Comparative Analysis of Pulsed EPR Distance Measurements in an E. coli Cobalamin Transporter in Cells Versus Isolated Outer Membranes Reveals Novel Conformational Changes Dependent on the Native Environment

Abstract

Gram negative bacteria are reliant on TonB dependent transporters for the movement of scarce compounds across their outer membranes. These proteins consist of an outer β-barrel surrounding an internal hatch domain. The substrates, including iron siderophores and cobalamin, are large and would necessitate significant conformational rearrangement of the hatch domain to facilitate their movement. In vitro work on the E. coli cobalamin transporter, BtuB, using the pulsed EPR technique double electron-electron resonance (DEER) in our lab has thus far failed to produce evidence of such conformational changes within the hatch domain. Recently, we have focused on the development of techniques to study the protein in intact systems, including whole cells and isolated outer membranes. While investigating hatch-barrel mutant pairs in these systems, we have seen evidence for conformational changes in the whole cells that are induced by the presence of substrate, and that are absent or greatly suppressed in the isolated membrane alone. These results may reveal elements of the transport process, as well as highlighting the importance of studying proteins in their native environments. This work was supported by NIGMS, GM035215.