Structure of a PEGylated protein reveals a highly porous double-helical assembly.

PEGylated proteins are a mainstay of the biopharmaceutical industry. Although the use of poly(ethylene glycol) (PEG) to increase particle size, stability and solubility is well-established, questions remain as to the structure of PEG-protein conjugates. Here we report the structural characterization of a model β-sheet protein (plastocyanin, 11.5 kDa) modified with a single PEG 5,000. An NMR spectroscopy study of the PEGylated conjugate indicated that the protein and PEG behaved as independent domains. A crystal structure revealed an extraordinary double-helical assembly of the conjugate, with the helices arranged orthogonally to yield a highly porous architecture. Electron density was not observed for the PEG chain, which indicates that it was disordered. The volume available per PEG chain in the crystal was within 10% of the calculated random coil volume. Together, these data support a minimal interaction between the protein and the synthetic polymer. Our work provides new possibilities for understanding this important class of protein-polymer hybrids and suggests a novel approach to engineering protein assemblies.