Enzymatic characterization of a PIB-ATPase from the psychrophilic bacterium Bizionia argentinensi

NOELIA I. BURGARDT; NOELIA A. MELIAN; JULIAN LESCANO; EZEQUIEL SARAVIA CASTRO; F. LUIS GONZÁLEZ FLECHA

Salto

Congreso; Latin American Crosstalk In Biophysics And Physiology; 2015

PIB-ATPases are integral membrane proteins which actively transport copper through the cell membrane in association with ATP hydrolysis1. These proteins have at least four highly conserved domains: a cytoplasmatic Cu(I) binding domain, an actuator domain, an ATP binding domain, and a transmembrane domain where it is located the Cu(I) transmembrane binding site. Our group choose this protein family as a model to study the relationships between membrane protein folding, function and stability. Previously, we investigated the unfolding of the thermophilic PIB-ATPase from Archaeglobus fulgidus by thermal and guanidinium hydrochloride denaturation2,3. Now, we decided to perform a comparative study with a PIB-ATPase from a psychrophilic organism. We select a potential cooper transport ATPase from the recently discovered psychrophilic bacteria Bizionia argentinensis (JUB59-T) isolated from the Argentine Antarctic4. This protein was cloned and expressed in S. cerevisiae as a fusion protein with GFP and a His-tag at the C-terminus end (BaCopA-GFP). Membrane proteins were solubilized with 0.5 % sodium deoxycholate and BaCopA-GFP was purified by IMAC chromatography with 0.05 % C12E10. In this work we present the results of ATP hydrolysis assays performed to identify the optimal conditions for BaCopA-GFP catalytic function. We tested pH, temperature, and different concentrations of each reaction mix component (Mg+2, asolectin, NaCl, ATP-Mg+2). BaCopA-GFP catalyze ATP hydrolysis at moderate temperatures (20-28 °C), low ionic strength, and basic pH, being magnesium essential for activity. The ATP apparent affinity is 0.15 mM, similar to the reported for other PIB-ATPases. The characterization of this psychrophilic PIB-ATPase and the comparison with mesophilic and thermophilic homologues will be helpful to identify the mechanisms of cold adaptation in membrane proteins.References:[1] Rensing C, et al. J Bacteriol 181:5891-5897, 1999.[2] Cattoni DI, et al. Arch Biochem Biophys 471:198-206, 2008.[3] Roman EA, et al. J Mol Biol 397:550-559, 2010.[4] Bercovich A, et al. Int J Syst Evol Microbiol 58:2363-2367, 2008.