Steric trapping reveals a cooperativity network in the intramembrane protease GlpG.
Steric trapping reveals a cooperativity network in the intramembrane protease GlpG.
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DOI：
10.1038/nchembio.2048
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发表时间：
2016-05
影响因子：
14.8
通讯作者：
Hong H
中科院分区：
文献类型：
--
作者：
Guo R;Gaffney K;Yang Z;Kim M;Sungsuwan S;Huang X;Hubbell WL;Hong H
膜蛋白通过蛋白质、脂质和水之间的平衡相互作用组装。研究它们的折叠，同时保持天然的脂质环境是必要的，但具有挑战性。在这里，我们提出了分析膜蛋白折叠的关键因素，包括热力学稳定性，在自然条件下的折叠状态和折叠的协同性的紧凑性的方法。所述方法基于空间捕获，其将双生物素化蛋白质的解折叠与单价链霉亲和素（mSA）的结合偶联。我们通过开发具有通过mSA结合或蛋白质解折叠敏化的光谱报告物的多功能生物素探针，进一步推进了该技术的一般应用。通过将这些方法应用于大肠杆菌的膜内蛋白酶GlpG，我们阐明了一个广泛解开的未折叠状态，亚全局展开的区域涵盖的活性位点，和网络的合作和本地化的相互作用，以保持稳定性。这些发现为膜蛋白的折叠能量景观提供了重要的见解。
Membrane proteins are assembled through balanced interactions among protein, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements in membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping which couples unfolding of a doubly-biotinylated protein to binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to an intramembrane protease GlpG of Escherichia coli, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain the stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.