Abstract
The acquisition and transport of ferrous iron (Fe2+) is essential for the survival and the virulence of many infectious prokaryotes. While bacteria possess several methods to acquire Fe2+, the ferrous iron transport (Feo) system is the most important Fe2+ transport complex with strong ties to bacterial virulence. Despite this significance, an understanding of the structure‐function relationship of the transporter FeoB is limited due to its intrinsically insoluble nature that renders it generally intractable outside of a lipid bilayer. Traditionally, detergents have been used to solubilize membrane proteins such as FeoB; however, detergent micelles can disrupt protein folding, lead to protein instability, and even inhibit important and essential protein‐protein interactions. In lieu of traditional amphipathic detergents, styrene‐maleic acid (SMA) copolymers have been demonstrated to solubilize membrane proteins into a nanodisc while encapsulating native lipids, thereby preventing protein misfolding and preserving protein‐protein interaction sites. In this work, we describe the first successful solubilization and purification of Escherichia coli FeoB (EcFeoB) in SMA nanodiscs. This approach has afforded our ability to obtain highly pure, monomeric EcFeoB‐SMA nanodiscs. In addition, we compare the NTPase activity of detergent‐solubilized EcFeoB to that of SMA‐solubilized EcFeoB nanodiscs to probe nucleotide‐hydrolysis rates in the presence of an artificial micelle compared to a lipid bilayer. This approach represents a promising first step in understanding the structure and the mechanism of the FeoB transporter in its native environment.
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