Figure 4 - uploaded by Peter Slavny
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5. Analytical ultracentrifugation analysis of the NifL PAS2 domain. Sedimentation equilibrium profiles of the wild-type PAS2 domain (triangles) and the "locked-on" variant PAS2-L175A (circles) at a rotor speed of 23,000 rpm. Lower panels: (A) 100 μM PAS2 and 70 μM PAS2-L175A measured at 275 nm. (B) 10 μM PAS2 and 7 μM PAS2-L175A measured at 230 nm. The lines represent a fit to both data sets for each sample using a 20 kDa monomer-dimer equilibrium model and K d values of 34 and 120 μM for the wild type domain and L175A variant respectively. Upper panels: residual absorbance between the experimental data and the fitted lines. This Figure was kindly provided by Dr. Thomas A. Clarke, UEA.
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Citations
... The PAS1 domain includes a solvent-accessible FAD cofactor that is readily oxidized by intracellular oxygen (13)(14)(15) and is the only part of NifL with a known structure (16). Oxidation of the PAS1 FAD causes reorganization of hydrogen bonds within the FAD-binding pocket that leads to reorientation of the non-FAD containing PAS2 domain to stimulate NifL binding to NifA, inhibiting nitrogenase expression in oxidizing conditions (17)(18)(19)(20). The carboxy-terminal (C-terminal) kinase-like DH and GHKL domains (21) perceive energy and fixed nitrogen signals. ...
... To refine the nascent PAS1-aligned NifL dimer model, we proceeded to refine the predicted oligomeric interfaces in the other domains of NifL. Moving sequentially from the N-terminal PAS1 domain, we found a set of residues in PAS2 (I153, V157, V166, L175, L177, R240, R262) that were identified as essential for dimerization in previous mutagenesis studies of the isolated PAS2 domain (13,18). Mapping these residues onto the NifL peptide model revealed that most were localized to the βsheet interface of PAS2, consistent with the conserved dimerization motif of PAS domains (35,38,39). ...
NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ54-dependent activator NifA to control the transcription of nitrogen fixation (nif) genes in response to intracellular oxygen, cellular energy, or nitrogen availability. The NifL-NifA two-component system is the master regulatory system for nitrogen fixation. NifL serves as a sensory protein, undergoing signal-dependent conformational changes that modulate its interaction with NifA, forming the NifL-NifA complex, which inhibits NifA activity in conditions unsuitable for nitrogen fixation. While NifL-NifA regulation is well understood, these conformationally flexible proteins have eluded previous attempts at structure determination. In work described here, we advance a structural model of the NifL dimer supported by a combination of scattering techniques and mass spectrometry (MS)-coupled structural analyses that report on the average structure in solution. Using a combination of small angle X-ray scattering-derived electron density maps and MS-coupled surface labeling, we investigate the conformational dynamics responsible for NifL oxygen and energy responses. Our results reveal conformational differences in the structure of NifL under reduced and oxidized conditions that provide the basis for a model for modulating NifLA complex formation in the regulation of nitrogen fixation in response to oxygen in the model diazotroph, Azotobacter vinelandii.
