The N-Terminal Domain of a TonB-Dependent Transporter Undergoes a Reversible Stepwise Denaturation

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• 作者：Ricardo H. Flores Jim茅nez ; David S. Cafiso
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：May 1, 2012
• 年：2012
• 卷：51
• 期：17
• 页码：3642-3650
• 全文大小：426K
• 年卷期：v.51,no.17(May 1, 2012)
• ISSN：1520-4995

文摘

Gram-negative bacteria contain a family of outer membrane transport proteins that function in the uptake of rare nutrients, such as iron and vitamin B₁₂. 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 B₁₂ 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.