Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes.
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 [C4mim]Cl and [C2OHmim]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.
REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal.
Ana Rita
AR
Otrelo-Cardoso
Universität Potsdam, Institut für Biochemie and Biologie, Molekulare Enzymologie, Golm, Germany.
Viola
V
Schwuchow
REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal.
David
D
Rodrigues
REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal.
Eurico J
EJ
Cabrita
Universität Potsdam, Institut für Biochemie and Biologie, Molekulare Enzymologie, Golm, Germany.
Silke
S
Leimkühler
REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal.
Maria João
MJ
Romão
REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal.
Teresa
T
Santos-Silva
D002850:Chromatography, Gel; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D052578:Ionic Liquids / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D008982:Molybdenum / Q000737:chemistry* / Q000378:metabolism; D000090006:Molybdenum Cofactors; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D011621:Pteridines / Q000737:chemistry / Q000378:metabolism; D014414:Tungsten / Q000737:chemistry / Q000378:metabolism
D003067:Coenzymes; D029968:Escherichia coli Proteins; D052578:Ionic Liquids; D008667:Metalloproteins; D018832:Molecular Chaperones; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D010088:Oxidoreductases; D011621:Pteridines; D014414:Tungsten; C000592540:PaoD protein, E coli; C022261:molybdopterin; C100768:dimethylsulfide - acceptor oxidoreductase