The main objective of this work is to understand the role of some molecular determinants (size of internal cavities and disulphide bridges) for the pressure sensitivity of globular proteins. To this end a single protein has been selected (azurin from Pseudomonas aeruginosa) and the importance of the above mentioned molecular parameters has been assessed through the construction of a an apposite series of mutant proteins in which one or more parameters have been specifically varied. Changes in the flexibility of the proteins matrix were monitored by the intrinsic phosphorescence lifetime. Pressure denaturation has been determined following the red-shift of the fluorescence spectrum and the changes in the phosphorescence intensity. The results of experiments conducted up to 7 kbar have demonstrated that pressure induces a reversible unfolding of azurin. The coincidence of fluorescence and phosphorescence transitions for each protein indicates that there are not intermediates in the unfolding equilibrium. Both removal of disulphide bridge and introduction of internal cavities bring to a consistent increase of the conformational freedom of the macromolecule and reduce the thermodynamic stability toward unfolding at atmospheric pressure. In spite of the cavities created by the mutations, ?V0, the volume reduction on unfolding, is practically unchanged. This implies that the cavities are not empty but occupied by water molecules, not observed in the X-ray structure, and therefore the destabilizing influence of pressure remains largely unchanged. The implication of these findings for the thermodynamic stability of proteins under pressure are discussed.
Effects of disulphide bridge removal and cavity forming mutations on the pressure stability of azurin
P Cioni;G B Strambini
2005
Abstract
The main objective of this work is to understand the role of some molecular determinants (size of internal cavities and disulphide bridges) for the pressure sensitivity of globular proteins. To this end a single protein has been selected (azurin from Pseudomonas aeruginosa) and the importance of the above mentioned molecular parameters has been assessed through the construction of a an apposite series of mutant proteins in which one or more parameters have been specifically varied. Changes in the flexibility of the proteins matrix were monitored by the intrinsic phosphorescence lifetime. Pressure denaturation has been determined following the red-shift of the fluorescence spectrum and the changes in the phosphorescence intensity. The results of experiments conducted up to 7 kbar have demonstrated that pressure induces a reversible unfolding of azurin. The coincidence of fluorescence and phosphorescence transitions for each protein indicates that there are not intermediates in the unfolding equilibrium. Both removal of disulphide bridge and introduction of internal cavities bring to a consistent increase of the conformational freedom of the macromolecule and reduce the thermodynamic stability toward unfolding at atmospheric pressure. In spite of the cavities created by the mutations, ?V0, the volume reduction on unfolding, is practically unchanged. This implies that the cavities are not empty but occupied by water molecules, not observed in the X-ray structure, and therefore the destabilizing influence of pressure remains largely unchanged. The implication of these findings for the thermodynamic stability of proteins under pressure are discussed.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
