Thermal Stability of a Thermophilic P-type ATPase

F. LUIS GONZÁLEZ FLECHA; DIEGO I. CATTONI; ATIN K. MANDAL; DIPTI SHARMA; GERMANO S. IANNACCHIONE; JOSÉ M. ARGÜELLO

Woods Hole, Massachusetts

Conferencia; Fifty-ninth Annual Meeting of the Society of General Physiologists; 2005

Society of General Physiologists

<!-- @page { size: 21.59cm 27.94cm; margin: 2cm } P { margin-bottom: 0.21cm } --> Protein stability is the result of a delicate balance betweenstabilizing and destabilizing interactions. While thermal denaturation of globular proteins is a well-characterized process, little is known about the thermal stability of membrane proteins. In addition, the lack of information on the stability of thermophilic membrane proteins is remarkable. The aim of this work was to initiate the characterization of the thermal denaturation process of CopA, a thermophilic PIB-type Cu-ATPase from Archaeoglobusfulgidus. CopA was heterologously expressed in Escherichia coli, solubilized in dodecylmaltoside (DDM), and affinity purified. The resulting enzyme retained thermophilic characteristics with maximum activity at 75°C and an Ea = 103 kJ/mol. DSC analysis of CopA showed a thermal transition at 81°C, a value significantlyhigher than that determined for mesophilic P-type ATPases. Thepresence of ATP-Mg further stabilized the protein, shiftingits Tm to 105°C. As expected, CopA denaturation was found to be much slower than that of mesophilic P-type ATPases in similar conditions. The enzyme preparation incubated at 75°C showed an irreversible exponential decrease in enzyme activity and intrinsic fluorescence intensity. This inactivation was not associated with either fragmentation or formation of SDS-stable aggregates of the protein. Moreover, the first-order rate of thermal inactivation suggests a two-state process involving only fully active and inactive molecules. CopA reconstitution in mixed micelles of asolectin and DDM before the inactivationfurther increased the enzyme stability. These results indicatethat thermophilic membrane proteins are more stable than theirmesophilic counterparts, that they retain their stability evenwhen heterologously expressed, and therefore, that their stability appears to depend largely on intramolecular interactions. [Supported by NSF grants MCM-0235165 and OISE-0436435 to J.M. Argüello, and DMR-0092786 to G.S. Iannacchione, and ANPCyT grant PICT-11138 to F.L.G. Flecha.]