Conserved Protein–Polymer Interactions across Structurally Diverse Polymers Underlie Alterations to Protein Thermal Unfolding

[Image: see text] Protein–polymer conjugates are widely used in many clinical and industrial applications, but lack of experimental data relating protein–polymer interactions to improved protein stability prevents their rational design. Advances in synthetic chemistry have expanded the palette of polymer designs, including development of nonlinear architectures, novel monomer chemical scaffolds, and control of hydrophobicity, but more experimental data are needed to transform advances in chemistry into next generation conjugates. Using an integrative biophysical approach, we investigated the molecular basis for polymer-based thermal stabilization of a human galectin protein, Gal3C, conjugated with polymers of linear and nonlinear architectures, different degrees of polymerization, and varying hydrophobicities. Independently varying the degree of polymerization and polymer architecture enabled delineation of specific polymer properties contributing to improved protein stability. Insights from NMR spectroscopy of the polymer-conjugated Gal3C backbone revealed patterns of protein–polymer interactions shared between linear and nonlinear polymer architectures for thermally stabilized conjugates. Despite large differences in polymer chemical scaffolds, protein–polymer interactions resulting in thermal stabilization appear conserved. We observed a clear relation between polymer length and protein–polymer thermal stability shared among chemically different polymers. Our data indicate a wide range of polymers may be useful for engineering conjugate properties and provide conjugate design criteria.