Osmolytes Modulate Conformational Transitions in Solvent-Exposed Regions of Two Outer Membrane Proteins

Abstract

Electron paramagnetic resonance (EPR) spectroscopic studies using site-directed spin labeling (SDSL) have been used to investigate local structure and conformational exchange in different regions of two E. coli outer-membrane TonB-dependent transporters: BtuB and FecA. It is known that the TonB-binding region of these transporters, called the Ton box, undergoes dramatic structural changes from an immobile to a mobile configuration upon substrate binding. However, this change is not observed in the crystal structures of BtuB. When SDSL is performed with protecting osmolytes such as polyethylene glycols (PEGs), which are contained in the buffers used in crystallization, this order-disorder transition of the Ton box is abolished in BtuB. Here we find that the substrate-dependent Ton box transition in FecA is also modulated by PEGs, and we show that the five residues N-terminal to the Ton box of BtuB are in conformational exchange between folded and unfolded states. Upon addition of PEGs, this equilibrium is shifted towards the folded state. Larger molecular weight PEGs produce a larger apparent shift in the conformational free energy, consistent with the finding that PEGs are excluded from protein surfaces as a result of steric interactions. The observation of conformational exchange in the N-terminus provides an explanation for differences seen between the in surfo and the in meso crystal structures of BtuB and suggests that each of these structures represents one conformational substate among a family of substates that are normally sampled by the protein. This work illustrates how SDSL and osmolytes may be used to characterize and quantitate conformational equilibria in membrane proteins.