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Role of Protein Cavities on Unfolding Volume Change and on Internal Dynamics under Pressure

Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Pisa 56100, Italy

Correspondence: Address reprint requests to Patrizia Cioni, E-mail: patrizia.cioni{at}pi.ibf.cnr.it.

The effects of two single point cavity forming mutations, F110S and I7S, on the unfolding volume change (V⁰) of azurin from Pseudomonas aeruginosa and on the internal dynamics of the protein fold under pressure were probed by the fluorescence and phosphorescence emission of Trp-48, deeply buried in the compact hydrophobic core of the macromolecule. Pressure-induced unfolding, monitored by the shift of the center of mass of the fluorescence spectrum, showed that V⁰ is in the range of 60–70 mL/mol, not significantly different between cavity mutants and compact azurin species such as the wild-type and the mutant C3A/C26A, in which the superficial disulphide has been removed. The lack of extra volume in F110S and I7S proves that the engineered cavities, 40 ų in I7S and 100 ų in F110S, are filled with water molecules. Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime (₀). The application of pressure in the predenaturation range initially decreases the internal flexibility of azurin, the trend eventually reverting on approaching unfolding. The main difference between compact folds, wild-type and C3A/C26A, and cavity mutants is that the inversion point is powered from 3 kbar to 1.5 kbar for F110S and <0.1 kbar for I7S, meaning that in the latter species pressure-induced internal hydration dominates very early over any compaction of the globular fold resulting from the reduction of internal free volume. The similar response between wild-type and the significantly less-stable C3A/C26A mutant suggests that thermodynamic stability per se is not the dominant factor regulating pressure-induced internal hydration of proteins.