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Mol Microbiol. 2008 Sep;69(6):1358-72. doi: 10.1111/j.1365-2958.2008.06355.x. Epub 2008 Jul 9.

System-level mapping of Escherichia coli response regulator dimerization with FRET hybrids.

Author information

1
Center for Advanced Biotechnology and Medicine, Howard HughesMedical Institute, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.

Abstract

Two-component signal transduction, featuring highly conserved histidine kinases (HKs) and response regulators (RRs), is one of the most prevalent signalling schemes in prokaryotes. RRs function as phosphorylation-activated switches to mediate diverse output responses, mostly via transcription regulation. As bacterial genomes typically encode multiple two-component proteins for distinct signalling pathways, the sequence and structural similarities of RR receiver domains create significant challenges to maintain interaction specificity. It is especially demanding for members of the OmpR/PhoB subfamily, the largest RR subfamily, which share a conserved dimerization interface for phosphorylation-mediated transcription regulation. We developed a strategy to investigate RR interaction by analysing Förster resonance energy transfer (FRET) between cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-fused RRs in vitro. Using the Escherichia coli RR PhoB as a model system, we were able to observe phosphorylation-dependent FRET between fluorescent protein (FP)-PhoB proteins and validated the FRET method by determining dimerization affinity and dimerization-coupled phosphorylation kinetics that recapitulated values determined by alternative methods. Further application of the FRET method to all E. coli OmpR/PhoB subfamily RRs revealed that phosphorylation-activated RR interaction is indeed a common theme for OmpR/PhoB subfamily RRs and these RRs display significant interaction specificity. Weak hetero-pair interactions were also identified between several different RRs, suggesting potential cross-regulation between distinct pathways.

PMID:
18631241
PMCID:
PMC2586830
DOI:
10.1111/j.1365-2958.2008.06355.x
[Indexed for MEDLINE]
Free PMC Article

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