Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus

Archives of Biochemistry and Biophysics
Diego I CattoniJosé M Argüello

Abstract

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.

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Citations

Sep 6, 2008·The Journal of Biological Chemistry·Manuel González-GuerreroJosé M Argüello
Jun 23, 2012·PloS One·Martín M Dodes TraianF Luis González Flecha
Jun 28, 2014·Biomolecules·Ernesto A Roman, F Luis González Flecha
Aug 18, 2009·Biochimica Et Biophysica Acta·Diego I CattoniF Luis González Flecha
May 12, 2009·Biochemical and Biophysical Research Communications·Aramis A VillafaneElisabetta Bini
Feb 2, 2010·Journal of Molecular Biology·Ernesto A RomanF Luis González Flecha
Apr 19, 2016·Biochimica Et Biophysica Acta·Luis M Bredeston, F Luis González Flecha
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Apr 27, 2017·The Journal of Physical Chemistry. B·M Agueda PlacentiRodolfo M González Lebrero
Sep 19, 2017·Biophysics Reviews·F Luis González Flecha
Dec 1, 2020·Biophysical Journal·Shahar Seifer, Michael Elbaum
Jan 31, 2012·The Journal of Physical Chemistry. B·Sergio B KaufmanRodolfo M González-Lebrero

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