title: The N-terminal domain of a TonB-dependent transporter undergoes a reversible stepwise denaturation.
abstract: Gram-negative bacteria contain a family of outer membrane transport proteins that function in the uptake of rare nutrients, such as iron and vitamin B(12). These proteins are termed TonB-dependent because transport requires an interaction with the inner-membrane protein TonB. Using a combination of site-directed spin labeling and chemical denaturation, we examined the site-specific unfolding of regions of the Escherichia coli vitamin B(12) transporter, BtuB. The data indicate that a portion of the N-terminal region of the protein, which occupies the lumen of the BtuB barrel, denatures prior to the unfolding of the barrel and that the free energy of folding for the N-terminus is smaller than that typically seen for globular proteins. Moreover, the data indicate that the N-terminal domain does not unfold in a single event but unfolds in a series of independent steps. The unfolding of the N-terminus is reversible, and removal of denaturant restores the native fold of the protein. These data are consistent with proposed transport mechanisms that involve a transient rearrangement or unfolding of the N-terminus of the protein, and they provide evidence of a specific protein conformation that might be an intermediate accessed during transport.
authors.affiliation: Department of Chemistry, Biophysics Program, and Center for Membrane Biology, University of Virginia, Charlottesville, Virginia 22904-4319, United States.
authors.forename: Ricardo H
authors.initials: RH
authors.lastname: Flores Jiménez
authors.forename: David S
authors.initials: DS
authors.lastname: Cafiso
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D001693:Biological Transport, Active; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D014805:Vitamin B 12 / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; C043426:inner membrane protein, E coli; C489181:tonB protein, E coli; D014805:Vitamin B 12