Kinetic thermal stability of CopA, a thermophilic membrane protein from Archeoglobus fulgidus

DIEGO I. CATTONI; JOSÉ M ARGÜELLO; F. LUIS GONZÁLEZ-FLECHA

Salt Lake, Utha, USA

Congreso; Biophysical Society 50th Annual Meeting; 2006

Biophysical Society

Thermal stability of membrane proteins is poorly understood, and remarkably little is known about the stability of thermophilic membrane proteins. The aim of this work was to characterize the kinetic thermal stability of CopA, a thermophilic PIB-type Cu-ATPase from Archaeoglobus fulgidus. CopA was heterologously expressed in E. coli, solubilized in dodecylmaltoside (DDM), and affinity purified. When reconstituted in asolectin-DDM micelles, the enzyme retained thermophilic characteristics with maximum activity at 75ºC and Ea=103 kJ/mol. The purified enzyme was preincubated at different temperatures and the ATPase activity was measure at 75°C as a function of the preincubation time. Our results showed an exponential irreversible decrease in the enzyme activity with a kinact=0.013 min-1 at 75°C and Ea=190 kJ/mol. As expected, CopA inactivates much slower than mesophilic P-ATPases (e.g. the plasma membrane calcium pump) in similar conditions, but surprisingly the Ea for the inactivation process are in the same order. We also determined the binding of ANS to the protein as a function of the preincubation time at 75°C and found that ANS fluorescence intensity irreversibly decay to a constant value (I∞=0.7Io), following the same kinetics than inactivation. Controls show that inactivation was not associated with either fragmentation or formation of SDS-stable protein aggregates. These results indicate that thermophilic membrane proteins are more stable than their mesophilic counterparts and retain their stability even when heterologously expressed. The first order rates suggest a two-state process involving only fully active and inactive molecules, and the similarity between the kinetics for inactivation and binding of ANS suggest that inactivation leads to a conformation that still conserves large hydrophobic regions (probably the transmembrane domain).