Solvent Reorganization in Stabilized Protein-Polymer Conjugates Visualized by 2D IR and NMR Spectroscopy

PEGylation is essential for effective function of biologics, shielding them from rapid degradation and clearance in the complex environment of the human body. Despite its significance, a mechanistic understanding of PEGylation’s role in enhancing protein stability is incomplete, limiting the ability to design PEGylated proteins with predictable properties. Solvation, a well-known driving force in protein folding and stability, is hypothesized to play a central role in protein stabilization via PEGylation, but molecular mechanisms underlying solvent-driven stabilization are not well understood. Here, we investigated solvent dynamics and the interactions of solvent with the PEGylated carbohydrate recognition domain of human Galectin-3 (Gal3C) in aqueous solutions. Two-dimensional infrared (2D IR) spectroscopy, which captures subpicosecond molecular ensembles, revealed polymer-length dependent differences in protein dynamics and solvent dynamics for PEGylated Gal3C. Slower solvent dynamics correlated with increased conjugate thermal stability. Complementing these data, multi-dimensional NMR spectroscopy provided evidence that Gal3C conjugated to longer PEG forms a non-covalent interaction “shroud”, which correlated with changes in dynamics of the solvent and protein backbone. Molecular dynamics (MD) simulations supported an interpretation of the experimental results that PEGylation did not reduce the protein’s solvent accessible surface area. The integration of these data challenge the idea that PEGylation stabilizes conjugated proteins by dehydrating a protein’s surface. Instead, these data support a mechanism whereby PEGylation improves protein stability by stabilizing the protein’s solvation shell. These insights offer guidance for optimizing polymer length to achieve the desired thermal stability in biologics.