INVESTIGADORES
GONZALEZ FLECHA Francisco Luis
artículos
Título:
Ice-induced partial unfolding and aggregation of an integral membrane protein
Autor/es:
IONA P GARABER COHEN; PABLO R. CASTELLO; F. LUIS GONZÁLEZ FLECHA
Revista:
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
Editorial:
ELSEVIER SCIENCE BV
Referencias:
Lugar: Burlington, MA; Año: 2010 vol. 1798 p. 2040 - 2047
ISSN:
0005-2736
Resumen:
Although the deleterious effects of ice on water-soluble proteins are
well established, little is known about the freeze stability of membrane
proteins. Here we explore this issue through a combined kinetic and
spectroscopic approach using micellar-purified plasma membrane calcium
pump as a model. The ATPase activity of this protein significantly
diminished after freezing using a slow-cooling procedure, with the
decrease in the activity being an exponential function of the storage
time at 253 K, with t½ = 3.9 ± 0.6 h. On the contrary, no
significant changes on enzyme activity were detected when a fast cooling
procedure was performed. Regardless of the cooling rate, successive
freeze?thaw cycles produced an exponential decrease in the Ca2+-ATPase
activity, with the number of cycles at which the activity was reduced
to half being 9.2 ± 0.3 (fast cooling) and 3.7 ± 0.2 (slow cooling).
PAGE analysis showed that neither degradation nor formation of
SDS-stable aggregates of the protein takes place during protein
inactivation. Instead, the inactivation process was found to be
associated with the irreversible partial unfolding of the polypeptide
chain, as assessed by Trp fluorescence, far UV circular dichroism, and
1-anilino-naphtalene-8-sulfonate binding. This inactive protein
undergoes, in a later stage, a further irreversible transformation
leading to large aggregates.