ArticleOsmolytes modulate conformational exchange in solvent-exposed regions of membrane proteins |
| Ricardo H. Flores Jiménez, Marie-Ange Do Cao, Miyeon Kim a, David S. Cafiso * |
Department of Chemistry and Biophysics Program, University of Virginia, Charlottesville, Virginia
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| email: David S. Cafiso (cafiso@virginia.edu) |
*Correspondence to David S. Cafiso, Department of Chemistry, McCormick Road University of Virginia, Charlottesville, VA 22904-4319
aCurrent address: The Jules Stein Eye Institute, UCLA school of Medicine, Los Angeles, CA 90095
Funded by:
National Institutes of Health, NIGMS; Grant Number: GM 035215
| site-directed spin labeling TonB-dependent transport EPR spectroscopy protein dynamics |
| 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. |
Received: 23 September 2009; Revised: 18 November 2009; Accepted: 4 December 2009
10.1002/pro.305
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