This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.105.078246v1 90/8/2922    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, M. Articles by Cafiso, D. S. Search for Related Content PubMed PubMed Citation Articles by Kim, M. Articles by Cafiso, D. S.

Solutes Modify a Conformational Transition in a Membrane Transport Protein

Miyeon Kim ^*, Qi Xu ^*, Gail E. Fanucci  and David S. Cafiso ^*

^* Department of Chemistry and Biophysics Program, University of Virginia, Charlottesville, Virginia; and Department of Chemistry, University of Florida, Gainesville, Florida

Correspondence: Address reprint requests to David S. Cafiso, Dept. of Chemistry, University of Virginia, Charlottesville, VA 22904-4319. Tel.: 434-924-3067; E-mail: cafiso{at}virginia.edu.

The bacterial outer-membrane vitamin B₁₂ transporter, BtuB, undergoes a dramatic order-to-disorder transition in its N-terminal energy-coupling motif (Ton box) upon substrate binding. Here, site-directed spin labeling (SDSL) is used to show that a range of solutes prevents this conformational change when ligand is bound to BtuB, resulting in a more ordered Ton box structure. For each solute examined, the data indicate that solutes effectively block this conformational transition through an osmotic mechanism. The molecular weight dependence of this solute effect has been examined for a series of polyethylene glycols, and a sharp molecular weight cutoff is observed. This cutoff indicates that solutes are preferentially excluded from a cavity within the protein as well as the protein surface. Furthermore, the sensitivity of the conformational change to solution osmolality is consistent with a structural model predicted by SDSL. When the Ton box is unfolded by detergents or mutations (rather than by ligand binding), solutes, such as polyethylene glycols and salts, also induce a more structured compacted conformation. These results suggest that conformational changes in this class of outer membrane transporters, which involve modest energy differences and changes in hydration, may be modulated by a range of solutes, including solutes typically used in protein crystallization.

This article has been cited by other articles:

				C. B. Stanley and H. H. Strey Osmotically Induced Helix-Coil Transition in Poly(Glutamic Acid) Biophys. J., June 1, 2008; 94(11): 4427 - 4434. [Abstract] [Full Text] [PDF]

				B. Knierim, K. P. Hofmann, O. P. Ernst, and W. L. Hubbell Sequence of late molecular events in the activation of rhodopsin PNAS, December 18, 2007; 104(51): 20290 - 20295. [Abstract] [Full Text] [PDF]

				M. Kim, G. E. Fanucci, and D. S. Cafiso Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved PNAS, July 17, 2007; 104(29): 11975 - 11980. [Abstract] [Full Text] [PDF]

				P. J. Bond, J. P. Derrick, and M. S. P. Sansom Membrane Simulations of OpcA: Gating in the Loops? Biophys. J., January 15, 2007; 92(2): L23 - L25. [Abstract] [Full Text] [PDF]

				N. Van Eps, W. M. Oldham, H. E. Hamm, and W. L. Hubbell Structural and dynamical changes in an {alpha}-subunit of a heterotrimeric G protein along the activation pathway PNAS, October 31, 2006; 103(44): 16194 - 16199. [Abstract] [Full Text] [PDF]