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Cavity-Creating Mutations in Pseudomonas aeruginosa Azurin: Effects on Protein Dynamics and Stability

Edi Gabellieri ^*, Ettore Balestreri ^*, Alvaro Galli  and Patrizia Cioni ^*

^* Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 56124 Pisa, Italy; and Istituto di Fisiologia Clinica, Consiglio Nazionale delle Ricerche, 56124 Pisa, Italy

Correspondence: Address reprint requests to Dr. Patrizia Cioni, Istituto di Biofisica, CNR Via G. Moruzzi, 1, 56124 Pisa, Italy. Tel.: 39-050-315-3051; Fax: 39-050-315-2760; E-mail: patrizia.cioni{at}pi.ibf.cnr.it.

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 (₀) 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.