Ve c). As shown, when excited at 280 nm, the 88495-63-0 Formula emission spectrum is dominated by emission at low wavelengths. Since the efficiency of fluorescence power transfer among donor and acceptor groups is strongly dependent around the distance in between the groups, 9 this suggests that fluorescence emission at low wavelengths corresponds to Dauda bound directly to KcsA, for which Trp-dansyl distances might be shorter than for Dauda positioned in the lipid bilayer element of the membrane. Fluorescence emission spectra with the dansyl group have the shape of a skewed Gaussian (eq 7).13 The emission spectrum for Dauda in water (Figure 2A) was match 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 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 745833-23-2 In stock component fixed in the values listed in Table 1, providing 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 towards the sum of 3 skewed Gaussians, with the parameters for the lipid-bound and aqueous components fixed at 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 two have been fit to 1 or much more skewed Gaussians (eq 7) as described inside the text. max could be the wavelength in 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. Lastly, the spectra obtained at 0.three and two M Dauda with excitation at 345 nm (curves a and b, Figure 2B) were match towards the sum of 3 skewed Gaussians with all the parameters fixed in the values offered in Table 1; the good 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 best fits for 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 consistent with weak binding of Dauda to DOPC, described by an effective dissociation continuous (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 competition experiments with TBA. A single TBA ion binds in the central cavity of KcsA,2,three and 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 within a decreased fluorescence emission at lowwavelengths, exactly where the spectra are dominated by the KcsAbound component, with no effects at larger wavelengths; the effects of TBA boost with increasing concentration as expected for straightforward competition among Dauda and TBA for binding for the central cavity in KcsA. Addition of oleic acid also benefits in a reduce in intensity for the 469 nm element (Figure 3B), displaying that binding of Dauda and oleic acid towards the central cavity is also competitive. Quantity of Binding Sites for Dauda on KcsA.
