The characterization of the behavior of lipid-attached spin probes in a bilayer is of fundamental importance for correct interpretation of the results of both EPR and fluorescence studies of protein-membrane interactions. The knowledge of the immersion depth of TEMPO spin probe attached to lipid headgroup in TEMPO-PC is critical for the determination of the transverse location of fluorescence probes attached to proteins and peptides. The question of bilayer penetration of TEMPO moiety in TEMPO-PC has recently came into prominence in two studies of interfacial solvation (Cheng et al. in Biophys J 109:330-339, 2015; Lee et al. in Biophys J 111:2481-2491, 2016). Here, we re-examine the arguments on TEMPO penetration using the cross-validation of MD simulations and depth-dependent fluorescence-quenching experiments, which confirms that TEMPO in TEMPO-PC penetrates below the level of phosphate groups. The proper analysis of fluorescence quenching requires the use of Tempo position below the level of phosphate groups; and failure to do so will result in substantial systematic errors in determining the penetration of the labeled site on a membrane protein.

中文翻译：

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TEMPO-PC在脂质双层中的位置：荧光猝灭的含义。

双层中附着脂质的自旋探针的行为表征对于正确解释EPR和蛋白质膜相互作用的荧光研究的结果至关重要。TEMPO-PC中与脂质头基相连的TEMPO旋转探针的浸入深度的知识对于确定与蛋白质和肽相连的荧光探针的横向位置至关重要。TEMPO-PC在TEMPO-PC中双层渗透的问题最近在两项界面溶剂化研究中受到关注（Cheng等人在Biophys J 109：330-339，2015; Lee等人在Biophys J 111：2481- 2491，2016）。在这里，我们使用MD模拟和深度相关的荧光猝灭实验的交叉验证来重新检查关于TEMPO穿透的争论，这证实了TEMPO-PC中的TEMPO渗透到磷酸盐基团的水平以下。要正确分析荧光猝灭，需要使用低于磷酸根水平的Tempo位置。如果不这样做，将导致在确定标记的蛋白在膜蛋白上的渗透方面存在重大的系统错误。