TAILIEUCHUNG - Báo cáo khóa học: Insight into the activation mechanism of Bordetella pertussis adenylate cyclase by calmodulin using fluorescence spectroscopy

The interaction of the adenylate cyclase catalytic domain (AC) of the Bordetella pertussismajor exotoxin with its activator calmodulin (CaM) was studied by time-resolved fluorescence spectroscopy using three fluorescent groups located indifferent regions ofAC: tryptophan residues (W69 and W242), a nucleotide analogue (3¢-anthraniloyl-2¢-deoxyadenosine 5¢-triphosphate, Ant-dATP) and a cysteine-specific probe (acrylodan). | Eur. J. Biochem. 271 821-833 2004 FEBS 2004 doi Insight into the activation mechanism of Bordetella pertussis adenylate cyclase by calmodulin using fluorescence spectroscopy Jacques Gallay1 Michel Vincent1 Ines M. Li de la Sierra2 Helene Munier-Lehmann2 Madalena Renouard1 Hiroshi Sakamoto2 Octavian Barzu2 and Anne-Marie Gilles2 1 Laboratoire pour I Utilisation du Rayonnement Electromagnetique UMR 130 du CNRS Universite Paris-Sud Orsay France 2Laboratoire de Chimie Structurale des Macromolecules URA 2185 du CNRS Institut Pasteur Paris cedex France The interaction of the adenylate cyclase catalytic domain AC of the Bordetella pertussis major exotoxin with its activator calmodulin CaM was studied by time-resolved fluorescence spectroscopy using three fluorescent groups located in different regions of AC tryptophan residues W69 and W242 a nucleotide analogue 3 -anthraniloyl-2 -deoxyadenosine 5 -triphosphate Ant-dATP and a cysteinespecific probe acrylodan . CaM binding elicited large changes in the dynamics of W242 which dominates the fluorescence emission of both AC and AC-CaM similar to that observed for isolated CaM-binding sequences of different lengths Bouhss A. Vincent M. Munier H. Gilles . Takahashi M. Barzu O. Danchin A. Gallay J. 1996 Eur. J. Biochem. 237 619-628 . In contrast Ant-dATP remains completely immobile and inaccessible to the solvent in both the AC and AC-CaM nucleotide-binding sites. As AC contains no cysteine residue a single-Cys mutant at position 75 was constructed which allowed labeling of the catalytic domain with acrylodan. Its environment is strongly apolar and rigid and only slightly affected by CaM. The protein s hydrodynamic properties were also studied by fluorescence anisotropy decay measurements. The average Brownian rotational correlation times of AC differed significantly according to the probe used 19 ns for W242 25 ns for Ant-dATP and 35 ns for acrylodan suggesting an elongated protein shape axial

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