Structural characterization and the role of EPR spectroscopy for protein-polymer conjugates

Protein-polymer conjugates (PPCs) are an important class of therapeutic macromolecules offering improvements in protein stability, circulation time, and immunogenicity. Despite progress in conjugation strategies and polymer design it remains a challenge to understand how polymer attachment reshapes protein conformation, dynamics, and interactions, and how these features relate to biological function. PPCs are inherently flexible and often heterogeneous systems whose function is dictated by their structural and dynamical arrangement. Accordingly, capturing and understanding such features including conformational dynamics, higher-order structures and molecular interactions, is crucial for modulating their function, particularly at physiologically relevant conditions. This review places PPCs in a macromolecular and structural framework examining how conjugation strategy, grafting density, polymer properties and linker chemistry influence protein structure, interactions and nanoscale organization. Additionally, summarizes the current experimental toolbox used to characterize PPCs in vitro, in cellulo, and in vivo highlighting strengths and limitations to resolve structural heterogeneity and dynamics. Here, Electron Paramagnetic Resonance (EPR) spectroscopy combined with site-directed spin labeling is introduced as a powerful but underutilized approach for probing local dynamics, conformational ensembles and heterogeneity, as well as nanometer scale distances both in solution and in living cells. Integration of EPR with established biochemical and biophysical techniques is expected to strengthen structure-function relationships and support the rational design and optimization of next-generation PPC therapeutics.