title: Heat stability of Proteobacterial PII protein facilitate purification using a single chromatography step.
abstract: The P(II) proteins comprise a family of widely distributed signal transduction proteins that integrate the signals of cellular nitrogen, carbon and energy status, and then regulate, by protein-protein interaction, the activity of a variety of target proteins including enzymes, transcriptional regulators and membrane transporters. We have previously shown that the P(II) proteins from Azospirillum brasilense, GlnB and GlnZ, do not alter their migration behavior under native gel electrophoresis following incubated for a few minutes at 95°C. This data suggested that P(II) proteins were either resistant to high temperatures and/or that they could return to their native state after having been unfolded by heat. Here we used (1)H NMR to show that the A. brasilense GlnB is stable up to 70°C. The melting temperature (Tm) of GlnB was determined to be 84°C using the fluorescent dye Sypro-Orange. P(II) proteins from other Proteobacteria also showed a high Tm. We exploited the thermo stability of P(II) by introducing a thermal treatment step in the P(II) purification protocol, this step significantly improved the homogeneity of A. brasilense GlnB and GlnZ, Herbaspirillum seropedicae GlnB and GlnK, and of Escherichia coli GlnK. Only a single chromatography step was necessary to obtain homogeneities higher than 95%. NMR(1) and in vitro uridylylation analysis showed that A. brasilense GlnB purified using the thermal treatment maintained its folding and activity. The purification protocol described here can facilitate the study of P(II) protein family members.
authors.affiliation: Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, Curitiba-PR 81531-990, Brazil.
authors.forename: Vivian R
authors.initials: VR
authors.lastname: Moure
authors.affiliation: Centro Nacional de Ressonância Magnética Nuclear, Universidade Federal do Rio de Janeiro, Instituto de Bioquímica Médica, Rio de Janeiro, Brazil.
authors.forename: Guilherme
authors.initials: G
authors.lastname: Razzera
authors.affiliation: Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, Curitiba-PR 81531-990, Brazil.
authors.forename: Luíza M
authors.initials: LM
authors.lastname: Araújo
authors.affiliation: Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, Curitiba-PR 81531-990, Brazil.
authors.forename: Marco A S
authors.initials: MAS
authors.lastname: Oliveira
authors.affiliation: Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, Curitiba-PR 81531-990, Brazil.
authors.forename: Edileusa C M
authors.initials: ECM
authors.lastname: Gerhardt
authors.affiliation: Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, Curitiba-PR 81531-990, Brazil.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Müller-Santos
authors.affiliation: Centro Nacional de Ressonância Magnética Nuclear, Universidade Federal do Rio de Janeiro, Instituto de Bioquímica Médica, Rio de Janeiro, Brazil.
authors.forename: Fabio
authors.initials: F
authors.lastname: Almeida
authors.affiliation: Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, Curitiba-PR 81531-990, Brazil.
authors.forename: Fabio O
authors.initials: FO
authors.lastname: Pedrosa
authors.affiliation: Centro Nacional de Ressonância Magnética Nuclear, Universidade Federal do Rio de Janeiro, Instituto de Bioquímica Médica, Rio de Janeiro, Brazil.
authors.forename: Ana P
authors.initials: AP
authors.lastname: Valente
authors.affiliation: Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, Curitiba-PR 81531-990, Brazil.
authors.forename: Emanuel M
authors.initials: EM
authors.lastname: Souza
authors.affiliation: Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de Nitrogênio, Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, Curitiba-PR 81531-990, Brazil. Electronic address: huergo@ufpr.br.
authors.forename: Luciano F
authors.initials: LF
authors.lastname: Huergo
mesh_terms: D015806:Azospirillum brasilense / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D051922:PII Nitrogen Regulatory Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D044366:Transition Temperature
chemical_list: D001426:Bacterial Proteins; C100965:GlnZ protein, Azospirillum brasilense; D051922:PII Nitrogen Regulatory Proteins; D011994:Recombinant Proteins; C404143:PIID regulatory protein, Bacteria