Schematic model of the TonB-receptor interaction. Structures of TonB interacting with the siderophore receptor FhuA (36) and vitamin B₁₂ receptor BtuB (37) have been solved. In this example, FhuA is shown in the outer membrane (OM). Its β-barrel is colored yellow, with residues 181–480 cut away for clarity and to reveal its N-terminal plug domain, shown here in red. Residues 8–18 (which contain the TonB box, residues 8–13) are colored green and shown interacting with the C terminus of TonB (colored solid blue). The ribbon structures of FhuA and the C terminus of TonB are taken from PDB file 2GRX (36). The rest of the figure is a diagram representation, and therefore is not representative of actual structures. The inner membrane is shown with ExbB, ExbD and the inner-membrane-anchored part of TonB depicted as cylinders, with a dashed blue line connecting the TonB inner-membrane cylinder to its C-terminal domain from 2GRX. The yellow lightning bolt indicates that the system is energy-driven.

Current traces obtained when membranes exposed to the specified TonB-dependent receptors were repeatedly cycled between cis 4 M urea and cis buffer exposure. Greater than 60% loss of conductance was consistently observed upon removal of urea (by perfusion) in the case of FhuA and Cir. With BtuB, we saw little reversal, generally <20%. For BtuB and FhuA, the transporters were added to the cis compartment (first arrows) with 4 M urea already perfused in. For Cir, the urea was perfused into the cis compartment (first arrow) 5 min after the addition of Cir. All traces conform to the same time and current scale, shown at the lower right. “+ Urea” indicates times when the receptor began its exposure to urea (via perfusion), whereas “− Urea” indicates the beginning of urea removal (by buffer perfusion). In all traces, perfusions were into the cis compartment at a rate of 2 mL/min over a period of 8 min. Breaks in the current traces are positions where voltage was switched to 0 or + 20 mV; those resulting current traces have been removed to simplify the figure. Note that with FhuA, there is some loss of conductance after such a protocol. The inset shows a magnification of the early minutes of the Cir current trace to display an example of the single channel conductances observed in the system for all receptors reported. Except for the short breaks in the traces, the voltage was held at cis −20 mV. The concentrations of Cir, FhuA, and BtuB in the cis compartment were approximately 14 nM, 1.4 nM, and 13 nM, respectively. Note also that after the initial exposure to urea, the free receptor in the cis compartment is washed out along with the urea; consequently, there is no free receptor in solution for the remainder of the experiment.

In the absence of an osmotic gradient, cis 4 M urea has little effect on membrane conductance. With 4 M urea in the cis solution, if FhuA is introduced to the cis compartment (final concentration ≈1.4 nM) in the presence of trans 4 M urea (A) or trans 3 M glycerol (B), little or no conductance increase is observed across the membrane. (In A, FhuA was introduced 15 min before the start of the record.) However, when the trans urea or glycerol is replaced by buffer, thus establishing an osmotic gradient across the membrane, a rapid rise in conductance across the membrane occurs. This conductance is reversible, as shown when the substitution of 4 M urea in the cis solution with buffer results in a decrease in conductance across the membrane. The breaks in the record indicate preparations for perfusing the opposite compartment of the chamber. Except in B, where the voltage polarity was switched twice for ≈1 min early in the record, the voltage was held at cis −20 mV.

An osmotic gradient is needed for proper insertion of the β-barrel proteins into the membrane; 3 M glycerol can create the osmotic gradient necessary to insert and orient FhuA. (A) Before the start of the record, FhuA (1.4 nM) was added to and then perfused out of the cis compartment, followed by the addition of T5 (1 × 10¹¹ particles, ≈55 pM) to the same compartment, producing no conductance. The trace begins (50 min after the addition of FhuA) with the perfusion (at a rate of 2 mL/min for 8 min) of 3 M glycerol into the cis compartment, and we have focused on the early minutes of the glycerol perfusion to allow easy visualization of the single channel events. Although perfusion of the cis compartment with 3 M glycerol simultaneously removes T5 phage from that compartment, it has not removed all of the phage. Current trace was obtained at −20 mV. (B) Addition of FhuA (1.4 nM) to the cis compartment with 3 M glycerol already present, and then removal of the glycerol (and FhuA) by perfusion, produced very little increase in conductance. However, addition of phage T5 (1 × 10¹¹ particles, ≈55 pM) to the trans solution caused a rapid increase in the conductance. (In comparable experiments in which the 3 M glycerol was omitted, trans T5 phage had no effect.) Arrows indicate changes to the cis solution unless otherwise specified. Current trace was obtained at cis voltages shown on the chart. These data in A and B indicate that FhuA was inserted into the membrane, with its T5 phage binding site facing either solution.

Three molar glycerol can insert FhuA to allow urea to induce conductance. The current trace begins after FhuA (1.4 nM) was added to the cis compartment with 3 M glycerol present. Later, the cis compartment was perfused with buffer, removing both FhuA and glycerol. The first arrow of the trace is the beginning of a trans 4 M urea perfusion; the rise in conductance indicates that FhuA was inserted into the membrane. The second arrow is the beginning of the urea perfusion out of the system, again demonstrating the reversibility of the conductance. (Similar results were obtained if 4 M urea was perfused into the cis rather than the trans compartment). The experiment was performed at cis +20 mV. Here, cis positive voltage was used to keep the side with the urea at negative voltage, to be consistent with the rest of the experiments shown. All perfusions in this experiment were 8 min long with a flow rate of 2 mL/min.

Cir is responsive to its cognate colicin. The current trace begins with the addition of Cir (≈14 nM) to 4 M urea. The 4 M urea-induced Cir-mediated membrane conductance is seen to be halted soon after addition of trans colicin Ia (≈15 nM). The experiment was performed with the cis compartment held at −20 mV.

FhuA is responsive to its natural ligand, ferric-ferrichrome. The current trace begins after FhuA (1.4 nM) was added to the cis compartment and washed out 10 min later by perfusion with buffer. Then, at the first arrow, 4 M urea was perfused into the cis compartment (at a rate of 2 mL per min for 8 min) and removed by a buffer perfusion (second arrow). Urea was then reintroduced, and following the second urea perfusion, the addition of ferric-ferrichrome (330 nM) to the trans compartment resulted in partial conductance decrease. The current shown was obtained at cis −20 mV, and the break in the trace indicates the time during which the voltage had been switched from −20 mV to + 20 mV.