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Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli
Author(s) -
Tworzydło Magdalena,
Polit Agnieszka,
Mikołajczak Jan,
Wasylewski Zygmunt
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04540.x
Subject(s) - camp receptor protein , dna binding domain , quenching (fluorescence) , biophysics , dna , conformational change , binding domain , allosteric regulation , förster resonance energy transfer , chemistry , escherichia coli , creb1 , biochemistry , biology , microbiology and biotechnology , binding site , receptor , fluorescence , gene , promoter , gene expression , transcription factor , physics , quantum mechanics , creb
Cyclic AMP receptor protein (CRP) regulates the expression of more then 100 genes in Escherichia coli . It is known that the allosteric activation of CRP by cAMP involves a long‐distance signal transmission from the N‐terminal cAMP‐binding domain to the C‐terminal domain of CRP responsible for the interactions with specific sequences of DNA. In this report we have used a CRP mutant containing a single Trp13 located in the N‐terminal domain of the protein. We applied the iodide and acrylamide fluorescence quenching method in order to study how different DNA sequences and cAMP binding induce the conformational changes in the CRP molecule. The results presented provide evidence for the occurrence of a long‐distance conformational signal transduction within the protein from the C‐terminal DNA‐binding domain to the N‐terminal domain of CRP. This conformational signal transmission depends on the promoter sequence. We also used the stopped‐flow and Förster resonance energy transfer between labeled Cys178 of CRP and fluorescently labeled DNA sequences to study the kinetics of DNA–CRP interactions. The results thus obtained lead to the conclusion that CRP can exist in several conformational states and that their distribution is affected by binding of both the cAMP and of specific DNA sequences.