Folding Proteins Outside Their Native Environment: Folding the Innards of an Outer Membrane Transporter

Abstract

During metabolite transport, TonB dependent receptors, found in the outer membranes of many bacterial species, are hypothesized to unfold their N-terminal “plug” domain. The 130 aa plug normally is folded inside a ∼450-550 aa, 22-strand β barrel, and must undergo significant rearrangement to allow passage of large (∼1kDa) substrates such as vitamin B12. In the crystal structures, the interface between the plugs and the inside surface of the barrels is well hydrated and is composed primarily of H-bonded, charged, and polar contacts, despite being located inside the membrane plane. Here, we present CD, NMR, and SAXS results indicating that several such plug domains are unfolded in solution whereas those from BtuB and CirA adopt a partial globular fold consistent with their crystal structures. In the crystal structure, the plugs have internal void volumes, suggesting that they can unfold non-cooperatively, as has been shown in urea denaturation EPR as well as recent AFM experiments. This is supported by our kinetic folding data and pressure denaturation NMR experiments, which has implications for folding pathways as well as the transport function. NMR studies of the BtuB & CirA plug domains will be presented, in which we take a divide and conquer approach to finding the minimal folding subunit of the plug.