From the GRAM-Negative Bacterial Extracellular Space to Periplasmic Space with EPR: Exploring the Escherichia Coli Vitamin B12 Transporter, BtuB, in Whole Cells

Abstract

Outer membrane (OM) biogenesis processes are pivotal for Gram-negative bacterial survival. These sophisticated and well-coordinated pathways are involved in synthesis, maturation and translocation of OM lipids and OM proteins such as lipopolysaccharides (LPS), lipoproteins (LP), and outer membrane proteins (OMPs). Assembly of LPS and OMPs within the OM can impact the structure and function of OMPs within bacteria. Additionally, function of some OMPs mediated by inner membrane (IM) and periplasmic proteins. For example, the active transport of TonB dependent transporters (TBDTs) in the OM is coupled with IM proton motive force (PMF) via TonB complex in the IM. Thereby, hindering the studies of TBDTs’ transport process in isolated systems compared to their native environment, whole cells. Recently, we demonstrated an effective approach to study a TBDT, BtuB, in Escherichia coli via EPR spectroscopy (CW EPR and DEER). This approach has recently been further modified to provide more physiological conditions and is utilized to explore the dynamics of BtuB loops and core regions exposed to the extracellular space. Based on DEER analysis, we were able to detect new substrate-dependent conformational states between sites T188 (loop) and V90 (core) which are associated with the previously characterized molecular switch, R14-D3161. Additionally, we have successfully overcome the periplasmic reduction potential and spin-labeled a range of sites within the periplasmic space, which permitted potential exploration of the transport process. In the isolated systems Ton box, a folded BtuB core region in the periplasmic side, undergoes substrate induced unfolding, but we have observed novel unfolded states of apo BtuB Ton box BtuB in whole cells. [1] Lukasik, S. M., David Ho, K. W., and Cafiso, D. S. (2007), Journal of molecular biology 370, 807-811.