Spectroscopic studies on the stability and nucleation-independent fibrillation of partially-unfolded proteins in crowded environment

Abstract

Fibril formation of globular proteins is driven by attaining an appropriate partially-unfolded conformation. Excluded volume effect exerted by the presence of other macromolecules in the solution, as found in the cellular interior, might affect the conformational state of proteins and alter their fibril formation process. The change in structure, stability and rate of fibril formation of aggregation-prone partially-unfolded states of lysozyme (Lyz) and α-lactalbumin (ALA) in the presence of different sizes of polyethylene glycol (PEG) is examined using spectroscopic methods. Thermal denaturation and far-UV CD studies suggest that Lyz is stabilized by PEGs and the stability increases with increasing concentration of PEGs. However, the stability of ALA depends on the size and concentration of PEG. The change in enthalpy of unfolding indicates the existence of soft-interactions between the proteins and PEG along with excluded volume effect. Fibrillation rate of Lyz is not significantly altered in the presence of lower concentrations of PEGs suggesting that the crowding effect dominates the viscosity-induced retardation of protein association whereas at higher concentrations the rates are reduced. In case of ALA, the rate of fibrillation is drastically reduced; however, there is a marginal increase with the increasing concentration of PEG. The results suggest that the fibril formation is influenced by change in initial conformation of the partially-unfolded states of the proteins and their stability in the presence of the crowding agent. Further, the size and concentration of the crowding agent, and the soft-interaction between the proteins and PEG also affects the fibrillation.