Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus.
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.
Cattoni
Diego I
DI
Instituto de Química y Fisicoquímica Biológicas, Universidad de Buenos Aires-CONICET, Junín 956 - C1113AAD, Buenos Aires, Argentina.
González Flecha
F Luis
FL
Argüello
José M
JM
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D019611:Archaeoglobus fulgidus / Q000201:enzymology*; D001665:Binding Sites; D027682:Cation Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D000073840:Copper-Transporting ATPases; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D029968:Escherichia coli Proteins; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D014364:Tryptophan; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases; C000612460:copA protein, E coli