Insights into Protein Unfolding under pH, Temperature,and Shear Using Molecular Dynamics Simulations

Protein biologics hold immense potential in therapeutic applications, but their ephemeral nature has hindered widespread application. The effects of different stressors on protein folding have long been studied, but whether these stressors induce protein unfolding through different pathways remains unclear. Here, we conduct all-atom molecular dynamics simulations to investigate the unfolding of bovine serum albumin (BSA) under three distinct stressors: high temperature, acidic pH, and shear stress. Our findings reveal that each stressor induces unique unfolding patterns in BSA, indicating stressor-specific unfolding pathways. Structural analyses show that high temperature significantly disrupts the protein’s secondary structure, while acidic pH causes alternations in the tertiary structure, leading to domain separation. Shear stress initially perturbs the tertiary structure, initiating structural rearrangements, which causes a loss of secondary structure similar to temperature. These distinct unfolding behaviors suggest that different stabilization strategies are required to enhance protein stability under different denaturation conditions.