Solvation of the folding‐transition state in Pseudomonas aeruginosa azurin is modulated by metal

The role of water in protein folding, specifically its presence or not in the transition‐state structure, is an unsolved question. There are two common classes of folding‐transition states: diffuse transition states, in which almost all side chains have similar, rather low phi (φ) values, and polarized transition states, which instead display distinct substructures with very high φ‐values. Apo‐and zinc‐forms of Pseudomonas aeruginosa azurin both fold in two‐state equilibrium and kinetic reactions; while the apo‐form exhibits a polarized transition state, the zinc form entails a diffuse, moving transition state. To examine the presence of water in these two types of folding‐transition states, we probed the equilibrium and kinetic consequences of replacing core valines with isosteric threonines at six positions in azurin. In contrast to regular hydrophobic‐to‐alanine φ‐value analysis, valine‐to‐threonine mutations do not disrupt the core packing but stabilize the unfolded state and can be used to assess the degree of solvation in the folding‐transition state upon combination with regular φ‐values. We find that the transition state for folding of apo‐azurin appears completely dry, while that for zinc‐azurin involves partially formed interactions that engage water molecules. This distinct difference between the apo‐and holo‐folding nuclei can be rationalized in terms of the shape of the free‐energy barrier.