The extracellular loops of bacterial outer membrane (OM) transporters are thought to sample a range of conformations in the apo state but to undergo a gating motion and assume a more defined conformation upon the binding of substrate. Here, we use pulse electron paramagnetic resonance to examine the conformations of the extracellular loops of BtuB, the Escherichia coli TonB-dependent vitamin B12 transporter, in whole cells. Unlike previous measurements carried out in vitro, the loops assume well-defined configurations in situ that closely match the in surfo crystal structures. Moreover, there is no evidence that the loops undergo significant gating motions upon the binding of substrate. The results demonstrate that the structure of BtuB is dependent upon an intact native OM environment, in which a critical component is likely to be the extracellular lipopolysaccharide. In general, this work indicates that measurements on OM proteins in reconstituted membrane systems may not reflect the native state of the protein in vivo.

中文翻译：

---

原生细胞环境限制大肠杆菌钴胺素转运蛋白 BtuB 中的环结构

细菌外膜 (OM) 转运蛋白的细胞外环被认为在 apo 状态下对一系列构象进行采样，但会进行门控运动并在与底物结合时呈现更明确的构象。在这里，我们使用脉冲电子顺磁共振来检查 BtuB（大肠杆菌 TonB 依赖性维生素 B12 转运蛋白）在整个细胞中的细胞外环的构象。与之前在体外进行的测量不同，这些环在原位采用明确定义的配置，与表面晶体结构密切匹配。此外，没有证据表明环在结合基底时会发生显着的门控运动。结果表明 BtuB 的结构依赖于完整的原生 OM 环境，其中关键成分可能是细胞外脂多糖。一般而言，这项工作表明，对重构膜系统中 OM 蛋白的测量可能无法反映蛋白质在体内的天然状态。