Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism
| العنوان: | Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism |
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| المؤلفون: | Nicole Reynolds, Chin Chuan Wei, Ya Ping Yang, Dennis J. Stuehr, Tiffany Boyle, Christina Palka, Kristen Wetherell, Zhi Qiang Wang |
| المصدر: | Chemistry Central Journal Chemistry Central Journal, Vol 6, Iss 1, p 29 (2012) |
| سنة النشر: | 2012 |
| مصطلحات موضوعية: | Conformational change, Circular dichroism, Chemistry(all), 030303 biophysics, chemistry.chemical_element, Nanotechnology, Calcium, EF-hand, Fluorescence, 03 medical and health sciences, chemistry.chemical_compound, Protein secondary structure, QD1-999, 030304 developmental biology, 0303 health sciences, EF hand, Superoxide, Isothermal titration calorimetry, General Chemistry, 3. Good health, Chemistry, chemistry, NADPH Oxidase 5, Biophysics, Calcium binding, Binding domain, Research Article |
| الوصف: | Background Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD) of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1st and 2nd EF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants. Results The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca2+ binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 105 and 5.0 × 102 M-1 with ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1st non-canonical EF-hand is largely enhanced by the binding of Ca2+ to the 2nd canonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca2+ binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its α-helix secondary structure content. All measurements exclude Mg2+-binding in NCaBD. Conclusions We demonstrated that the 1st non-canonical EF-hand of NOX5 has very weak Ca2+ binding affinity compared with the 2nd canonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca2+ binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca2+ specific. Graphical abstract |
| تدمد: | 1752-153X |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::e98ed2db5b8275b33088e58c234a0180Test https://pubmed.ncbi.nlm.nih.gov/22490336Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....e98ed2db5b8275b33088e58c234a0180 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 1752153X |
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