Ve c). As shown, when excited at 280 nm, the emission spectrum is dominated by emission at low wavelengths. Because the efficiency of fluorescence power transfer amongst donor and acceptor groups is strongly dependent on the distance in 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 probably be shorter than for Dauda situated within the lipid bilayer component of your membrane. Fluorescence emission spectra of 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, giving the parameters listed in Table 1. The emission spectrum for Dauda within the presence of DOPC (Figure 2A) was then match for the sum of two skewed Gaussians, corresponding to Dauda in water and bound within the lipid bilayer, with all the parameters for the aqueous element fixed at the values listed in Table 1, giving the values for Dauda in the lipid bilayer (Table 1). The emission spectrum for Dauda in the presence of KcsA with excitation at 280 nm was then fit for the sum of 3 skewed Gaussians, using the parameters for the lipid-bound and aqueous elements fixed at the values listed in Table 1, giving 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 fit to a single or additional skewed Gaussians (eq 7) as described in the text. max may be the wavelength in the peak maximum, the peak width at half-height, and b the skew parameter.values for the KcsA-bound component once more listed in Table 1. Finally, the spectra obtained at 0.three and two M Dauda with excitation at 345 nm (curves a and b, Figure 2B) had been match to the sum of three skewed Gaussians using the parameters fixed at the values offered in Table 1; the great fits obtained show that the experimental emission spectra can indeed be represented by the sum of KcsA-bound, lipid-bound, and aqueous elements. The amplitudes in the KcsA-bound, lipid-bound, and aqueous elements providing the very best fits towards the emission spectra excited at 345 nm had 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 component is consistent with weak 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 towards the central cavity of KcsA comes from competition experiments with TBA. A single TBA ion binds in the central cavity of KcsA,2,3 and also the effects of fatty acids and tetraalkylammonium ions on channel function are competitive.7 As shown in Figure 3A, incubation of KcsA with TBA benefits in a decreased fluorescence emission at lowwavelengths, where the spectra are dominated by the KcsAbound element, with no effects at higher wavelengths; the effects of TBA 99489-94-8 Epigenetics increase with growing concentration as expected for easy competition among Dauda and TBA for binding to the central cavity in KcsA. Addition of oleic acid also final results inside a decrease 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 Internet sites for Dauda on KcsA.