Ve c). As shown, when excited at 280 nm, the emission 1141777-14-1 supplier spectrum is dominated by emission at low wavelengths. Because the 169590-42-5 custom synthesis efficiency of fluorescence power transfer amongst donor and acceptor groups is strongly dependent on the distance among the groups, 9 this suggests that fluorescence emission at low wavelengths corresponds to Dauda bound straight to KcsA, for which Trp-dansyl distances might be shorter than for Dauda positioned inside the lipid bilayer component on the 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 match to this equation, providing the parameters listed in Table 1. The emission spectrum for Dauda within the presence of DOPC (Figure 2A) was then fit to the sum of two skewed Gaussians, corresponding to Dauda in water and bound in the lipid bilayer, with the parameters for the aqueous component fixed at the values listed in Table 1, providing the values for Dauda in the lipid bilayer (Table 1). The emission spectrum for Dauda within the presence of KcsA with excitation at 280 nm was then match for the sum of three skewed Gaussians, with the parameters for the lipid-bound and aqueous components fixed at the values listed in Table 1, providing | 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 three 78 2 b 0.20 0.01 0 0.37 0.Articlea Fluorescence emission spectra shown in Figure 2 were match to one or much more skewed Gaussians (eq 7) as described within the text. max would be the wavelength at the peak maximum, the peak width at half-height, and b the skew parameter.values for the KcsA-bound component again listed in Table 1. Ultimately, the spectra obtained at 0.3 and two M Dauda with excitation at 345 nm (curves a and b, Figure 2B) have been fit for the sum of three skewed Gaussians together with the parameters fixed at the values offered in Table 1; the good fits obtained show that the experimental emission spectra can indeed be represented by the sum of KcsA-bound, lipid-bound, and aqueous components. The amplitudes in the KcsA-bound, lipid-bound, and aqueous components providing the very best fits for the emission spectra excited at 345 nm had been two.14 0.01, 0 0.01, and 0.36 0.01, respectively, at 0.three M Dauda and 3.40 0.01, 0.39 0.02, and 2.97 0.01, respectively, at two.0 M Dauda. The low intensity for the lipid-bound element is constant with weak binding of Dauda to DOPC, described by an effective dissociation constant (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 competitors experiments with TBA. A single TBA ion binds in the central cavity of KcsA,two,three plus 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 inside a decreased fluorescence emission at lowwavelengths, exactly where the spectra are dominated by the KcsAbound element, with no effects at greater wavelengths; the effects of TBA enhance with increasing concentration as expected for straightforward competitors between Dauda and TBA for binding to the central cavity in KcsA. Addition of oleic acid also outcomes inside a reduce in intensity for the 469 nm component (Figure 3B), displaying that binding of Dauda and oleic acid to the central cavity is also competitive. Variety of Binding Web sites for Dauda on KcsA.