Analysis of Activation-induced Conformational Changes in p47 phox Using Tryptophan Fluorescence Spectroscopy

• Published in: The Journal of biological chemistry 1997-11, Vol.272 (47), p.29502-29510
• Main Authors: Swain, Steve D., Helgerson, Sam L., Davis, Angela R., Nelson, Laura K., Quinn, Mark T.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.47.29502

Activation of the neutrophil NADPH oxidase requires translocation of cytosolic proteins p47 phox , p67 phox , and Rac to the plasma membrane or phagosomal membrane, where they assemble with membrane-bound flavocytochrome b. During this process, it appears that p47 phox undergoes conformational changes, resulting in the exposure of binding sites involved in assembly and activation of the oxidase. In the present study, we have directly evaluated activation-induced conformational changes in p47 phox using tryptophan fluorescence and circular dichroism spectroscopy. Treatment of p47 phox with amphiphilic agents known to activate the NADPH oxidase (SDS and arachidonic acid) caused a dose-dependent quenching in the intrinsic tryptophan fluorescence of p47 phox , whereas treatment with a number of other amphiphilic agents that failed to activate the oxidase had no effect on p47 phox fluorescence. In addition, the concentration range of activating agents required to induce changes in fluorescence correlated with the concentration range of these agents that induced maximal NADPH oxidase activity in a cell-free assay system. We next determined if activation by phosphorylation caused the same type of conformational changes in p47 phox . Protein kinase C phosphorylation of p47 phoxin vitro resulted in comparable quenching of fluorescence, which also correlated directly with NADPH oxidase activity. Finally, the circular dichroism (CD) spectrum of p47 phox was significantly changed by the addition of SDS, whereas treatment with a non-activating detergent had no effect on the CD spectrum. These results support the conclusion that activation by amphiphilic agents results in changes in the secondary structure of p47 phox . Thus, our studies provide direct evidence linking conformational changes in p47 phox to the NADPH oxidase activation/assembly process and also further support the hypothesis that amphiphile-mediated activation of the NADPH oxidase induces changes in p47 phox that are similar to those mediated by phosphorylation in vivo.