A Thermal Unfolding Study of Plastocyanin from the Thermophilic CyanobacteriumPhormidium laminosum

Abstract

The thermal unfolding of the plastocyanin from Phormidium laminosum, a thermophilic cyanobacterium, is herein described. The main objective of this work is to identify structural factors responsible for the higher stability observed in proteins from thermophilic organisms. With the aid of fluorescence spectroscopy, EPR, and NMR, the factors influencing the unfolding process of the protein were investigated, and procedures for its study have been standardized. The different spectroscopic techniques used provided consistent results showing that the thermal unfolding of plastocyanin is irreversible under all the conditions investigated and that this irreversibility does not appear to be related to the presence of oxygen. The oxidized plastocyanin species has proven to be more stable than the reduced one, with respect to both the required temperature for protein unfolding (up to a 9 degrees C difference between the two forms) and the kinetics of the process. The behavior of this plastocyanin contrasts with that of other cupredoxins whose unfolding had previously been studied. The unfolding pH dependence and kinetic studies indicate a process with a tight control around the physiological pH in which plastocyanin plays its redox role and the protein's isoelectric point (5.2), suggesting a close compromise between function and stability.

Modulation of copper site properties by remote residues determines the stability of plastocyanins

“…2 shows normalised thermal unfolding curves for the oxidised and reduced forms of Pho-WT and P49G/G50P species. The T m values of Pho-WT agree with those previously reported by Feio et al [15]. The melting temperatures of the P49G/G50P mutant are lower than those of Pho-WT, and the differences ca.…”

“…Moreover, the redox state of the metal modulates their thermal stability. In fact, oxidised species of azurin [4], rusticyanin [14], and Pc from thermophilic cyanobacteria [15] are more thermally stable than the reduced ones, whereas the opposite occurs in Pc from plants [16–18] or non‐thermophilic cyanobacteria [19]. Notably, the T m values of oxidised species span in a range of 20° among organisms, whereas those of the reduced forms vary in a range of only 10° [15–19].…”

Voltammetric study of the adsorbed thermophilic plastocyanin from Phormidium laminosum up to 90°C

“…Wild type plastocyanin from P. laminosum was expressed in Escherichia coli and purified as described previously [13]. Azurin from P. aeruginosa was purchased from Sigma-Aldrich, and was used without further purification.…”

Residue-level NMR View of the Urea-driven Equilibrium Folding Transition of SUMO-1 (1-97): Native Preferences Do Not Increase Monotonously

“…The local preferences could vary, depending upon the environmental conditions. In view of this, there have been several efforts to characterize the unfolded states in vitro and the intermediates created by different means, which include chemical denaturation, [14][15][16][17][18][19][20][21] pH denaturation, [22][23][24] thermal denaturation, 25,26 etc.…”