Effects of Macromolecular Crowding on Oligomeric Protein Unfolding: Case Study with Human Co-Chaperonin Protein 10 (cpn10)

Abstract

In vivo, proteins fold and associate in highly crowded environments. Previousexperiments on several monomeric proteins have shown that macromolecular crowding stabilizes towards heat perturbation and also can modulate native-state structure. To assess the effects of macromolecular crowding on unfolding of an oligomeric protein, where denaturation involves both unfolding and dissociation, we here tested the effects of the synthetic crowding agent Ficoll 70 on cpn10, a heptameric protein consisting of seven identical β-barrel subunits assembling into a ring. The stability of the heptamer is dominated by subunit-subunit interactions and the individual subunits have low stability on their own. Using far-UV circular dichroism (CD), tyrosine fluorescence and cross-linking experiments, we investigated thermal and chemical stability without and with crowding agent. We find that cpn10 is thermally stabilized by about 5°C in 300 mg/ml Ficoll 70 as compared to in buffer. Whereas there is a large effect of Ficoll 70 on the urea-induced unfolding reaction (midpoint shifts by 2 M), a smaller effect is seen when GuHCl is used as the chemical denaturant (midpoint shifts by 0.6 M). Kinetic unfolding experiments show that the higher equilibrium stability found for cpn10 in crowded conditions can be explained in part by slower unfolding rates.