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...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Ana Rita Otrelo-Cardoso, Viola Schwuchow, David Rodrigues, Eurico J Cabrita, Silke Leimkühler, Maria João Romão, Teresa Santos-Silva
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c1261c4a38884b709db8fd5c8cb646fa
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:c1261c4a38884b709db8fd5c8cb646fa
record_format	dspace
spelling	oai:doaj.org-article:c1261c4a38884b709db8fd5c8cb646fa2021-11-18T08:34:36ZBiochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes.1932-620310.1371/journal.pone.0087295https://doaj.org/article/c1261c4a38884b709db8fd5c8cb646fa2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24498065/?tool=EBIhttps://doaj.org/toc/1932-6203Molybdenum 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.Ana Rita Otrelo-CardosoViola SchwuchowDavid RodriguesEurico J CabritaSilke LeimkühlerMaria João RomãoTeresa Santos-SilvaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 1, p e87295 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ana Rita Otrelo-Cardoso Viola Schwuchow David Rodrigues Eurico J Cabrita Silke Leimkühler Maria João Romão Teresa Santos-Silva Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes.
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.
format	article
author	Ana Rita Otrelo-Cardoso Viola Schwuchow David Rodrigues Eurico J Cabrita Silke Leimkühler Maria João Romão Teresa Santos-Silva
author_facet	Ana Rita Otrelo-Cardoso Viola Schwuchow David Rodrigues Eurico J Cabrita Silke Leimkühler Maria João Romão Teresa Santos-Silva
author_sort	Ana Rita Otrelo-Cardoso
title	Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes.
title_short	Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes.
title_full	Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes.
title_fullStr	Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes.
title_full_unstemmed	Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes.
title_sort	biochemical, stabilization and crystallization studies on a molecular chaperone (paod) involved in the maturation of molybdoenzymes.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/c1261c4a38884b709db8fd5c8cb646fa
work_keys_str_mv	AT anaritaotrelocardoso biochemicalstabilizationandcrystallizationstudiesonamolecularchaperonepaodinvolvedinthematurationofmolybdoenzymes AT violaschwuchow biochemicalstabilizationandcrystallizationstudiesonamolecularchaperonepaodinvolvedinthematurationofmolybdoenzymes AT davidrodrigues biochemicalstabilizationandcrystallizationstudiesonamolecularchaperonepaodinvolvedinthematurationofmolybdoenzymes AT euricojcabrita biochemicalstabilizationandcrystallizationstudiesonamolecularchaperonepaodinvolvedinthematurationofmolybdoenzymes AT silkeleimkuhler biochemicalstabilizationandcrystallizationstudiesonamolecularchaperonepaodinvolvedinthematurationofmolybdoenzymes AT mariajoaoromao biochemicalstabilizationandcrystallizationstudiesonamolecularchaperonepaodinvolvedinthematurationofmolybdoenzymes AT teresasantossilva biochemicalstabilizationandcrystallizationstudiesonamolecularchaperonepaodinvolvedinthematurationofmolybdoenzymes
_version_	1718421645363773440

Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes.

Ejemplares similares