Impact of polyethylene glycol molecular weight on C-phycocyanin stability

Protein stability depends on both subunit folding and inter-subunit interactions, which are modulated by environmental conditions and cosolutes. This study examined how polyethylene glycols (PEG) of different molecular weights affect the structural stability of the multimeric protein C-phycocyanin (CPC). Using differential scanning calorimetry (DSC), fluorescence spectroscopy, and rheology, we analyzed CPC in the presence of PEG 4 000 g/mol (PEG4) and PEG 35 000 g/mol (PEG35). Fluorescence spectroscopy showed that both PEGs increased the emission intensity of CPC without shifting the emission maximum, indicating changes without major alterations of the global structure. DSC revealed a marked decrease in the enthalpy of unfolding, particularly with PEG35, despite only minor changes in denaturation temperatures. Rheology demonstrated effects of CPC on PEG solution viscosity. The relative increase of the viscosity of PEG4 solution upon addition of CPC is almost two times larger than that of the PEG35. These results suggest that the smaller size and the lower hydration of PEG4 allow it to penetrate more ultimately the hydration shell of CPC, while the larger molecular weight and higher hydration number of PEG35 induce protein–protein association and loss of solubility. Altogether, these results show that PEG molecular weight governs CPC stability: PEG4 may destabilize CPC via crowding and hydration-shell disruption, whereas PEG35 likely reduces CPC solubility through depletion-driven aggregation without altering its folded structure.