Structural and Functional Studies of Ferrous Iron Transporter B

Abstract

The transmembrane bacterial iron importer FeoB has been shown to be the key component for ferrous iron acquisition for most pathogenic bacteria and has been shown to be a viable antimicrobial target. The molecular mechanisms for FeoB’s iron transport are not well understood with the most significant finding being active transport via a cytosolic GTPase. The lack of understanding is the result of inherent difficulties in obtaining pure monodispersed full length FeoB for structural and biochemical analysis. Truncated FeoB GTPase domains (termed NFeoB) from various species have been deposited in the PDB with and without bound GTP analogues. The key to furthering the knowledge of bacterial ferrous iron transport involves improving techniques in expression and purification of the full length FeoB protein. To obtain more monodispersed and pure protein, expression of FeoB was trialled with a TEV cleavable GST affinity tag, purified with the reducing agent, Sodium dithionite as well as, with and without GDP. Another construct of FeoB was tested with a small fused flexible C-terminal region (to allow for oligomerisation using an 8 histidine affinity tag). Expression was also trialled within an FtsH (a FeoB specific protease) E. coli knockout. Sodium dithionite evoked a more pure FeoB sample, compared to purification without, as did the flexible C-terminal construct, however producing no protein crystals from either experiment. FeoB expressed in FtsH knockout cells was less degraded compared to the cell’s isogenic paired counterpart. However degradation was not less than our lab’s standard membrane expression cell line ‘C41’. Important outcomes from this work include; purification with Sodium dithionite proved vitally important in stabilising FeoB during purification as well as expression (i.e. stabilised oligomerisation) and also improved expression systems with FtsH knockout cells which are all promising directions to take to obtain a more pure and monodispersed protein.