Polyethylene Glycol Size and Protein Stability

Abstract

Concentrated solutions of synthetic polymers are often used to stabilize proteins, yet there are few systematic studies on the crowding effect of polymer molecular weight. Crowding effects arise from two types of crowder-test protein interactions: hard-core repulsions and soft interactions. Simple hard-core repulsions are only stabilizing, while soft (chemical) interactions can be stabilizing (if repulsive) or destabilizing (if attractive). Most synthetic polymers are assumed to interact with test proteins via hard-core repulsions, but polymers are often not inert. We investigated the temperature dependence of protein stability in polyethylene glycols as a function of polymer size and concentration. We used the 7-kDa metastable N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) as the test protein. SH3 was fluorine labeled at its single tryptophan enabling observation of the folded and unfolded species using 19F nuclear magnetic resonance spectroscopy. The temperature dependence of the free energy of unfolding (ΔGu°′) was used to obtain the entropy (ΔSu°′) and enthalpy (ΔHu°′) to aid in the separation of hardcore repulsions and soft interactions effects.