Proline concentration driven thermostability and hydration properties of ubiquitin

Abstract

Among the amino acids, proline belongs to the category that acts as a promising excipient for biotherapeutic protein stabilization under environmental stress, prevention for protein aggregation, fibrillation, etc. Understanding the molecular mechanisms by which proline solutions affect protein structures under thermal stress is essential for applications in bioinformatics, pharmaceutical industries, and the design of new excipients. This study aimed to elucidate the role of proline solution on protein structure and dynamics under thermal stress conditions. Atomistic molecular dynamics simulations explored how varying proline concentrations influence a model protein ubiquitin’s thermal unfolding or structural stability. The results revealed that at higher proline concentrations (≥2.5 M) the native-like folded state of ubiquitin is maintained through the structural contacts over the simulation period. In contrast, at lower concentrations (2 M), the stability time is reduced, and the pure water fails to prevent thermal stabilization of the protein and promote unfolding. Free energy profiles and unfolding pathways identified different intermediate states, suggesting that proline modulates protein stability, alters the solvation dynamics, and affects energy barriers for structural transitions compared to that in pure water. This study highlights the concentration-driven dual role of proline solutions; its preferential exclusion from the protein surface enhances hydration, while its self-association impacts the dynamics and structuring of water in the solvation shell. These findings highlight the concentration-dependent osmoprotective behavior of proline and its implications for protein stability, offering insights relevant to designing stabilization strategies for biomolecules under stress conditions.