Investigating Improved Protein Stability in Random Heteropolymer/Protein Mixtures Using Molecular Dynamics Simulations

Yinhao Jia, Janani Sampath

Department of Chemical Engineering, University of Florida

Protein function diminishes outside their native environments, posing substantial challenges across different fields. Polymers have been shown to enhance protein performance under varying environmental conditions and can be conjugated or mixed with proteins. Recent advances in the development of random heteropolymers (RHPs), made up of four different methacrylate-based monomers, offer a new avenue in protein stabilization by simply forming a polymer-protein hybrid without chemical conjugation, giving proteins improved activity and stability under non-native environments. Though the design of RHPs with controlled statistical monomer composition has shown a lot of promise, it is still unclear whether these sequences are the most effective option or whether custom-designed RHPs for different proteins would yield better protection. Meanwhile, even with only four monomer types, the latent space for RHPs design is vast. A deeper mechanistic understanding of how these polymers stabilize proteins could facilitate rational design of RHPs to accommodate diverse proteins.

In this study, we perform molecular dynamics simulations to evaluate how RHPs can maintain the native structure of lysozyme at elevated temperatures and to elucidate how RHPs stabilize proteins under these conditions. We begin with a known RHP composition and ratio – 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 molar ratio with a polymerization degree of 80 – and produce simulated polymer sequences using the Composition Drift software. We then assess the protein's stability using a high-temperature unfolding protocol and track the structural changes of lysozyme. The simulations reveal a significant stabilizing effect on lysozyme, with RHPs facilitating protein secondary structure at elevated temperatures. Further examination of the interactions between protein residues and RHP monomers provided insights into the stabilization mechanisms, suggesting potential pathways for strategic RHP design. Future exploration will utilize the existing knowledge of protein's weak points to tailor the RHP designs for larger therapeutic proteins.