Objective
"Two-component" regulatory systems and sigma54-dependent transcription control many aspects of bacterial metabolism of medical, agronomic, and biotechnological importance. Structural information would greatly enhance our understanding of these control mechanisms and our ability to manipulate them. We propose to use X-ray crystallography to determine high-resolution structures of 7 proteins involved in such systems; the rhizobial FixLJ proteins, the NtrBC and NifLA proteins, and sigma54 itself. NtrC and NifA are sigma54 dependent transcriptional activators, and our ultimate objective is to generate dynamic models of these sensory transduction pathways from sensory input to transcriptional initiation.
NMR will be used principally to characterise various conformational changes involved in sensory transduction, namely i) changes in NifL resulting from nucleotide binding or oxidation of FAD that affect its interaction with NifA, information that we wish to relate to the overall form of the NifL:NifA complex, and ii) changes in conformation of the receiver domains of NtrC and FixJ resulting from phosphorylation.
We further wish to determine the structure of interfaces between pairs of sensory transduction proteins and within the activated sigma54 based transcription complex. Where proteins form stable complexes (eg the NifL:NifA pair) we will attempt to crystallise these to obtain such information directly. In other cases X-ray and neutron scattering of biochemically stabilised complex will be used to provide envelope structures into which high-resolution models o constituent proteins can be incorporated using software developed within the partnership, an approach to structure determination we intend to develop further to cope with the sigma54-based transcription complex. Solution scattering techniques will also be used to determine the quaternary structure of the oligomeric forms of NtrC, NifA and FixJ which are the transcriptionally active species.
Structural information will be used in the rational design of chimeric sensory transduction proteins which allow one regulatory system to be subjected to the sensory inputs of another. If successful this will pave the way to manipulating the expression of entire metabolic pathways in response to chosen environmental stimuli; we identify potential industrial applications. Molecular biological approaches will, in addition, identify protein interfaces to assist modelling of complexes, seek to stabiliseable entities for structure determination, and identify tractable material for structure determination where this is not already available.
Fields of science (EuroSciVoc)
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CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques.
- natural sciencescomputer and information sciencessoftware
- natural scienceschemical scienceselectrochemistryelectrolysis
- natural sciencesearth and related environmental sciencesgeologymineralogycrystallography
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteins
- natural sciencesbiological sciencesgeneticsnucleotides
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Topic(s)
Call for proposal
Data not availableFunding Scheme
CSC - Cost-sharing contractsCoordinator
NR4 7UH Norwich
United Kingdom