Mechanistic insights into the stabilization of srcSH3 by PEGylation

Protein PEGylation (attaching PEG chains to proteins) has been widely used in pharmaceuticals and nanotechnology. Although it is widely known that PEGylation can increase the thermodynamic stability of proteins, the underlying mechanism remains elusive. In this Article, we studied the effect of PEGylation on the thermodynamic and kinetic stability of a protein, SH3. We show that the thermodynamic stability of SH3 is enhanced upon PEGylation, mainly due to the slowing of the unfolding rate. Moreover, PEGylation can decrease the solvent-accessible surface area of SH3, leading to an increase of the m-value (the change in free energy with respect to denaturant concentration, which is a measure of the transition cooperativity between corresponding states). Such an effect also causes an enhancement of the thermodynamic stability. We quantitatively measured how the physical properties of PEG, such as the molecular weight and the number of PEGylation sites, affect the stabilization effect. We found that the stabilization effect is largely dependent on the number of PEGylation sites but only has a weak correlation with the molecular weight of the attached PEG. These experimental findings inspire us to derive a physical model based on excluded volume effect, which can satisfactorily describe all experimental observations. This model allows quantitatively calculating the free energy change upon PEGylation based on the change of water excluded zone on the protein surface. Although it is still unknown whether such a mechanism can be extended to other proteins, our work represents a key step toward the understanding of the nature of protein stabilization upon PEGylation.