NADPH Oxidase-5 (NOX5) is a member of the NADPH Oxidase (NOX) family that produces superoxide in cells to enhance cellular differentiation and proliferation. NOX5 also exhibits pathological effects when produced in excess. Studies have suggested that binding of Ca2+ to its Ca 2+ binding domain (CaBD) is the key factor that activates NOX5 to generate superoxide. However, the mechanism of activation of NOX5 by Ca 2+ still remains largely unknown. Previous studies carried out in our laboratory have reported that the two domains of NOX5, N-terminal domain (NCaBD) and C-terminal domain (CCaBD) have different Ca2+ binding properties. Stopped-flow spectrometry revealed a fast Ca2+ dissociation from NCaBD followed by slow Ca2+ dissociation from CCaBD and chemical stability studies suggested higher Ca2+ binding affinity and higher chemical stability for CCaBD than for NCaBD. In order to obtain a better insight on the difference between these two domains, here we characterized the Ca2+ binding specifics of CaBD and its mutant, CaBD-Q3Q4 using Fluorescence spectroscopy, Isothermal Titration Calorimetry (ITC) and Differential Scanning Calorimetry (DSC). Also, we carried out fluorescence experiments to investigate the interaction between the flavoprotein domain and CaBD upon Ca2+ binding. We hope that the results obtained will help us to have a better understanding on NOX5 activation by Ca2+ binding.
ANS studies using fluorescence spectroscopy and ITC both suggested higher surface hydrophobicity for CaBD than for CaBD-Q3Q4 upon Ca2+ binding. TCSPC fluorescence lifetime studies of CaBD revealed the presence of two different types of Trp residues with lifetimes τ1 = 2.01 ns and τ2 = 5.50 ns. DSC data revealed a higher melting temperature difference (ΔTm) value for CCaBD than for NCaBD, suggesting the C-terminal domain has higher thermostability and hence higher Ca2+ binding affinity than N-terminal domain. This result corresponds to the stability data obtained from chemical denaturation experiments in the past. The significant increase in ΔTm value for NCaBD upon increasing salt concentration suggested the role of ionic interactions and hydrogen-bonding for the stability of NCaBD upon Ca2+ binding, whereas a comparatively lower increase in ΔTm value for CCaBD indicated the primary involvement of hydrophobic interactions for stability. From the fluorescence studies of the refolded flavoprotein and AEDANS labelled CaBD, we concluded that Ca2+ binding to CaBD brings about a conformational change that exposes the hydropbibic residues in CaBD, which serves as an interface to interact with the flavoprotein domain.
|Commitee:||Luesse, Sarah, O'Brien, Leah, Wei, Chin Chuan|
|School:||Southern Illinois University at Edwardsville|
|School Location:||United States -- Illinois|
|Source:||MAI 53/06M(E), Masters Abstracts International|
|Keywords:||CaBD, Calcium binding domain, NOX5, NaDPH oxidase-5, Superoxide|
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