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>Analysis of protein and peptide penetration into membranes by depth-dependent fluorescence quenching: theoretical considerations.
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Analysis of protein and peptide penetration into membranes by depth-dependent fluorescence quenching: theoretical considerations.
Depth-dependent fluorescence quenching in membranes is playing an increasingly important role in the determination of the low resolution structure of membrane proteins. This paper presents a graphical way of visualizing membrane quenching caused by lipid-attached bromines or spin labels with the help of a depth-dependent fluorescence quenching profile. Two methods are presently available to extract information on membrane penetration from quenching: the parallax method (PM; ) and distribution analysis (DA; A. S. Biophys. J. 64:290a (Abstr.); A. S. Methods Enzymol. 278:462-473). Analysis of various experimental and simulated data by these two methods is presented. The effects of uncertainty in the local concentration of quenching lipids (due to protein shielding or nonideality in lipid mixing), the existence of multiple conformations of membrane-bound protein, incomplete binding, and uncertainty in the fluorescence in nonquenching lipid are described. Regardless of the analytical form of the quenching profile (Gaussian function for DA or truncated parabola for PM), it has three primary characteristics: position on the depth scale, area, and width. The most important result, not surprisingly, is that one needs three fitting parameters to describe the quenching. This will keep the measures of the quenching profile independent of each other resulting in the reduction of systematic errors in depth determination. This can be achieved by using either DA or a suggested modification of the PM that introduces a third parameter related to quenching efficiency. Because DA utilizes a smooth fitting function, it offers an advantage for the analysis of deeply penetrating probes, where the effects of transleaflet quenching should be considered.
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机译：膜中依赖深度的荧光猝灭在确定膜蛋白的低分辨率结构中起着越来越重要的作用。本文提出了一种图形化的方法，借助依赖于深度的荧光淬灭曲线，可视化由脂质附着的溴或自旋标记物引起的膜淬灭。目前有两种方法可用于从淬灭中提取有关膜渗透的信息：视差方法（PM;）和分布分析（DA; AS Biophys。J. 64：290a（Abstr。）; AS Methods Enzymol。278：462-473） 。介绍了通过这两种方法对各种实验和模拟数据的分析。描述了猝灭脂质的局部浓度不确定性的影响（由于蛋白质屏蔽或脂质混合中的非理想性），膜结合蛋白的多种构象的存在，结合不完全以及非猝灭脂质中荧光的不确定性。不管淬火曲线的分析形式（DA的高斯函数或PM的截断抛物线），它都具有三个主要特征：深度标度上的位置，面积和宽度。毫不奇怪，最重要的结果是，需要三个拟合参数来描述淬灭。这将使淬火曲线的测量结果彼此独立，从而减少了深度确定中的系统误差。这可以通过使用DA或建议的PM修改（引入与淬灭效率相关的第三个参数）来实现。因为DA利用了平滑的拟合功能，所以它在分析深穿透探针时具有优势，在这种情况下，应考虑经跨叶淬灭的影响。
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