Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824.
Department of Chemistry, Michigan State University, East Lansing, MI 48824.
Proc Natl Acad Sci U S A. 2022 Jan 4;119(1). doi: 10.1073/pnas.2109169119.
Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of () in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy. These data, along with our simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. 's lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.
定义变性状态集合（DSE）和无规蛋白对于理解折叠、伴侣蛋白作用、降解和易位至关重要。与水溶性蛋白相比，膜蛋白的 DSE 特征要少得多。在这里，我们测量了 () 螺旋膜蛋白 GlpG 的天然类似脂质双层中的 DSE。DSE 是使用我们的位阻捕获方法获得的，该方法将双生物素化 GlpG 的变性与两个链霉亲和素分子的结合偶联。使用有限的蛋白水解和质谱法探测螺旋和环，而使用我们的顺磁生物素衍生物和双电子-电子共振光谱法确定尺寸。这些数据以及我们的模拟确定 DSE 是高度动态的，涉及一些跨膜（TM）螺旋的拓扑变化和展开。与天然状态相比，DSE 会扩展，但仅扩展到完全展开状态的 15%至 75%。扩展程度取决于局部蛋白质包装和脂质组成。'的脂质双层促进了 DSE 中 TM 螺旋的缔合，并且可能通常促进了螺旋间相互作用。这种趋势可能是细胞膜内蛋白质普遍疏水性的结果。
