Cavity-creating mutations in Pseudomonas aeruginosa azurin: effects on protein dynamics and stability

Changes in flexibility and structural stability of Pseudomonas aeruginosa azurin in response to cavity-creating mutations were probed by the phosphorescence emission of Trp-48, which was deeply buried in the compact hydrophobic core of the macromolecule, and by measurements of guanidinum hydrochloride unfolding, respectively. Replacement of the bulky side chains Phe-110, Phe-29, and Tyr-108 with the smaller Ala introduced cavities at different distances from the hydrophobic core. The phosphorescence lifetime (τ₀) of Trp-48, buried inside the protein core, and the acrylamide quenching rate constant (k_q) were used to monitor local and global flexibility changes induced by the introduction of the cavity. The results of this work demonstrate the following: 1), the effect on core flexibility of the insertion of cavities is not correlated readily to the distance of the cavity from the core; 2), the protein global flexibility results are related to the cavity distance from the packed core of the macromolecule; and 3), the increase in protein flexibility does not correspond necessarily to a comparable destabilizing effect of some mutations.