O'Neil Pierce T
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, 66160, USA.
J Membr Biol. 2022 Feb;255(1):123-127. doi: 10.1007/s00232-021-00199-6. Epub 2021 Oct 25.
Determining the topology of membrane-inserted proteins and peptides often relies upon indirect fluorescent measurements. One such technique uses NBD, an environmentally sensitive fluorophore that can be covalently linked to proteins. Relative to a hydrophilic environment, NBD in a hydrophobic environment shows an increase in emission intensity and a shift to shorter wavelengths. To gain further insight, NBD fluorescence can be chemically quenched using dithionite. As dithionite is an anion, it is only expected to penetrate the outer leaflet interfacial region and should be excluded from the hydrocarbon core, the inner leaflet, and the lumen of LUV. This assumption holds at neutral pH, where a large number of NBD/dithionite experiments are carried out. Here, we report control experiments in which LUV were directly labeled with NBD-PE to assess dithionite quenching in acidic conditions. Results showed that at acidic pH, dithionite moved more freely across the bilayer to quench the inner leaflet. For the buffer conditions used, dithionite exhibited a sharp change in behavior between pH 5.5 and 6.0. Therefore, in acidic conditions, dithionite could not differentiate in which leaflet the NBD resided.
确定膜插入蛋白和肽的拓扑结构通常依赖于间接荧光测量。一种这样的技术使用NBD，一种对环境敏感的荧光团，它可以与蛋白质共价连接。相对于亲水环境，疏水环境中的NBD发射强度增加且波长向更短方向移动。为了进一步深入了解，NBD荧光可以用连二亚硫酸盐进行化学淬灭。由于连二亚硫酸盐是一种阴离子，预计它只会穿透外层小叶界面区域，并且应该被排除在烃核、内层小叶和大单层囊泡（LUV）的内腔之外。这个假设在中性pH下成立，大量的NBD/连二亚硫酸盐实验都是在这个条件下进行的。在这里，我们报告了对照实验，其中用NBD-PE直接标记LUV以评估酸性条件下连二亚硫酸盐的淬灭情况。结果表明，在酸性pH下，连二亚硫酸盐更自由地穿过双层膜以淬灭内层小叶。对于所使用的缓冲条件，连二亚硫酸盐在pH 5.5和6.0之间表现出行为上的急剧变化。因此，在酸性条件下，连二亚硫酸盐无法区分NBD位于哪一层小叶中。
