Heat stability of Proteobacterial P II protein facilitate purification using a single chromatography step

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 (T m ) of GlnB was determined to be 84 °C using the fluorescent dye Sypro-Orange. P II proteins from other Proteobacteria also showed a high T m. 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 1Abbreviations used: NMR, nuclear magnetic resonance; 2-OG, 2-oxoglutarate. 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.