Osmolytes modulate conformational exchange in solvent-exposed regions of membrane proteins.
Site-directed spin labeling (SDSL) was used to investigate local structure and conformational exchange in two bacterial outer-membrane TonB-dependent transporters, BtuB and FecA. Protecting osmolytes, such as polyethylene glycols (PEGs) are known to modulate a substrate-dependent conformational equilibrium in the energy coupling motif (Ton box) of BtuB. Here, we demonstrate that a segment that is N-terminal to the Ton box in BtuB, is in conformational exchange between ordered and disordered states with or without substrate. Protecting osmolytes shift this equilibrium to favor the more ordered, folded state. However, a segment of BtuB that is C-terminal to the Ton box that is not solvent exposed is insensitive to PEGs. Protecting osmolytes also modulate a conformational equilibrium in the Ton box of FecA, with larger molecular weight PEGs producing the largest shifts in the conformational free energy. These data indicate that solvent-exposed regions of these transporters undergo conformational exchange and that regions of these transporters that are involved in protein-protein interactions sample multiple conformational substates. The sensitivity to solute provides an explanation for differences seen between two high-resolution structures of BtuB, which each likely represent one conformation from a subset of states that are normally sampled by the protein. This work also illustrates how SDSL and osmolytes may be used to characterize and quantitate conformational equilibria in membrane proteins.
Flores Jiménez
Ricardo H
RH
Department of Chemistry and Biophysics Program, University of Virginia, Charlottesville, Virginia 22904-4319, USA.
Do Cao
Marie-Ange
MA
Kim
Miyeon
M
Cafiso
David S
DS
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009995:Osmosis; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry*; D013379:Substrate Specificity
D001425:Bacterial Outer Membrane Proteins; D026901:Membrane Transport Proteins; D012997:Solvents