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

Insight into the Activation Mechanism of Bordetella Pertussis Adenylate Cyclase by Calmodulin Using Fluorescence Spectroscopy

Authors: Jacques Gallay, Michel Vincent, Inès M. Li de la Sierra, Hélène Munier-Lehmann, Madalena Renouard, Hiroshi Sakamoto, Octavian Bârzu, and Anne-Marie Gilles

Field: Biochemistry, Biophysics

Document Content: This study investigates the mechanism by which calmodulin (CaM) activates the catalytic domain (AC) of the Bordetella pertussis major exotoxin. Using time-resolved fluorescence spectroscopy with various fluorescent probes (tryptophan residues, Ant-dATP, and acrylodan), researchers examined changes in the protein’s dynamics and hydrodynamic properties upon CaM binding. The findings suggest that CaM binding induces significant alterations in the AC’s conformation, leading to a more elongated shape and rigidifying the CaM-binding sequence. The study also explores the dynamics of the nucleotide-binding site and the catalytic domain, providing insights into how CaM binding facilitates the activation of adenylate cyclase activity.

Detailed Table of Contents:

• Introduction
• Materials and Methods (Chemicals, Bacterial strains, plasmids and growth conditions, Purification of the recombinant proteins, Synthesis of the acrylodan conjugate of the AC-Y75C mutant, Steady-state and time-resolved fluorescence measurements, Other analytical procedures)
• Results (Biochemical characterization of the AC, CaM and AC-CaM proteins, Dynamics of the AC nucleotide-binding site as probed by Ant-dATP, Dynamics of the AC catalytic domain as probed by acrylodan, Dynamics of the CaM-binding domain probed by W242)
• Discussion
• Hydrodynamic properties of the AC and of the AC-CaM complex
• Conclusion
• References