Zolidinyl-N-oxyl)stearic acid (14-SASL) to KcsA.8 We Cefminox (sodium) Description observed a strongly immobilized signal that weReceived: July ten, 2012 Revised: September 10, 2012 Published: September 12,dx.doi.org/10.1021/bi3009196 | Biochemistry 2012, 51, 7996-Biochemistry attributed to fatty acid bound within the cavity but have been unable to establish the amount of binding web pages per channel; assuming 1 web-site per channel gave a binding constant in the range of 0.1-1 M.8 The observation that 14-SASL was strongly immobilized on KcsA recommended that it could possibly also be feasible to study fatty acid binding using fluorescent analogues of fatty acids, simply because Fluorescence emission spectra might be sensitive to environmental mobility too as to environmental polarity.9 In particular, the fluorescence emission spectrum of the dansyl probe shows a marked time dependence around the nanosecond fluorescence time scale, because of solvent relaxation around the excited state dansyl group, resulting inside a shift from the emission spectrum to longer wavelengths with rising times after excitation.ten The extent to which solvent can relax about a dansyl group through the time it remains inside the excited state depends upon the mobility on the solvent; massive shifts inside the fluorescence emission spectrum to long wavelengths are expected when the solvent is mobile, but only compact shifts are expected for any rigid solvent. The atmosphere of a dansyl group bound to a website on a protein will consist of, no less than in aspect, amino acid residues whose mobility is likely to become restricted on the nanosecond fluorescence time scale; in contrast, a dansyl group embedded in a lipid bilayer will practical experience an environment with considerably higher mobility. This suggests that the fluorescence emission spectrum for a dansyl-containing probe bound to a reconstituted membrane protein might include separate components due to protein-bound and lipid-bound probe. We show here that this really is the case for 11-dansylaminoundecanoic acid (Dauda) bound to KcsA and that Dauda is often utilized to characterize the fatty acid binding web page in the cavity of KcsA.ArticleDauda;9 the fluorescence intensity of NADH (ten M) was measured in the absence and presence of KcsA with excitation and emission wavelengths of 345 and 450 nm, respectively, in addition to a set of correction aspects was generated by comparing the measured fluorescence intensity in the presence of a given concentration of KcsA to that within the absence of KcsA. It was also necessary to right for the inner filter effect9,12 observed at higher Dauda concentrations. Fluorescence intensities were measured for Dauda solutions in methanol as a function of Dauda concentration, with excitation and emission wavelengths of 345 and 450 nm, respectively. At low Dauda concentrations, fluorescence intensities elevated GSK1016790A Technical Information linearly with an escalating Dauda concentration, but at higher concentrations, the fluorescence intensity was lowered due to the inner filter effect; comparison of the observed fluorescence intensities at high concentrations with these anticipated by extrapolation on the values observed at low concentrations gave the required set of correction factors. The reported fluorescence intensities represent averages of triplicate measurements from two or three separate reconstitutions. Analysis of Fluorescence Titrations. As described later, titrations measuring fluorescence intensities of Dauda at 450 nm were match for the sum of a saturable and a nonsaturable component, corresponding to binding for the cavity of K.