Using CW-EPR to Explore Substrate Binding and the Mechanism of Tonb-Dependent Transport in BtuB

Abstract

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. During the coupling of the Ton box to TonB, transport is thought to involve a transient unfolding or rearrangement of the N-terminal core promoting the release of the substrate to the periplasm. Utilizing a combination of site-directed spin labeling (SDSL) and chemical denaturation we have examined the thermal stability of the core domain in the Escherichia coli vitamin B12 transporter, BtuB, as well as the effects of substrate binding on the stability of the core. The data indicate that core unfolds in a series of steps and that substrate, which alters the stability of the Ton box, also alters the thermal stability of the core. Pulse EPR methods are being used to determine the steps that occur during transport and to determine the position and binding sites for the substrate within the transporter.This work was supported by NIGMS, GM035215.