Ve c). As shown, when excited at 280 nm, the emission spectrum is dominated by emission at low wavelengths. Because the efficiency of ABMA Autophagy Fluorescence power transfer amongst donor and acceptor groups is strongly dependent on the distance between the groups, 9 this suggests that fluorescence emission at low wavelengths corresponds to Dauda bound straight to KcsA, for which Trp-dansyl distances will likely be shorter than for Dauda located within the lipid bilayer element in the membrane. Fluorescence emission spectra in the dansyl group have the shape of a skewed Gaussian (eq 7).13 The emission spectrum for Dauda in water (Figure 2A) was fit to this equation, providing the parameters listed in Table 1. The emission spectrum for Dauda inside the presence of DOPC (Figure 2A) was then fit towards the sum of two skewed Gaussians, corresponding to Dauda in water and bound inside the lipid bilayer, with all the parameters for the aqueous component fixed at the values listed in Table 1, providing the values for Dauda within the lipid bilayer (Table 1). The emission spectrum for Dauda in the presence of KcsA with excitation at 280 nm was then fit towards the sum of three skewed Gaussians, together with the parameters for the lipid-bound and aqueous elements fixed in the values listed in Table 1, providing thedx.doi.org/10.1021/bi3009196 | Biochemistry 2012, 51, 7996-Biochemistry Table 1. Fluorescence Emission Parameters for Daudaacomponent water DOPC KcsA max (nm) 557 three 512 1 469 1 (nm) 102 1 84 3 78 2 b 0.20 0.01 0 0.37 0.Articlea Fluorescence emission spectra shown in Figure 2 have been match to one or far more skewed Gaussians (eq 7) as described in the text. max could be the wavelength at the peak maximum, the peak width at half-height, and b the skew parameter.values for the KcsA-bound element once again listed in Table 1. Finally, the spectra obtained at 0.3 and 2 M Dauda with excitation at 345 nm (curves a and b, Figure 2B) have been match towards the sum of 3 skewed Gaussians with all the parameters fixed at the values given in Table 1; the excellent fits obtained show that the experimental emission spectra can certainly be represented by the sum of KcsA-bound, lipid-bound, and aqueous components. The amplitudes from the KcsA-bound, lipid-bound, and aqueous elements providing the top fits towards the emission spectra excited at 345 nm have been two.14 0.01, 0 0.01, and 0.36 0.01, respectively, at 0.3 M Dauda and three.40 0.01, 0.39 0.02, and two.97 0.01, respectively, at two.0 M Dauda. The low intensity for the lipid-bound element is constant with weak DL-Tropic acid Epigenetic Reader Domain binding of Dauda to DOPC, described by an efficient dissociation continual (Kd) of 270 M.14 Confirmation that the blue-shifted peak centered at 469 nm arises from binding of Dauda to the central cavity of KcsA comes from competitors experiments with TBA. A single TBA ion binds inside the central cavity of KcsA,2,3 along with the effects of fatty acids and tetraalkylammonium ions on channel function are competitive.7 As shown in Figure 3A, incubation of KcsA with TBA results inside a decreased fluorescence emission at lowwavelengths, exactly where the spectra are dominated by the KcsAbound component, with no effects at greater wavelengths; the effects of TBA improve with growing concentration as anticipated for uncomplicated competitors among Dauda and TBA for binding for the central cavity in KcsA. Addition of oleic acid also benefits in a lower in intensity for the 469 nm component (Figure 3B), showing that binding of Dauda and oleic acid to the central cavity is also competitive. Quantity of Binding Web-sites for Dauda on KcsA.