Ve c). As shown, when excited at 280 nm, the emission spectrum is dominated by emission at low wavelengths. Since the efficiency of Fluorescence energy transfer between donor and acceptor groups is strongly dependent on the distance amongst the groups, 9 this suggests that fluorescence emission at low wavelengths corresponds to Dauda bound directly to KcsA, for which Trp-dansyl distances will probably be shorter than for Dauda located inside the lipid bilayer component with the membrane. Fluorescence emission spectra of your 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 1492-18-8 MedChemExpress equation, giving 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, 4-Aminosalicylic acid Protocol corresponding to Dauda in water and bound in the lipid bilayer, using the parameters for the aqueous component fixed at the values listed in Table 1, giving the values for Dauda inside the lipid bilayer (Table 1). The emission spectrum for Dauda in the presence of KcsA with excitation at 280 nm was then match for 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 three 78 2 b 0.20 0.01 0 0.37 0.Articlea Fluorescence emission spectra shown in Figure 2 had been match to 1 or additional skewed Gaussians (eq 7) as described in the text. max is definitely 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. Lastly, the spectra obtained at 0.three and two M Dauda with excitation at 345 nm (curves a and b, Figure 2B) were fit to the sum of 3 skewed Gaussians with the parameters fixed at the values offered in Table 1; the very 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 of your KcsA-bound, lipid-bound, and aqueous components giving the ideal 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 3.40 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 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 competition experiments with TBA. A single TBA ion binds in the central cavity of KcsA,two,3 as well as the effects of fatty acids and tetraalkylammonium ions on channel function are competitive.7 As shown in Figure 3A, incubation of KcsA with TBA outcomes 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 boost with rising concentration as anticipated for easy competitors between 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), showing that binding of Dauda and oleic acid for the central cavity is also competitive. Quantity of Binding Web sites for Dauda on KcsA.