TAILIEUCHUNG - Báo cáo khoa học: Steady-state and time-resolved fluorescence studies of conformational changes induced by cyclic AMP and DNA binding to cyclic AMP receptor protein from Escherichia coli

cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes inEscheri-chia coli. As binding of cAMP leads to undefined conform-ational changes in CRP, we performed a steady-state and time-resolved fluorescence study to showhow the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single trypto-phan residue at position 85 or 13, and fluorescently labeled with 1,5-I-AEDANS attached toCys178. | Eur. J. Biochem. 270 1413-1423 2003 FEBS 2003 doi Steady-state and time-resolved fluorescence studies of conformational changes induced by cyclic AMP and DNA binding to cyclic AMP receptor protein from Escherichia coli Agnieszka Polit Urszula Btaszczyk and Zygmunt Wasylewski Department of Physical Biochemistry Faculty of Biotechnology Jagiellonian University Krakow Poland cAMP receptor protein CRP allosterically activated by cAMP regulates the expression of several genes in Escherichia coli. As binding of cAMP leads to undefined conformational changes in CRP we performed a steady-state and time-resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single tryptophan residue at position 85 or 13 and fluorescently labeled with 1 5-I-AEDANS attached to Cys178. Binding of cAMP in the CRP- cAMP 2 complex leads to changes in the Trp13 microenvironment whereas its binding in the CRP- cAMP 4 complex alters the surroundings of Trp85. Time-resolved anisotropy measurements indicated that cAMP binding in the CRP- cAMP 2 complex led to a substantial increase in the rotational mobility of the Trp13 residue. Measurement of fluorescence energy transfer FRET between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP- cAMP 2 complex caused a substantial increase in FRET efficiency. This indicates a decrease in the distance between the two domains of the protein from A in apo-CRP to A in the CRP- cAMP 2 complex. The binding of cAMP in the CRP- cAMP 4 complex resulted in only a very small increase in FRET efficiency. The average distance between the two domains in CRP-DNA complexes possessing lac gal or ICAP sequences shows an increase as evidenced by the increase in the average distance between Cys178 and Trp85 to w 20 A. The spectral changes observed provide new structural information about the cAMP-induced allosteric .

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