INVESTIGADORES
GONZALEZ FLECHA Francisco Luis
artículos
Título:
Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus.
Autor/es:
DIEGO I. CATTONI; F. LUIS GONZÁLEZ FLECHA; JOSÉ M. ARGÜELLO
Revista:
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Editorial:
Elsevier
Referencias:
Año: 2008 vol. 471 p. 198 - 206
ISSN:
0003-9861
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.