Effect of molecular crowding on native Cytochrome C: A Time-Dependent study

The interior of the cell is crowded with various types of macromolecules that can effectively interact with proteins and alter their native conformation, consequently resulting in protein aggregation. Protein aggregation has been linked to various pathological conditions such as Alzheimer’s and Parkinson’s disease. In this study, we analyze the effect of macromolecular crowding on the native structure of Cytochrome C using polyethylene glycol of different molecular weights (PEG 4000 and PEG 6000) at a constant concentration of 200 mg/mL. Time-dependent conformational alterations were analyzed over a 32-hour incubation period at room temperature using turbidity, thioflavin T fluorescence (ThT), Soret absorption and fluorescence microscopy. The notable increase in turbidity at 350 nm suggested crowder-induced aggregation. Increased ThT fluorescence further confirmed the formation of amyloid-like fibrillar assemblies in the presence of PEG. Furthermore, the kinetic analysis revealed a nucleation-dependent mechanism of cytochrome C aggregation, specified by an initial lag phase of 8 hours, followed by a rapid growth phase, and finally a saturation phase at 32 hours, marking the presence of mature fibril-like structures. The red shift of 4 and 9 nm in the presence of PEG 4000 and PEG 6000, with increased Soret absorbance, confirmed the exposure of the heme group to the solvent as a result of structural distortions. Fluorescence microscopy confirms the formation of fibrillar assemblies by direct visualization, with a more pronounced fibrillation in the presence of PEG 6000. Altogether, these results exhibit that macromolecular crowding alters the native structure of cytochrome C and drives the protein toward fibril formation, suggesting a stronger aggregation-promoting effect of higher molecular weight crowders along with increased incubation time. Therefore, this study emphasizes the importance of the size of the crowding agent and the time of incubation in promoting the conformational perturbations of globular proteins, providing insights into protein aggregation in the crowded microenvironment of the cell.