(515g) Stabilizing Insulin and Lysozyme Against Thermal Denaturation through Pegylation: Insights from Molecular Dynamics Simulations

Protein-based therapeutics, also known as "biologics," represent one of the most rapidly expanding categories of therapies approved by the FDA. However, the inherent instability of proteins poses a significant challenge to their utilization as standalone therapeutic agents. During storage and administration, proteins may unravel and lose their secondary structure due to various factors, such as fluctuations in temperature, solution pH, shearing forces, and other external stressors. Understanding the relationship between temperature and protein structure is critical in developing effective strategies to prevent protein misfolding and aggregation. Meanwhile, to prolong their lifetime and enhance in vivo performance, many protein drugs currently on the market are conjugated with polyethylene glycol (PEG). PEGylation improves protein therapeutics' stability, solubility, and pharmacokinetic properties, allowing for reduced dosing frequency and potentially minimizing side effects. Despite the widespread use of PEGylation, the specific mechanisms of polymers that stabilize proteins remain largely unknown. A comprehensive understanding of the destabilizing effects triggered by external stressors and the subsequent stabilizing interactions is essential for designing more stable and effective protein therapeutics.
In this study, we employed all-atom molecular dynamics (MD) simulations to examine the protein unfolding process of hen egg-white lysozyme (well-characterized protein) and insulin (widely used therapeutic protein) under elevated temperatures. We first assessed the accuracy of two forcefields - CHARMM36 and Amber ff99SB-ILDN for modeling the high-temperature unfolding process. By accessing the global structural change and local residue level details, we are able to capture the unfolding process and identify residues that degrade first, which we refer to as the proteins' "weak links." Subsequently, we conjugated the proteins with PEG to understand how PEG helps preserve the protein structure at higher temperatures. We find that PEG displaces water molecules from the protein, leading to the preservation of native contact and the stabilization of the secondary structure of the protein. Results from this work will help us design novel polymer conjugates for protein therapeutic applications.