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. Author manuscript; available in PMC: 2022 Feb 2.
Published in final edited form as: Methods Enzymol. 2021 Feb 2;649:341–370. doi: 10.1016/bs.mie.2020.12.016

Fig. 6.

Fig. 6

Application of Distribution Analysis (DA) to depth-dependent fluorescence quenching of shallow penetrating probe in P378C-NBD mutant [Kyrychenko et al., 2018]. (A) Fluorescence steady-state (triangles) and time-resolved (squares) quenching profiles plotted against the depth of the following quenchers (from right to left): Tempo—PC and n-Doxyl—PCs with n= 5, 7, 10, 12, and 14. (B) Static QP calculated from the data in panel A as QP(h) = (F0/F(h))/(τ(h)/τ0). Solid lines represent the fit of the corresponding datasets using DA equation from Fig. 5C. The shaded areas represent a Gaussian function for the single leaflet quenching profiles. The sum of the shaded area, G(h), and its mirror image, G(−h), represents the total fitting function of DA, with the total of three fitting parameters: hm, σ and S. (C) Support-plane analysis of the robustness of the determination of the parameter for the most probable depth of the NBD dye (see text for details).