Solutes modify a conformational transition in a membrane transport protein.
The bacterial outer-membrane vitamin B(12) 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.
Kim
Miyeon
M
Department of Chemistry and Biophysics Program, University of Virginia, Charlottesville, 22904-4319, USA.
Xu
Qi
Q
Fanucci
Gail E
GE
Cafiso
David S
DS
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003681:Dehydration; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D011092:Polyethylene Glycols / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014805:Vitamin B 12 / Q000737:chemistry
D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D014805:Vitamin B 12