Quantification of Excluded Volume Effects on the Folding Landscape of Pseudomonas aeruginosa Apoazurin In Vitro

Proteins fold and function inside cells that are crowded with macromolecules. Here, we address the role of the resulting excluded volume effects by in vitro spectroscopic studies of Pseudomonas aeruginosa apoazurin stability (thermal and chemical perturbations) and folding kinetics (chemical perturbation) as a function of increasing levels of crowding agents dextran (sizes 20, 40, and 70 kDa) and Ficoll 70. We find that excluded volume theory derived by Minton quantitatively captures the experimental effects when crowding agents are modeled as arrays of rods. This finding demonstrates that synthetic crowding agents are useful for studies of excluded volume effects. Moreover, thermal and chemical perturbations result in free energy effects by the presence of crowding agents that are identical, which shows that the unfolded state is energetically the same regardless of method of unfolding. This also underscores the two-state approximation for apoazurin’s unfolding reaction and suggests that thermal and chemical unfolding experiments can be used in an interchangeable way. Finally, we observe increased folding speed and invariant unfolding speed for apoazurin in the presence of macromolecular crowding agents, a result that points to unfolded-state perturbations. Although the absolute magnitude of excluded volume effects on apoazurin is only on the order of 1–3 kJ/mol, differences of this scale may be biologically significant.