TAILIEUCHUNG - Báo cáo khoa học: 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 inEscherichia coli. It is known that the allosteric activation of CRP by cAMP involves a long-distance signal transmission from the N-ter-minal 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. | ềFEBS Journal Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli Magdalena Tworzydto Agnieszka Polit Jan Mikotajczak and Zygmunt Wasylewski Department of PhysicalBiochemistry Faculty of Biotechnology Jagiellonian University Krakow Poland Keywords cAMP receptor protein CRP CRP-DNA interactions fluorescence quenching FRET fast kinetics Correspondence Z. Wasylewski Department of Physical Biochemistry Faculty of Biotechnology Jagiellonian University ul. Gronostajowa 7 30-387 Krakow Poland Fax 48 12 66 46 902 Tel 48 12 66 46 122 E-mail wasylewski@ Received 29 July 2004 revised 22 November 2004 accepted 21 December 2004 doi 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-ter-minal 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 longdistance 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 .

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