The covalent attachment of poly(ethylene glycol) (PEG) to a protein surface (known as PEGylation), has been demonstrated to increase the serum half-life of therapeutic proteins by reducing kidney clearance and immunogenicity and by protecting against proteolysis. Theses beneficial effects could be further enhanced if PEGylation consistently increased protein conformational stability (i.e. the difference in free energy between the folded and unfolded states). However, the effects of PEGylation on protein conformational stability are unpredictable; PEGylation has been reported to increase, decrease, or have no effect on the conformational stability of medicinal proteins.This thesis details the results of two studies aimed at discovering the structural determinants which influence the thermodynamic impact of PEGylation on the WW domain, a small model protein. Chapter 1 is a brief introduction to protein therapeutics and protein PEGylation. Chapter 2 describes a study which demonstrates that the thermodynamic impact of PEGylation is strongly dependent on the site to which PEG is conjugated. The studies described in Chapter 3 elaborate on this site dependence, and demonstrate that PEG stabilizes the WW domain through interactions with the surface of the folded peptide, and that two factors – the orientation of the PEG chain (relative to the protein surface) and the identity of nearby side chains – play a critical role in determining the thermodynamic impact of PEGylation.



College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry



Date Submitted


Document Type





PEGylation, Therapeutic Proteins, Thermodynamic Stability, Circular Dichroism, beta-sheet, D-Amino Acids