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  • Purification and Biochemical Characterization of the DNA Binding Domain of the Nitrogenase Transcriptional Activator NifA from Gluconacetobacter diazotrophicus
Title: Purification and Biochemical Characterization of the DNA Binding Domain of the Nitrogenase Transcriptional Activator NifA from Gluconacetobacter diazotrophicus
Abstract NifA is a σ54activator 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-proteobacteriumGluconacetobacter 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. TheKdof NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 µM. TheKdof 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 ofGd-NifA does not regulate DNA binding or domain dimerization in a redox dependent manner.  more » « less
Award ID(s):
1905399
PAR ID:
10571626
Author(s) / Creator(s):
; ;
Publisher / Repository:
Springer
Date Published:
Journal Name:
The Protein Journal
Volume:
42
Issue:
6
ISSN:
1572-3887
Page Range / eLocation ID:
802 to 810
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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