Molecular crowding induced loss of native conformation and aggregation of α -chymotrypsinogen A

Conformational characterization of the folding and unfolding pathway is the key to studying protein-macromolecular interactions. Also, we know that unfolding mediated protein aggregation leads to many disorders like Alzheimer's, Parkinson's etc. of which complete cure and effective drug development have been a milestone. It will be difficult to create effective treatments against protein aggregation-related disorders without a thorough grasp of unfolding and aggregation mechanisms. Because a variety of environmental conditions influence protein native conformation in vivo and in vitro to varying degrees, it's crucial to investigate the impact of diverse substances on the protein unfolding mechanism. Keeping the above situation in mind we carefully designed in vitro experiments to analyze the molecular crowding effect on the conformational changes taking place in α-chymotrypsinogen A ( α Cgn-A) at pathologically elevated temperature. Molecular crowding effect analysis, was done in the presence of two globular proteins i.e. human lysozyme (HLZ), hemoglobin (Hb) and 2, 2, 2-trifluoroethanol (TFE). Increasing concentrations (10–50%, v/v) of HLZ, Hb, and TFE were incubated along with the native α Cgn-A for 48 h at 40 °C (pH 7.4). In vitro analyses like ThT (Thioflavin T) fluorescence, ANS (8-anilino-1-naphthalenesulfonic acid) fluorescence, CR (congo red) assay etc. was performed to analyze the conformational changes in the native α Cgn-A and TEM (Transmission electron microscopy) was used for the visual characterization of the incubated samples. We observed that increasing the molecular crowding from 10 to 50% (v/v) at pathologically elevated temperature leads to a loss in the native protein conformation causing protein aggregation. The effect of molecular crowding leading to aggregation of α Cgn-A was more pronounced in the presence of 50% (v/v) Hb than HLZ at the same concentration, indicating that the effect is proportional to the molecular weight of the protein.