Fig. 5.

(A and B) Lipid-attached fluorescence quenchers used for depth-dependent measurements and their depth (the distances from bilayer center to the quencher) [Kyrychenko & Ladokhin, 2013; McIntosh & Holloway, 1987]. (A) Brominated phospholipids: 1-palmitoyl-2-(n,n-dibromo)stearoyl-sn-glycero-3-phosphocholine (n,n-Br-PC). (B) Spin-labeled phospholipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(TEMPO) choline (Tempo-PC), 1-palmitoyl-2-stearoyl-(n-doxyl)-sn-glycero-3-phosphocholine (n-doxyl-PC). (C) The DA methodology approximates the transverse quenching profile (QP(h)) with a sum of two mirror-image Gaussian functions (G(h)), where h_m—the center (mean) of the quenching profile, σ—the width of the distribution, and S—the area of the quenching profile [Ladokhin, 1997, 1999, 2014; London & Ladokhin, 2002]. (D) Top Right: Structure of NBD-PE and TEMPO-PC. Bottom Left: An MD snapshot of a lipid bilayer composed of Tempo-PC and POPC with a ratio of that correspond to 11 mol% of bilayer spin-labeling, respectively. The POPC phosphorus atoms are shown as blue balls. POPC and Tempo-PC are shown in stick representations in olive and red, respectively, with the Tempo moieties shown using van der Waals representation Bottom Right: Comparison of the MD-simulated distribution of a NBD moiety [Kyrychenko, Rodnin, & Ladokhin, 2015] (shaded area) with those estimated from experimental data (red and green lines). Green profile corresponds to the result obtained when Tempo is assumed to reside above the phosphate level, as suggested by Schrader and Han [Schrader & Han, 2017], while red profile corresponds to the result obtained when position of Tempo is taken from our MD simulation ([Kyrychenko & Ladokhin, 2013] and central panel). The latter gives excellent correspondence to the MD-determined profile of the NBD (blue), thus validating the DA approach. Reprinted by permission from [Kyrychenko, A., Ladokhin, A.S. (2020) Location of TEMPO-PC in lipid bilayers: Implications for fluorescence quenching. The Journal of Membrane Biology, 253, 73–77.], COPYRIGHT 2020 from Springer