Department of Chemistry, Biophysics Program, and Center for Membrane Biology, University of Virginia, Charlottesville, Virginia 22904-4319, United States.
Biochemistry. 2012 May 1;51(17):3642-50. doi: 10.1021/bi300118a. Epub 2012 Apr 22.
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.
革兰氏阴性菌含有一组外膜转运蛋白，这些蛋白在摄取稀有营养物质（如铁和维生素 B(12)）方面发挥作用。这些蛋白被称为 TonB 依赖性，因为运输需要与内膜蛋白 TonB 相互作用。我们使用定点自旋标记和化学变性的组合，检查了大肠杆菌维生素 B(12)转运蛋白 BtuB 的特定区域的特异性展开。数据表明，该蛋白的 N 端部分占据了 BtuB 桶的内腔，在桶的展开之前变性，并且 N 端的折叠自由能小于通常看到的球状蛋白。此外，数据表明，N 端结构域不是以单个事件展开，而是以一系列独立的步骤展开。N 端的展开是可逆的，去除变性剂会恢复蛋白质的天然折叠。这些数据与涉及蛋白质 N 端瞬时重排或展开的提议的运输机制一致，并提供了可能是运输过程中涉及的特定蛋白质构象的证据。
