STRUCTURAL CHARACTERIZATION OF THERMAL INACTIVATION OF CopA: A THERMOPHILIC MEMBRANE PROTEIN

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

Rosario

Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica; 2006

Sociedad Argentina de Biofísica

INTRODUCTION Studies of the soluble proteins that allow life at extreme temperatures have produced relevant information on proteins stability, however, little is known about the factors that determine the stability of membrane proteins from hyperthermophilic organisms. CopA, is a thermophilic membrane Cu+-ATPase from Archaeoglobus fulgidus. It was heterologously expressed in E. coli, purified and finally reconstituted in asolectin/DDM mixed micelles. Purified CopA retains its high temperature dependence. In this work we attempt to characterize the structural changes involved in the spontaneous inactivation of purified CopA. RESULTS AND DISCUSION The kinetic of CopA thermal inactivation was studied by measuring Ag+-ATPase activity as a function of the preincubation time at 75ºC. This showed an irreversible exponential decrease with ki = 0.054 ± 0.01 min-1, suggesting a two-state process involving only fully active and inactive molecules In order to study structural changes produced by thermal inactivation, Trp fluorescence, far UV circular dichroism, SDS/PAGE and Blue Native Gel electrophoresis were carried on. The intensity of fluorescence decayed exponentially until a constant value as function of the incubation time, while the mass center of the fluorescence spectra for the inactivated CopA didn’t show significant changes. Exposure of the tryptophan residues to the solvent may explain this decay of fluorescence intensity. Circular dichroism spectra showed significant changes as a function of the incubation time and the elipticity data evaluated at 209 and 222 nm followed an exponential decrease until constant value (60 % of the initial elipticity value) revealing large changes in the secondary structure of CopA. Previous studies of the binding of ANS to the purified protein as a function of the incubation time at 75°C showed that ANS fluorescence intensity irreversibly decay to a constant value. The kinetic constant of the structural changes detected for each method of structural analysis were in the same range of the inactivation constant (ki). Native and inactivated CopA migrated as a single band after SDS/PAGE and as two well differentiated bands in Blue Native gel indicating that the inactivation was not associated with either fragmentation or formation of SDS-stable aggregates of the protein, neither changes in the oligomeric state of CopA. CONCLUSION The similarity between the kinetics for the decay of CopA Ag+-ATPase activity, Trp fluorescence intensity ANS fluorescence and Circular Dichroism elipticity suggest that inactivation is the result of protein unfolding. Our results indicate that thermally inactivated CopA still conserves large hydrophobic regions and a large amount of secondary structure as well. Supported by UBACyT, ANPCYT, and NSF/CONICET