Conformational States and kinetics of the calcium binding domain of NADPH oxidase 5.

The Open Biochemistry Journal
Chin-Chuan WeiDennis J Stuehr

Abstract

Superoxide generated by human NADPH oxidase 5 (NOX5) is of growing importance for various physiological and pathological processes. The activity of NOX5 appears to be regulated by a self-contained Ca(2+) binding domain (CaBD). Recently Bánfi et al. suggest that the conformational change of CaBD upon Ca(2+) binding is essential for domain-domain interaction and superoxide production. The authors studied its structural change using intrinsic Trp fluorescence and hydrophobic dye binding; however, their conformational study was not thorough and the kinetics of metal binding was not demonstrated. Here we generated the recombinant CaBD and an E99Q/E143Q mutant to characterize them using fluorescence spectroscopy. Ca(2+) binding to CaBD induces a conformational change that exposes hydrophobic patches and increases the quenching accessibilities of its Trp residues and AEDANS at Cys107. The circular dichroism spectra indicated no significant changes in the secondary structures of CaBD upon metal binding. Stopped-flow spectrometry revealed a fast Ca(2+) dissociation from the N-terminal half, followed by a slow Ca(2+) dissociation from the C-terminal half. Combined with a chemical stability study, we concluded that the C-terminal half of ...Continue Reading

Citations

Dec 21, 2011·Free Radical Biology & Medicine·Karen BedardKarl-Heinz Krause
Nov 3, 2010·Clinical Science·Augusto C MontezanoRhian M Touyz
Aug 4, 2016·Frontiers in Physiology·Feng ChenDavid J R Fulton
Dec 1, 2019·The FEBS Journal·Elisa Millana FañanásAndrea Mattevi

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Methods Mentioned

BETA
flow dialysis
PCR
protein assay
Circular Dichroism
Fluorescence
fluorescence spectroscopy

Software Mentioned

AEDANS
IT
SCAN
UN

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