects.