title: Temperature and pressure effects on C112S azurin: volume, expansivity, and flexibility changes.
abstract: The pressure-induced unfolding of the mutant C112S azurin from Pseudomonas aeruginosa was monitored both under steady state and dynamic conditions. The unfolding profiles were obtained by recording the spectral shift of the fluorescence emission as well as by phosphorescence intensity measurements. We evaluated the difference in free energy, ΔG, as a function of pressure and temperature. The dependence of ΔG on temperature showed concave profile at all pressures studied. A positive heat capacity change of about 4.3 kJ mol(-1) deg(-1) fitted all the curves. The volume change of the reaction showed a moderate dependence on temperature when compared with other proteins previously studied. The kinetic activation parameters (ΔV*, ΔH*, ΔS*) were obtained from upward and downward pressure-jump experiments and used to characterize the volumetric and energetic properties of the transition state between native and unfolded protein. Our findings suggest that the folding and unfolding reaction paths passed through different transition states. The change in the phosphorescence lifetime with pressure pointed out that pressure-induced unfolding occurred within two steps: the first leading to an increased protein flexibility, presumably caused by water penetration into the protein. Major structural changes of the tryptophan environment occurred in a second step at higher pressures.
authors.affiliation: Istituto di Biofisica, Sezione di Pisa, Area della Ricerca, CNR, via G. Moruzzi, 1, 56124, Pisa, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Cioni
authors.forename: Edi
authors.initials: E
authors.lastname: Gabellieri
authors.forename: Stéphane
authors.initials: S
authors.lastname: Marchal
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lange
mesh_terms: D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D008958:Models, Molecular; D011312:Pressure; D055550:Protein Stability; D058767:Protein Unfolding; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D014867:Water; D001400:Azurin
keywords: pressure unfolding; pressure-jump; protein dynamics; tryptophan fluorescence; tryptophan phosphorescence