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J. Biol. Chem., Vol. 279, Issue 7, 5528-5536, February 13, 2004

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Phosphorylation-induced Conformational Changes of Cystic Fibrosis Transmembrane Conductance Regulator Monitored by Attenuated Total Reflection-Fourier Transform IR Spectroscopy and Fluorescence Spectroscopy^*

Vinciane Grimard¶, Canhui Li||, Mohabir Ramjeesingh||, Christine E. Bear||, Erik Goormaghtigh**, and Jean-Marie Ruysschaert

From the Center for Structural Biology and Bioinformatics, Free University of Brussels, Campus Plaine CP206/2, 1050 Brussels, Belgium and the ^||Program in Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada

Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ABC protein superfamily. Phosphorylation of a regulatory domain of this protein is a prerequisite for activity. We analyzed the effect of protein kinase A (PKA) phosphorylation on the structure of purified and reconstituted CFTR protein. ¹H/²H exchange monitored by attenuated total reflection Fourier transform IR spectroscopy demonstrates that CFTR is highly accessible to aqueous medium. Phosphorylation of the regulatory (R) domain by PKA further increases this accessibility. More specifically, fluorescence quenching of cytosolic tryptophan residues revealed that the accessibility of the cytoplasmic part of the protein is modified by phosphorylation. Moreover, the combination of polarized IR spectroscopy with ¹H/²H exchange suggested an increase of the accessibility of the transmembrane domains of CFTR. This suggests that CFTR phosphorylation can induce a large conformational change that could correspond either to a displacement of the R domain or to long range conformational changes transmitted from the phosphorylation sites to the nucleotide binding domains and the transmembrane segments. Such structural changes may provide better access for the solutes to the nucleotide binding domains and the ion binding site.

Received for publication, October 7, 2003 , and in revised form, December 1, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A Research Fellow with the National Fund for Scientific Research, Belgium.

^¶ Present address: Max-Planck Institute of Molecular Biology and Genetics, Pfotenhauerstasse 108, 01307 Dresden, Germany.

^** Director of Research with the National Fund for Scientific Research, Belgium.

To whom correspondence should be addressed: Center of Structural Biology and Bioinformatics, Free University of Brussels, Campus Plaine CP206/2, 1050 Brussels, Belgium. Tel.: 32-2-650-53-77; Fax: 32-2-650-53-82; E-mail: jmruysss{at}ulb.ac.be.

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