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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 u...

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Published in:PloS one 2014-01, Vol.9 (1), p.e87295-e87295
Main Authors: Otrelo-Cardoso, Ana Rita, Schwuchow, Viola, Rodrigues, David, Cabrita, Eurico J, Leimkühler, Silke, Romão, Maria João, Santos-Silva, Teresa
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cited_by cdi_FETCH-LOGICAL-c593t-ad334074a1f84c6449b94d558fb5c30664288d944e8fe4e72d21b3e297646153
cites cdi_FETCH-LOGICAL-c593t-ad334074a1f84c6449b94d558fb5c30664288d944e8fe4e72d21b3e297646153
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container_title PloS one
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creator Otrelo-Cardoso, Ana Rita
Schwuchow, Viola
Rodrigues, David
Cabrita, Eurico J
Leimkühler, Silke
Romão, Maria João
Santos-Silva, Teresa
description 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.
doi_str_mv 10.1371/journal.pone.0087295
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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. 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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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>24498065</pmid><doi>10.1371/journal.pone.0087295</doi><oa>free_for_read</oa></addata></record>
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subjects Aggregates
Ammonium
Ammonium salts
Ammonium sulfate
Biology
Biosynthesis
Chaperones
Chromatography, Gel
Coenzymes - chemistry
Coenzymes - metabolism
Crystallization
Crystallography, X-Ray
E coli
Electrophoresis, Polyacrylamide Gel
Enzymes
Escherichia coli
Escherichia coli - genetics
Escherichia coli - metabolism
Escherichia coli Proteins - chemistry
Escherichia coli Proteins - genetics
Escherichia coli Proteins - metabolism
Freezing
Genetic aspects
Ionic liquids
Ionic Liquids - chemistry
Ligands
Magnetic resonance
Magnetic Resonance Spectroscopy - methods
Metalloproteins - chemistry
Metalloproteins - metabolism
Molecular Chaperones - chemistry
Molecular Chaperones - genetics
Molecular Chaperones - metabolism
Molecular Structure
Molybdenum
Molybdenum - chemistry
Molybdenum - metabolism
NMR
Nuclear magnetic resonance
Oxidoreductase
Oxidoreductases - chemistry
Oxidoreductases - metabolism
Physics
Polyethylene glycol
Precipitation (Meteorology)
Protein Binding
Protein Multimerization
Protein Stability
Proteins
Protons
Pteridines - chemistry
Pteridines - metabolism
Stabilization
Studies
Sulfates
Sulfur
Thawing
Tungsten
Tungsten - chemistry
Tungsten - metabolism
title Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes
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