Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli.
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 Forster 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.
Tworzydło
Magdalena
M
Department of Physical Biochemistry, Faculty of Biotechnology, Jagiellonian University, Kraków, Poland.
Polit
Agnieszka
A
Mikołajczak
Jan
J
Wasylewski
Zygmunt
Z
D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D014364:Tryptophan; D000242:Cyclic AMP