Zolidinyl-N-oxyl)stearic acid (14-SASL) to KcsA.8 We observed a strongly immobilized signal that weReceived: July 10, 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 ascertain the number of binding sites per channel; assuming one internet site per channel gave a binding constant in the selection of 0.1-1 M.eight The observation that 14-SASL was strongly immobilized on KcsA suggested that it may well also be possible to study fatty acid binding applying fluorescent analogues of fatty acids, since DBCO-NHS ester Purity & Documentation fluorescence emission spectra is often sensitive to environmental mobility at the same time as to environmental polarity.9 In specific, the fluorescence emission spectrum of your dansyl probe shows a marked time dependence on the nanosecond fluorescence time scale, because of solvent relaxation around the excited state dansyl group, resulting inside a shift of the emission spectrum to longer wavelengths with growing times soon after excitation.10 The extent to which solvent can loosen up around a dansyl group through the time it remains inside the excited state is dependent upon the mobility with the solvent; large shifts within the fluorescence emission spectrum to extended wavelengths are anticipated when the solvent is mobile, but only smaller shifts are expected for a rigid solvent. The atmosphere of a dansyl group bound to a web-site on a protein will consist of, a minimum of in portion, amino acid residues whose mobility is probably to be restricted on the nanosecond fluorescence time scale; in contrast, a dansyl group embedded inside a lipid bilayer will knowledge an atmosphere with substantially greater mobility. This suggests that the fluorescence emission spectrum for a dansyl-containing probe bound to a reconstituted membrane protein could include separate components because of protein-bound and lipid-bound probe. We show right here that this can be the case for 11-dansylaminoundecanoic acid (Dauda) bound to KcsA and that Dauda might be employed to characterize the fatty acid binding web page in the cavity of KcsA.ArticleDauda;9 the fluorescence intensity of NADH (10 M) was measured inside the absence and presence of KcsA with excitation and emission wavelengths of 345 and 450 nm, respectively, as well as a set of correction variables was generated by comparing the measured fluorescence intensity inside the presence of a offered concentration of KcsA to that within the absence of KcsA. It was also essential to appropriate for the inner filter effect9,12 observed at high Dauda concentrations. Fluorescence intensities have been 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 increased linearly with an escalating Dauda concentration, but at higher concentrations, the fluorescence intensity was reduced due to the inner filter effect; comparison on the observed fluorescence intensities at high concentrations with those anticipated by extrapolation in the values observed at low concentrations gave the essential set of correction variables. The reported fluorescence intensities represent averages of triplicate measurements from two or 3 separate reconstitutions. Evaluation of Fluorescence Titrations. As described later, titrations measuring fluorescence intensities of Dauda at 450 nm have been match for the sum of a saturable in addition to a nonsaturable element, corresponding to binding to the cavity of K.