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Conformational changes in truncated p47phox proteins monitored by fluorescent labeling.
1Department of Biochemistry, College of Natural Sciences, Kyung-pook National University, Taegu, Korea.
Journal of Protein Chemistry
|April 20, 2001
Summary
Researchers investigated the activation of leukocyte NADPH oxidase, a key enzyme in neutrophils. They found that the C-terminal region of p47phox undergoes conformational changes during activation, exposing its SH3 domains.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Leukocyte NADPH oxidase is a crucial membrane-bound enzyme in neutrophils, responsible for producing superoxide (O2-).
- Activation involves cytosolic components p47phox and p67phox, containing Src homology 3 (SH3) domains, migrating to the plasma membrane to assemble with cytochrome b558.
Purpose of the Study:
- To elucidate the conformational changes in p47phox during NADPH oxidase activation.
- To identify the specific region of p47phox responsible for SH3 domain masking in the resting state.
Main Methods:
- Utilized a cell-free system mimicking oxidase activation with anionic amphiphiles (e.g., sodium dodecyl sulfate).
- Employed fluorescence spectroscopy with a covalently labeled probe (IANBD) to monitor conformational changes in full-length and truncated p47phox variants.
Main Results:
- Anionic amphiphiles induced increased fluorescence in full-length p47phox and N-terminal-truncated p47Phox-(SH3)2-C, indicating SH3 domain exposure.
- C-terminal-truncated p47Phox-N-(SH3)2 and doubly truncated p47Phox-(SH3)2 showed no fluorescence alteration, suggesting the C-terminus masks the SH3 domains.
Conclusions:
- The C-terminal region of p47phox plays a primary role in masking the SH3 domains in the resting state of NADPH oxidase.
- Conformational changes in the p47phox C-terminus are critical for oxidase activation.