重氮葡萄球菌固氮酶转录激活剂NifA DNA结合域的纯化及生化性质研究。

IF 1.9 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY The Protein Journal Pub Date : 2023-10-03 DOI:10.1007/s10930-023-10158-w
Heidi G. Standke, Lois Kim, Cedric P. Owens
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引用次数: 0

摘要

NifA是一种σ54激活剂,在还原条件下和固定细胞氮水平较低时开启细菌固氮。NifA中的氧化还原传感机制尚不清楚。在α-和β-蛋白细菌中,氧化还原传感涉及蛋白质中心AAA+结构域内和紧挨着该结构域的两对Cys残基。在这项工作中,我们检测了一个额外的Cys对是否参与氧化还原传感,该Cys对是C(X)5C基序的一部分,位于α-蛋白细菌重氮葡萄球菌(Gd)的NifA的DNA结合结构域的正上游。我们假设Cys残基的氧化还原状态可能直接影响DNA结合结构域的DNA结合亲和力和/或改变蛋白质的寡聚状态。产生了两个DNA结合结构域构建体,一个较长的构建体(2C-DBD),由具有上游Cys对的DNA结合结构体组成,另一个较短的构建体缺乏Cys对。NC-DBD的同源DNA序列(nifH-UAS)的Kd等于20.0µM。当Cys对被氧化时,nifH UAS的2C-DBD的Kd为34.5µM。二硫键的还原不会改变DNA结合亲和力。另外的实验表明Cys残基的氧化还原状态不影响NifA DNA结合结构域的二级结构或低聚状态。总之,这些结果表明,Gd-NifA的DNA结合结构域上游的Cys对不以氧化还原依赖的方式调节DNA结合或结构域二聚化。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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Purification and Biochemical Characterization of the DNA Binding Domain of the Nitrogenase Transcriptional Activator NifA from Gluconacetobacter diazotrophicus

NifA is a σ54 activator that turns on bacterial nitrogen fixation under reducing conditions and when fixed cellular nitrogen levels are low. The redox sensing mechanism in NifA is poorly understood. In α- and β-proteobacteria, redox sensing involves two pairs of Cys residues within and immediately following the protein’s central AAA+ domain. In this work, we examine if an additional Cys pair that is part of a C(X)5 C motif and located immediately upstream of the DNA binding domain of NifA from the α-proteobacterium Gluconacetobacter diazotrophicus (Gd) is involved in redox sensing. We hypothesize that the Cys residues’ redox state may directly influence the DNA binding domain’s DNA binding affinity and/or alter the protein’s oligomeric sate. Two DNA binding domain constructs were generated, a longer construct (2C-DBD), consisting of the DNA binding domain with the upstream Cys pair, and a shorter construct (NC-DBD) that lacks the Cys pair. The Kd of NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 µM. The Kd of 2C-DBD for nifH-UAS when the Cys pair is oxidized is 34.5 µM. Reduction of the disulfide bond does not change the DNA binding affinity. Additional experiments indicate that the redox state of the Cys residues does not influence the secondary structure or oligomerization state of the NifA DNA binding domain. Together, these results demonstrate that the Cys pair upstream of the DNA binding domain of Gd-NifA does not regulate DNA binding or domain dimerization in a redox dependent manner.

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来源期刊
The Protein Journal
The Protein Journal 生物-生化与分子生物学
CiteScore
5.20
自引率
0.00%
发文量
57
审稿时长
12 months
期刊介绍: The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.
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