info:eu-repo/semantics/article
Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus
Fecha
2008-03Registro en:
Cattoni, Diego Ignacio; Gonzalez Flecha, Francisco Luis; Argüello, José M.; Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus; Elsevier Science Inc.; Archives of Biochemistry and Biophysics; 471; 2; 3-2008; 198-206
0003-9861
CONICET Digital
CONICET
Autor
Cattoni, Diego Ignacio
Gonzalez Flecha, Francisco Luis
Argüello, José M.
Resumen
Despite recent progress in understanding membrane protein folding, little is known about the mechanisms stabilizing these proteins. Here we characterize the kinetic thermal stability of CopA, a thermophilic P(IB)-type Cu+-ATPase from Archaeoglobus fulgidus. When heterologously expressed in Escherichia coli, purified and reconstituted in mixed micelles, CopA retained thermophilic characteristics with maximum activity at 75 degrees C. Incubation of CopA in the absence of substrates at temperatures in the 66-85 degrees C range led to an irreversible exponential decrease in enzyme activity suggesting a two-state process involving fully-active and inactive molecules. Although CopA inactivated much slower than mesophilic proteins, the activation energy was similar to that observed for mesophilic P-type ATPases. The inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, Phe UV spectroscopy, far UV circular dichroism, and 1-aniline-8-naphtalenesulfonate binding. However, the inactive thermally denatured protein still conserves large hydrophobic regions and considerable secondary structure.