Expression and characterization of potential Cu(I) transport ATPase from the psychrophilic bacterium Bizionia argentinensis

N.I. BURGARDT; F.L. GONZÁLEZ FLECHA

Chascomus

Congreso; IV Latin American Meeting on Biological Inorganic Chemistry; 2014

INTRODUCTION The relationships between membrane protein folding, function and stability are the main topics of research in our group1. To explore this subject we selected the PIB-ATPase family as a model. These integral membrane proteins actively transport copper through the cell membrane2. Recently, it was obtained the first crystal structure of a copper transporting PIB-ATPase (CopA) from Legionella pneumophila3 (Figure 1). We have previously demonstrated that the thermophilic copper transporting PIB-ATPase from Archaeglobus fulgidus undergoes irreversible thermal denaturation due to a partial unfolding of the aminoacidic chain mainly at the level of the cytoplasmatic domains4. Furthermore, we found that guanidinium hydrochloride induce a reversible unfolding of CopA, allowing the complete thermodynamic characterization of the folding process5. To further explore this issue we decided to perform a comparative study of the structure-folding-function relationships in a set of copper transporting PIB-ATPases from mesophilic and psychrophilic organisms. As the psychrophilic partner we select a potential cooper transport ATPase from the recently discovered psychrophilic bacteria Bizionia argentinensis (JUB59-T), isolated from the Argentine Antarctic6,7. Here we present the cloning, expression and preliminary characterization of this psychrophilic CopA (BaCopA). EXPERIMENTAL METHODS The genome of Bizionia argentinensis was analized by sequence homology search (BLAST) and the sequences obtained were checked to contain the characteristic features of copper transporting ATPases. The selected gene was cloned by homologous recombination, fusioned with GFP, and transformed in Saccharomyces cerevisiae8. Protein expression was induced by the addition of 2 % galactose, and protein expression was monitored by GFP fluorescence. Protein integrity was controlled by SDS-PAGE in-gel GFP fluorescence. After yeast cells disruption and membrane isolation, the protein was solubilized with detergents and purified by IMAC chromatography. Activity measurements were performed by ATPase9 and phosphatase assays10. RESULTS AND DISCUSSION The search of genes with copper transporting ATPase homology in the genome of Bizionia argentinensis was performed using the sequence of Archaeglobus fulgidus CopA as template. The screening revealed the presence of two proteins with high homology, which also contained the DKTG sequence characteristic of this protein family. One of them was cloned as a fusion with GFP and a His-tag at the C-terminus end. Protein expression and purification was followed by GFP fluorescence and SDS-PAGE. Two detergents, DDM and C12E10, were tested for membrane proteins solubilization and purification. Protein solubilization was done at one detergent concentration and at a fix time. The amount of solubilized protein obtained in these conditions was higher with DDM than C12E10, according to the GFP fluorescence measurements. Anyway, IMAC chromatography was performed with both detergents and the result was analyzed by in-gel GFP fluorescence. The data show that the protein obtained after the purification with DDM was degraded (Figure 2). In the other hand, the protein purified with C12E10 shows almost no degradation (Figure 2). Preliminary activity assays were done with BaCopA purified in the presence of C12E10. As the optimal activity conditions of this enzyme are unknown yet, the assays were performed at 25 ºC, pH 7.0 and low ionic strength. The results show the presence of copper-induced ATPase (1.3 nmol /h μg) and phosphatase activity (2.4 nmol /h μg). CONCLUSION This work presents the cloning and expression of a psychrophilic PIB-ATPase from Bizionia argentinensis. Preliminary activity assays indicated that this protein show copper-induced ATPase and phosphatase activity. Biochemical and biophysical characterization of BaCopA will be helpful for the understanding of cold adaptation mechanism in membrane proteins. REFERENCES 1. Roman E.A. et al., Kinetics and Thermodynamics of Membrane Protein Folding, Biomolecules (2014), 4:354-373. 2. Rensing C. et al., Families of soft-metal-ion-transporting ATPases, J Bacteriol. (1999), 181:5891-5897. 3. Gourdon P. et al, Crystal structure of a copper-transporting PIB-type ATPase, Nature (2011), 475:59-64. 4. Cattoni D.I. et al, Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus, Arch Biochem Biophys. (2008), 471:198-206. 5. Roman E.A. et al, Reversible unfolding of a thermophilic membrane protein in phospholipid/detergent mixed micelles, J Mol Biol. (2010), 397:550-559. 6. 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