Abstract
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.
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Acknowledgements
This research was supported by National University of Ireland Galway and Science Foundation Ireland grants 10/RFP/BIC2807 to P.B.C. and 12/IA/1255 to M. Caffrey (postdoctoral funding to L.V.). We acknowledge the Diamond Light Source (UK) for beam-time allocation and the staff of beam line I24 for assistance with the data collection. We thank M. Vignoles and G. Manning for mass spectrometry data. R. Pastor and H. Lee are acknowledged for helpful discussions and for providing a coarse-grained model of PEG 5,000.
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P.B.C. devised the research. G.C. performed the experiments. L.V. collected the synchrotron data and helped with data processing and analysis. G.C. and P.B.C. analysed the data. P.B.C. wrote the paper. All the authors commented on the manuscript.
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Cattani, G., Vogeley, L. & Crowley, P. Structure of a PEGylated protein reveals a highly porous double-helical assembly. Nature Chem 7, 823–828 (2015). https://doi.org/10.1038/nchem.2342
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DOI: https://doi.org/10.1038/nchem.2342
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