No effect of covalently linked poly(ethylene glycol) chains on protein internal dynamics

Abstract

Poly(ethylene glycol) or PEG is a hydrophilic polymer that covalently linked to therapeutical proteins may significantly increase their pharmacological properties. Despite the extensive production of PEG-conjugated proteins the effects of the polymer on the protein structure and dynamics is poorly understood, making the production of active biomaterials a largely unpredictable process. The present investigation examines the effects of 5 k and 20 k PEG on the internal flexibility of Ribonuclease T1, the mutant C112S of azurin from Pseudomonas aeruginosa, alcohol dehydrogenase and alkaline phosphatase, native and Zn-depleted. These systems encompass structural domains that range from rather superficial, flexible sites to deeply buried, rigid cores. The approach is based on three sensitive parameters related to the phosphorescence emission of internal Trp residues, namely, the intrinsic room-temperature phosphorescence lifetime ( τ 0) that reports on the local flexibility of the protein matrix around the chromophore and the bimolecular rate constant ( k q) for the quenching of phosphorescence by O 2 and by acrylamide in solution, which are related to the diffusion of these solutes through the protein fold. The results obtained by these three independent, intrinsic probes of protein structure-dynamics concur that mono-PEGylation does not detectably perturb the conformation and dynamics of the protein native fold, over a wide temperature range. The implication is that protein motions are essentially not coupled to the polymer and that adverse effects of chemical modification on biological function are presumably owed to steric hindrance by PEG units blocking the access to sites critical for molecular recognition.