(169ad) Customized Random Heteropolymer Design to Improve Protein Stability Using Molecular Dynamics Simulations

Protein activity is known to decline outside their native environments, posing significant challenges in various fields. Polymers are promising materials used to either conjugate or mix with proteins to boost their function when subjected to changes in environmental factors. The recent development of random heteropolymers (RHPs) containing four distinct methacrylate-based monomers provides a promising new avenue of soft materials that possess the ability to shield proteins when mixed even without chemical conjugation, contributing to the improved activity and stability of proteins outside their native environment. While the controlled statistical composition design of RHPs has been proven to be effective, questions still remain regarding the ultimate efficiency of these conjugates, and whether designing custom polymers for different proteins might be a better alternative. Meanwhile, even with four types of monomers, the design space of RHPs is enormous. Given the broad design space of these materials, a mechanistic understanding of the stabilization would be beneficial for the rational design to fit different proteins.

In this study, we employ molecular dynamics simulations to assess the capacity of RHPs to improve lysozyme stability at high temperatures and understand the stabilization effect of the RHPs on proteins in the protein/RHPs mixture. Starting with a previously identified RHP composition – methyl methacrylate (MMA), oligo(ethylene glycol) methacrylate (OEGMA), 2-ethylhexyl methacrylate (EHMA), and 3-sulfopropyl methacrylate potassium salt (SPMA) in a 50:25:20:5 mole ratio and a polymerization degree of 80 – we generated simulated polymer sequences via the Composition Drift program. The stability of the protein in the mixture was evaluated using a high-temperature unfolding protocol. We then monitor the structural evolution of lysozyme at high temperatures. A promising stabilization effect was observed for lysozyme with a relatively stable and long-lasting secondary structure. Further analysis of the contacts between the protein residues and RHP monomers gives insight into the stabilization mechanism, enabling us to strategically design RHPs. Leveraging existing knowledge of protein’s weak point, we further tailor RHP designs specifically for lysozyme, opening avenues for customized protein stabilization strategies.