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... Show moreA 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 degreesC at the scan rate of 60 degreesC/h, whereas for the mutant it is reduced to 58.0 degreesC. 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 degreesC, 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, Ile2lCys and Glu25Cys, introduces strain into the protein which offsets the stabilizing effect expected from the formation of a covalent crosslink. (C) 2004 Elsevier B.V. All rights reserved. Show less
Andolfi, L.; Bruce, D.; Cannistraro, S.; Canters, G.W.; Davis, J.J.; Hill, H.A.O.; ... ; Astier, Y. 2004
Site-specifically engineered disulphide or surface cysteine residues have been introduced into two blue copper proteins, Pseudomonas aeruginosa azurin and Populus nigra plastocyanin, in order to... Show moreSite-specifically engineered disulphide or surface cysteine residues have been introduced into two blue copper proteins, Pseudomonas aeruginosa azurin and Populus nigra plastocyanin, in order to facilitate protein chemisorption on gold electrodes. The subsequently formed well-defined protein monolayers gave rise to robust electrochemical responses and electron transfer rates comparable to those observed at modified electrode surfaces. Proximal probe characterisation confirms the presence, at high coverage, of well-ordered protein adlayers. Additionally, gold-metalloprotein affinity is such that molecular-level tunnelling and topographic analyses can be carried out under aqueous solution. The approaches outlined in this work can, in principal, be extended to the generation of arrays of any redox-active biomolecule. (C) 2003 Elsevier B.V. All rights reserved. Show less
Andolfi, L.; Canters, G.W.; Verbeet, M.P.; Cannistraro, S. 2004
The study of the electronic conduction through plastocyanin (PC) mutants assembled on a gold surface has been addressed by scanning tunneling spectroscopy. The two mutants exploit a single thiol... Show moreThe study of the electronic conduction through plastocyanin (PC) mutants assembled on a gold surface has been addressed by scanning tunneling spectroscopy. The two mutants exploit a single thiol group (PCSH) or a disulfide bridge (PCSS) to covalently bind at gold surface. The I-V measurements were performed by positioning the STM tip on top of a single molecule and sweeping the bias potential between +/- 1 V, under both ambient and controlled atmosphere. For PCSS, under ambient conditions, asymmetric I-V characteristics were obtained, which disappear under nitrogen atmosphere. PCSH, instead shows a symmetric I-V relation in air and under nitrogen environment. Here, as factors underlying this distinct electron conductive behaviour, a potential role for hydration water molecules and for copper redox levels are discussed. (C) 2003 Elsevier B.V. All rights reserved. Show less
Aiming to achieve stable immobilization for a redox-active cupredoxin protein onto a gold substrate and its consequent molecular level monitoring by Scanning Tunnelling Microscopy (STM), we... Show moreAiming to achieve stable immobilization for a redox-active cupredoxin protein onto a gold substrate and its consequent molecular level monitoring by Scanning Tunnelling Microscopy (STM), we introduced a disulphide bridge within poplar plastocyanin, while avoiding the perturbation of its active site. We selected and modified residues lle-21 to Cys and Glu-25 to Cys by structurally conservative mutagenesis. Optical absorption spectroscopy (UV-Vis), electron paramagnetic resonance (EPR), and resonance raman scattering (RRS) results indicate that the active site of the Ile21Cys, Glu25Cys plastocyanin (PCSS) to a large extent retains the spectroscopic properties of the wildtype protein. Furthermore, the redox midpoint potential of the couple CuII/CuI in PCSS, determined by cyclic voltammetry was found to be +348 mV close to the wild-type value. The STM images display self-assembled PCSS molecules immobilised onto gold substrate. Moreover, the fall potentiostatic control of the electron transfer reaction during STM imaging, suggests that the adsorbed molecule maintains essentially its native redox properties. (C) 2002 Elsevier Science (USA). Show less
