Deciphering the Unfolding Pathway of Bovine Serum Albumin Under Varying External Stressors: Insights from Molecular Dynamics Simulation

The therapeutic potential of proteins has revolutionized the treatment of a wide range of diseases, from diabetes to cancer. However, the effectiveness of these therapeutics is often compromised by the sensitivity of proteins to environmental factors during manufacturing, delivery, and administration. Environmental variables such as pH, temperature, and shear force can cause proteins to destabilize and unfold, leading to a loss of functionality and therapeutic efficacy. To address this challenge, it is crucial to engineer proteins with enhanced stability. A key aspect of this engineering process is understanding how proteins respond to external stressors and identifying the specific regions within the protein that need to be targeted to enhance stability. Thus, deciphering the mechanisms by which a stressor destabilizes a protein is pivotal for the targeted design of more stable therapeutic proteins.

In this study, we employed all-atom molecular dynamics simulations to elucidate the unfolding pathway of Bovine Serum Albumin (BSA) under varying external stressors, including temperature, pH, and shear stress. We characterized global changes in the protein during denaturation using a range of structural metrics, including root-mean-square deviation (RMSD), hydrogen bond number, and hydrophobic solvent accessible surface area (SASA), and validated these results through secondary structure assessment and representative simulation trajectory snapshots. Additionally, we divided BSA into three domains and tracked the sequential order of their destabilization to further explore the unfolding pathway. Our results revealed distinct denaturation patterns across different stressors, providing insights into the underlying mechanisms that can be leveraged to design novel strategies for enhancing the stability of therapeutic proteins.