Unfolding and refolding of a thermophilic membrane protein

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

Rosario

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

Sociedad Argentina de Biofísica

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 models to understand stability determinants . The aim of this work was to characterize chemical denaturation of CopA, a thermophilic PIB-type ATPase from Archaeglobus fulgidus. Methods: CopA was heterologously expressed in E. coli, solubilized in dodecylmaltoside, affinity purified, and reconstituted in a lipid/detergent micellar form. Isolated CopA retained thermophilic  characteristics with maximun activity at 75C. Results: In this work, 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 [θ]222nm. in a reversible manner. This decrease occurs within the first second. Cm values were 3.5 M. Both ΔG0H2O calculated are about 10 KJ/mol . Unfolding and refolding curves obtained when denaturant was added and diluted were similar. In addition to these changes, CopA was inactivated by GndHCl; this inactivation was reversed when denaturant was diluted. When CopA was incubated with SDS at concentrations below cmc, the activity fell to zero and then was recovered when SDS was diluted. The Trp fluorescence decreased within the first second. Unfolding and refolding curves were identical upon addition and dilution of the denaturant, respectively. The Cm value was 1.62 mM. ΔG0H2O calculated was about 8 KJ/mol. [θ]222nm showed no change when incubated with SDS.  Discussion Estimated unfolding ΔG0H2O were similar for both denaturants, although the unfolded states might differed in secondary structure. This finding is suggested since [θ]222nm changed with GndHCl but did not with SDS. This is in accord with 1Otzen et al who described an expanded native-like intermediate that unfolds when higher concentrations of SDS are added. In addition, observed GndHCl Cm values were higher than those for mesophilic membrane proteins. Concluding remarks 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. Supported by UBACyT, ANPCyT, and NSF-CONICET. References: [1] Otzen D., Oliverg M. J. Mol. Biol, 2002,315,1231-1240