A thermal unfolding study of plastocyanin from the thermophilic cyanobacterium Phormidium laminosum.
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.
Feio
Maria J
MJ
Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de La Cartuja, Universidad de Sevilla y CSIC, Av. Américo Vespucio 49, 41092 Sevilla, Spain.
Navarro
José A
JA
Teixeira
Miguel S
MS
Harrison
David
D
Karlsson
B Göran
BG
De la Rosa
Miguel A
MA
D000332:Aerobiosis; D000693:Anaerobiosis; D003300:Copper / Q000378:metabolism; D000458:Cyanobacteria / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D010970:Plastocyanin / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D003300:Copper; D010970:Plastocyanin