Reversible unfolding of CopA, a thermophilic PIB-type membrane ATPase from Archaeglobus fulgidus

ERNESTO A. ROMAN; JOSÉ M ARGÜELLO; F. LUIS GONZÁLEZ-FLECHA

Baltimore, MD, USA

Congreso; Biophysical Society Annual Meeting; 2007

Biophysical Society

Folding and stability are determinant elements of protein biological functions. The stability of membrane proteins is poorly understood. Highly stable polytopic membrane proteins from extremophilic organism might provide convenient models to understand stability determinants. The aim of this work was to characterize chemical denaturation of CopA, a thermophilic PIB-type Cu-ATPase from Archaeglobus fulgidus. CopA was heterologously expressed in Escherichia coli, solubilized in dodecylmaltoside, affinity purified, and obtained in a lipid/detergent micellar form. Isolated CopA retained thermophilic characteristics with maximum activity at 75°C. The effects of guanidinium chloride (GndHCl) and sodium dodecyl sulfate (SDS) on enzyme activity, Trp fluorescence, and circular dichroism were analyzed. Our results show that GndHCl decreases Trp fluorescence and [è]222nm in a reversible manner, both with Cm values of 3.5 M.  In parallel to these changes, CopA was reversibly inactivated. When CopA was incubated with SDS at concentrations below cmc, the activity and the Trp fluorescence decayed reversibly, with a Cm value of 1.62 mM. However, [è]222nm remained unchanged. Estimated unfolding ÄG0H2O were similar for both denaturants, although the unfolded states might differed in secondary structure. Observed GndHCl Cm values were higher than those reported for mesophilic membrane proteins. These results suggest that thermophilic membrane proteins are more stable than their mesophilic counterparts and retain their stability even when heterologously expressed. Moreover, these findings point out the suitability of extremophilic membrane proteins for thermodynamic folding studies.