The multidomain NADPH oxidase (NOX) protein defines a family of heme-containing enzymes dedicated to reactive oxygen species (ROS) production by transferring electrons across the membrane. As specialized ROS producers, NOX enzymes participate in many crucial physiological processes. A NOX misregulation can lead to a wide range of severe pathologies, including atherosclerosis, hypertension, diabetic nephropathy, lung fibrosis, cancer, or neurodegenerative diseases, giving this membrane protein a strong therapeutic interest.
In this context, it is crucial to elucidate the electron transfer mechanism occurring within the protein and more specifically, at the interface between the transmembrane and cytosolic domains. However, acquiring structural data is hampered by the multidomain features and the overall lack of stability associated with these proteins. Partially hydrophobic surfaces within the transmembrane region further limits insights using classical characterization techniques, which so far have only furnished information on the independent domains of NOX proteins.
Our work describes an innovative strategy specifically developed for the characterization of the entire NOX protein. In SANS, you have access to low-resolution information on the whole object. Membrane proteins need to be solubilized in detergent to be extracted from a membrane and studied. However, when studying the solubilized membrane protein embedded in a detergent belt, the SANS signal will be representative of the addition of the detergent belt and the membrane protein inside. In the end, it is hard to distinguish the specific features of the membrane protein. The detergent belt around the protein contributes to the SANS signal and hampers the collection of specific information on the solubilized membrane protein. We have been able to make it invisible, allowing access to structural information specific to the protein. Ultimately, this approach revealed an unexpected flexibility in the key region located at the hinge between the transmembrane and cytosolic NOX domains. this approach can be applied to any membrane protein system of interest for which study by SANS could reveal new structural or functional features.
The cover image for the August 4th issue of the Biophysical Journal is composed from a set of light dots, suggesting the technique, the search for structural information, and the overall shape of the protein. The image also evokes the interdomain flexibility implied by the results of this work.
The overall envelope showing the shape of the protein is predominant in the design. Here, the illustration has been elaborated in a minimalist style deliberately depicting the global shape of the protein on a dark background to support the strength of the methodology, which takes advantage of the detergent properties to erase the undesired micelle scattering contribution.
The use of light dots recalls the neutron beam scattered during SANS to determine the global envelope. These lights also appear in the background in more blurred shades in order to evoke the flexibility of the dehydrogenase domain of the protein compared to the transmembrane domain.
More generally, the "bokeh" and blurring type light points can be related to the search for structural information, which is often long and relatively complex to implement, in particular for membrane proteins.
For readers who want to know more about our research, visit https://www.ibs.fr/research/research-groups/membrane-and-pathogens-group-f-fieschi/
— Annelise Vermot (also cover art), Isabelle Petit-Härtlein, Cécile Breyton, Aline Le Roy, Michel Thépaut, Corinne Vivès, Martine Moulin, Michael Härtlein, Sergei Grudinin, Susan M.E. Smith, Christine Ebel, Anne Martel, Franck Fieschi