Thermal stability of wild type and disulfide bridge containing mutant of poplar plastocyanin.
A comparative study of the thermal stability of wild type poplar plastocyanin and of a mutant form containing a disulfide bridge between residues 21 and 25 was performed using differential scanning calorimetry and optical spectroscopic techniques. For wild type plastocyanin the transition temperature, determined from the calorimetric profiles, is 62.7 degrees C at the scan rate of 60 degrees C/h, whereas for the mutant it is reduced to 58.0 degrees C. In both cases, the endothermic peak is followed by an exothermic one at higher temperatures. The unfolding process monitored by optical absorption at 596 nm also reveals a reduced thermal stability of the mutated plastocyanin compared to the wild type protein, with transition temperatures of 54.8 and 58.0 degrees C, respectively. For both proteins, the denaturation process was found to be irreversible and dependent on the scan rate preventing the thermodynamic analysis of the unfolding process. In parallel, small conformational changes between wild type and mutant plastocyanin emerge from fluorescence spectroscopy measurements. Here, a difference in the interaction of the two proteins between the microenvironment surrounding the fluorophores and the solvent was proposed. The destabilization observed in the disulfide containing mutant of plastocyanin suggests that the double mutation, Ile21Cys and Glu25Cys, introduces strain into the protein which offsets the stabilizing effect expected from the formation of a covalent crosslink.
Guzzi
Rita
R
Dipartimento di Fisica and Unità INFM, Laboratorio di Biofisica Molecolare, Università della Calabria, Ponte P. Bucci, Cubo 31C, 87036 Rende, Italy. guzzi@fis.unical.it
Andolfi
Laura
L
Cannistraro
Salvatore
S
Verbeet
Martin Ph
MP
Canters
Gerard W
GW
Sportelli
Luigi
L
D002152:Calorimetry, Differential Scanning; D004220:Disulfides / Q000737:chemistry*; D006358:Hot Temperature; D009154:Mutation / Q000235:genetics*; D010970:Plastocyanin / Q000737:chemistry* / Q000235:genetics; D032107:Populus / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D004220:Disulfides; D010970:Plastocyanin