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.

Influence of the ADP/ATP ratio, 2-oxoglutarate and divalent ions on Azospirillum brasilense PII protein signalling

“…The A. brasilense GlnD, DraT, DraG and AmtB proteins were expressed and purified as N-terminally 66His-tagged proteins, as described previously (Huergo et al, 2007(Huergo et al, , 2009). The A. brasilense GlnB, GlnZ, GlnZ Q39K and GlnZ Q39E proteins were expressed and purified as described previously (Moure et al, 2012;Rajendran et al, 2011). The GlnZ Q39E mutant was obtained using the Quick-Change site-directed mutagenesis kit (Agilent) using the pMSA4 plasmid (Moure et al, 2012) as a template.…”

“…The A. brasilense GlnB, GlnZ, GlnZ Q39K and GlnZ Q39E proteins were expressed and purified as described previously (Moure et al, 2012;Rajendran et al, 2011). The GlnZ Q39E mutant was obtained using the Quick-Change site-directed mutagenesis kit (Agilent) using the pMSA4 plasmid (Moure et al, 2012) as a template. The HisDraTGlnB complex was purified as described previously (Huergo et al, 2009).…”

Purification of the Campylobacter jejuni Dps protein assisted by its high melting temperature

“…A real-time PCR device (Applied Biosystems Step One Plus) was used to monitor protein unfolding by the fluorescent dye Sypro Orange as previously described [24]. CjDps at the concentration of 10 lM was prepared in 50 mM Tris-HCl pH 7.0 containing Sypro Orange diluted to 20Â (Invitrogen).…”

Conserved histidine residues at the ferroxidase centre of the Campylobacter jejuni Dps protein are not strictly required for metal binding and oxidation

“…A real-time PCR thermocycler (StepOnePlus; Applied Biosystems) was used to monitor protein unfolding using the fluorescent dye Sypro Orange (Invitrogen) as described (Moure et al, 2012;Niesen et al, 2007). CjDps (10 mM) was kept in 50 mM Tris/HCl pH 7.0 containing Sypro Orange diluted |20.…”