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Characterization of EF‐hand motifs and Investigation of Domain‐Domain Interaction in NADPH Oxidase 5 and Duox Oxidase by Fluorescence Spectrometry and Isothermal Titration Calorimetry
Author(s) -
Wei ChinChuan,
Reynolds Nicole,
Wetherell Kristen,
Boyle Tiffany,
Reimer Michael
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.913.9
Subject(s) - isothermal titration calorimetry , chemistry , oxidase test , flavoprotein , conformational change , biophysics , biochemistry , enzyme , biology
The study of reactive oxygen species (ROS) generated by non‐phagocyte NADPH oxidase 5 (NOX5) and dual oxidase (DUOX) is of growing importance for vascular physiology and disease development. Their ROS activities appear to be regulated by their self‐contained EF‐hand domains (EFDs). Previously, we demonstrated that Ca 2+ induces conformational change on NOX5‐EFD and suggested that its N‐ and C‐terminal halves behave differently, including conformational change, kinetics of binding, and chemical stability. Here we extended the study by using separated half domain proteins and characterized their Ca 2+ and Mg 2+ binding using fluorescence and isothermal titration calorimetry. The key residues for Ca 2+ binding in each EF‐hand motif of the half domain proteins were mutated to determine the impacts on Ca 2+ and Mg 2+ binding. Since domain‐domain interaction is essential for ROS production, interaction of NOX5 EFD to flavoprotein was only observed in the presence of Ca 2+ . Furthermore, EFD enhances the cytochrome C reduction of flavoprotein. For DUOX‐EFD, the preliminary data suggests that a dramatic conformational change is not essential based on fluorescence data. Currently, we are performing experiments to identify the peptide sequence that interacts with its EFD and to investigate possible dimer formation that might play roles in DUOX ROS production. This work is supported in part by Cottrell College Science Award from Research Corporation.