| Abstract: | 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. |
