Analysis of protein and peptide penetration into membranes by depth-dependent fluorescence quenching: Theoretical considerations

Depth-dependent fluorescence quenching in membranes is playing an increasin gly important role in the determination of the low resolution structure of membrane proteins. This paper presents a graphical way of visualizing membr ane quenching caused by lipid-attached bromines or spin labels with the hel p of a depth-dependent fluorescence quenching profile. Two methods are pres ently available to extract information on membrane penetration from quenchi ng: the parallax method (PM; Chattopadhyay, A., and E. London, 1987. Bioche mistry. 26:39-45) and distribution analysis (DA; A. S. Ladokhin, 1993. Biop hys. J. 64:290a (Abstr.); A. S. Ladokhin, 1997. Methods Enzymol. 278:462-47 3). Analysis of various experimental and simulated data by these two method s is presented. The effects of uncertainty in the local concentration of qu enching lipids (due to protein shielding or nonideality in lipid mixing), t he existence of multiple conformations of membrane-bound protein, incomplet e binding. and uncertainty in the fluorescence in nonquenching lipid are de scribed. Regardless of the analytical form of the quenching profile (Gaussi an function for DA or truncated parabola for PM), it has three primary char acteristics: position on the depth scale, area, and width. The most importa nt result, not surprisingly, is that one needs three fitting parameters to describe the quenching. This will keep the measures of the quenching profil e independent of each other resulting in the reduction of systematic errors in depth determination. This can be achieved by using either DA or a sugge sted modification of the PM that introduces a third parameter related to qu enching efficiency. Because DA utilizes a smooth fitting function, it offer s an advantage far the analysis of deeply penetrating probes, where the eff ects of transleaflet quenching should be considered.