Moure Vivian R, Razzera Guilherme, Araújo Luíza M, Oliveira Marco A S, Gerhardt Edileusa C M, Müller-Santos Marcelo, Almeida Fabio, Pedrosa Fabio O, Valente Ana P, Souza Emanuel M, Huergo Luciano F
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.
Centro Nacional de Ressonância Magnética Nuclear, Universidade Federal do Rio de Janeiro, Instituto de Bioquímica Médica, Rio de Janeiro, Brazil.
Protein Expr Purif. 2012 Jan;81(1):83-88. doi: 10.1016/j.pep.2011.09.008. Epub 2011 Sep 22.
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.
P(II)蛋白构成了一类广泛分布的信号转导蛋白家族，它们整合细胞内氮、碳和能量状态的信号，然后通过蛋白质-蛋白质相互作用调节多种靶蛋白的活性，这些靶蛋白包括酶、转录调节因子和膜转运蛋白。我们之前已经表明，来自巴西固氮螺菌的P(II)蛋白GlnB和GlnZ在95°C孵育几分钟后，在天然凝胶电泳下其迁移行为没有改变。该数据表明，P(II)蛋白要么对高温具有抗性，要么在被热解折叠后能够恢复到其天然状态。在这里，我们使用核磁共振氢谱（¹H NMR）表明巴西固氮螺菌的GlnB在高达70°C时是稳定的。使用荧光染料Sypro-Orange测定GlnB的解链温度（Tm）为84°C。来自其他变形菌门的P(II)蛋白也显示出较高的Tm。我们通过在P(II)纯化方案中引入热处理步骤来利用P(II)的热稳定性，这一步骤显著提高了巴西固氮螺菌GlnB和GlnZ、血清草螺菌GlnB和GlnK以及大肠杆菌GlnK的均一性。仅需一个色谱步骤就能获得高于95%的均一性。核磁共振（¹H NMR）和体外尿苷酰化分析表明，使用热处理纯化的巴西固氮螺菌GlnB保持了其折叠状态和活性。这里描述的纯化方案有助于对P(II)蛋白家族成员进行研究。
