Biochemical, Stabilization and Crystallization Studies on a Molecular Chaperone (PaoD) Involved in the Maturation of Molybdoenzymes
| Autor: | David Rodrigues, Viola Schwuchow, Eurico J. Cabrita, Silke Leimkühler, Ana Rita Otrelo-Cardoso, Maria João Romão, Teresa Santos-Silva |
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| Rok vydání: | 2014 |
| Předmět: |
Protein Folding
Magnetic Resonance Spectroscopy Coenzymes Ionic Liquids Protein aggregation Crystallography X-Ray Biochemistry chemistry.chemical_compound Macromolecular Structure Analysis Metalloprotein Biomacromolecule-Ligand Interactions chemistry.chemical_classification 0303 health sciences Multidisciplinary Molecular Structure biology Protein Stability Physics Escherichia coli Proteins Pteridines 030302 biochemistry & molecular biology Molybdenum Chromatography Gel Medicine Electrophoresis Polyacrylamide Gel Crystallization Oxidoreductases Molybdenum cofactor Research Article Protein Binding Protein Structure Science Biophysics chemistry.chemical_element Tungsten Cofactor 03 medical and health sciences Oxidoreductase Metalloproteins Escherichia coli Protein Interactions Biology Institut für Biochemie und Biologie 030304 developmental biology Cofactor binding Cofactors Proteins Computational Biology Chaperone Proteins chemistry Chaperone (protein) biology.protein Protein Multimerization Molybdenum Cofactors Molecular Chaperones |
| Zdroj: | PLoS ONE, Vol 9, Iss 1, p e87295 (2014) PLoS ONE 'PloS One ', vol: 9, pages: e87295-1-e87295-9 (2014) |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0087295 |
| Popis: | Molybdenum and tungsten enzymes require specific chaperones for folding and cofactor insertion. PaoD is the chaperone of the periplasmic aldehyde oxidoreductase PaoABC. It is the last gene in the paoABCD operon in Escherichia coli and its presence is crucial for obtaining mature enzyme. PaoD is an unstable, 35 kDa, protein. Our biochemical studies showed that it is a dimer in solution with a tendency to form large aggregates, especially after freezing/thawing cycles. In order to improve stability, PaoD was thawed in the presence of two ionic liquids [C(4)mim]Cl and [C(2)OHmim]PF6 and no protein precipitation was observed. This allowed protein concentration and crystallization using polyethylene glycol or ammonium sulfate as precipitating agents. Saturation transfer difference - nuclear magnetic resonance (STD-NMR) experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process, showing a clear interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays also show a reduction of the overall size of the protein aggregates in presence of ionic liquids. Furthermore, cofactor binding studies on PaoD showed that the protein is able to discriminate between molybdenum and tungsten bound to the molybdenum cofactor, since only a Mo-MPT form of the cofactor remained bound to PaoD. |
| Databáze: | OpenAIRE |
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