Outer-membrane TonB-dependent transporters function in the uptake of essential nutrients, and are important for the success of many pathogenic bacteria These proteins consist of a 22 stranded β-barrel where the N-terminal 130 to 150 residues form a core domain that fills the barrel. During transport, these proteins undergo a cycle of binding and unbinding to the inner membrane protein TonB, through an interaction that is mediated by the Ton box, an energy coupling segment near the transporter N-terminus. Over 50 high-resolution crystal structures have been obtained for 12 different TonB-dependent transporters, however the mechanisms of substrate transport remain unclear. Determination of membrane protein structure or dynamics with high resolution in whole cells is an attractive way to solve transport mechanisms but yet to be demonstrated.
In this work, the cobalamin transporter BtuB was overexpressed and spin labelled in whole cells and outer membranes and interspin distances were measured to a spin labelled cobalamin using pulse EPR. This represents the first example of double electron-electron resonance (DEER) preformed in whole E. coli cells. A comparative analysis of the data reveals a similar interspin distance distribution between whole cells, outer membranes and synthetic vesicles. We then take advantage of these native lipid environments to study the conformational heterogeneity of BtuB outer loops. The data indicate that there is a strong calcium mediated ordering of the 1-2 loop. We also see evidence of allosteric loop modulation, the binding of TonB to the Ton box on the periplasmic surface of BtuB alters the configuration of these extracellular loops. This approach provides an elegant way to study conformational changes or protein-protein/ligand interactions for large outer membrane protein complexes in whole cells and native membranes, and provides a method to validate high-resolution structures of membrane proteins in their native environment.
Available at: http://works.bepress.com/arthur-sikora/15/