Characterization of intact FeoB in a lipid bilayer using styrene-maleic acid (SMA) copolymers.

The acquisition of ferrous iron (Fe(2+)) is crucial for the survival of many pathogenic bacteria living within acidic and/or anoxic conditions such as Vibrio cholerae, the causative agent of the disease cholera. Bacterial pathogens utilize iron as a cofactor to drive essential metabolic processes, and the primary prokaryotic Fe(2+) acquisition mechanism is the ferrous iron transport (Feo) system. In V. cholerae, the Feo system comprises two cytosolic proteins (FeoA, FeoC) and a complex, polytopic transmembrane protein (FeoB) that is regulated by an N-terminal soluble domain (NFeoB) with promiscuous NTPase activity. While the soluble components of the Feo system have been frequently studied, very few reports exist on the intact membrane protein FeoB. Moreover, FeoB has been characterize almost exclusively in detergent micelles that can cause protein misfolding, disrupt protein oligomerization, and even dramatically alter protein function. As many of these characteristics of FeoB remain unclear, there is a critical need to characterize FeoB in a more native-like lipid environment. To address this unmet need, we employ styrene-maleic acid (SMA) copolymers to isolate and to characterize V. cholerae FeoB (VcFeoB) encapsulated by a styrene-maleic acid lipid particle (SMALP). In this work, we describe the development of a workflow for the expression and the purification of VcFeoB in a SMALP. Leveraging mass photometry, we explore the oligomerization of FeoB in a lipid bilayer and show that the VcFeoB-SMALP is mostly monomeric, consistent with our previous oligomerization observations in surfo. Finally, we characterize the NTPase activity of VcFeoB in the SMALP and in a detergent (DDM), revealing higher NTPase activity in the presence of the lipid bilayer. When taken together, this report represents the first characterization of any FeoB in a native-like lipid bilayer and provides a viable approach for the future structural characterization of FeoB.