PMID:16401068

Citation	Fic, E, Polit, A and Wasylewski, Z (2006) Kinetic and structural studies of the allosteric conformational changes induced by binding of cAMP to the cAMP receptor protein from Escherichia coli. Biochemistry 45:373-80
Abstract	The cAMP receptor protein, allosterically activated by cAMP, regulates the expression of more than 100 genes in Escherichia coli. CRP is a homodimer of two-domain subunits. It has been suggested that binding of cAMP to CRP leads to a long-distance signal transduction from the N-terminal cAMP binding domain to the C-terminal domain of the protein responsible for interaction with specific sequences of DNA. In this study, the stopped-flow and time-resolved fluorescence lifetime measurements were used to observe the kinetics of the distance changes between the N-terminal and C-terminal domain of CRP induced by binding of cAMP to high-affinity binding sites. In these measurements, we used the constructed CRP heterodimer, which possesses a single Trp85 residue localized at the N-terminal domain of one CRP subunit, and fluorescently labeled by 1,5-I-AEDANS Cys178 localized at the C-terminal domain of the same subunit or at the opposite one. The Förster resonance energy transfer method has been used to study the distance changes, induced by binding of cAMP, between Trp85 (fluorescence donor) and Cys178-AEDANS (fluorescence acceptor) in the CRP structure. The obtained results show that the allosteric transitions of CRP at micromolar cAMP concentrations follow the sequential binding model, in which binding of cAMP to high-affinity sites causes a 4 A movement of the C-terminal domain toward N-terminal domains of the protein, with kinetics faster than 2 ms, and CRP adopts the "closed" conformation. This fast process is followed by the slower reorientation of both CRP subunits.
Links	PubMed Online version:10.1021/bi051586a
Keywords	Allosteric Regulation; Bacterial Adhesion; Circular Dichroism; Cyclic AMP/chemistry; Cyclic AMP/metabolism; Cyclic AMP Receptor Protein; Dimerization; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; Fluorescence Resonance Energy Transfer; Kinetics; Naphthalenesulfonates; Protein Binding; Protein Conformation; Protein Subunits/chemistry; Protein Subunits/metabolism; Receptors, Cell Surface/chemistry; Receptors, Cell Surface/metabolism; Transcription Factors/chemistry; Transcription Factors/metabolism; Tryptophan/chemistry; Tryptophan/metabolism
edit table

Significance

You can help EcoliWiki by summarizing why this paper is useful

Useful Materials and Methods

You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.

Annotations

<annotationlinks/>

EcoliWiki Links

Add links to pages that link here (e.g. gene, product, method pages)

References

See Help:References for how to manage references in EcoliWiki.