Zolidinyl-N-oxyl)stearic acid (14-SASL) to KcsA.8 We observed a strongly immobilized signal that weReceived: July 10, 2012 Revised: September ten, 2012 Published: September 12,dx.doi.org/10.1021/bi3009196 | Quinocetone Autophagy Biochemistry 2012, 51, 7996-Biochemistry attributed to fatty acid bound inside the cavity but were unable to figure out the amount of binding web sites per channel; assuming 1 internet site per channel gave a binding continuous in the range of 0.1-1 M.8 The observation that 14-SASL was strongly immobilized on KcsA suggested that it may also be doable to study fatty acid binding working with fluorescent analogues of fatty acids, for the reason that fluorescence emission spectra might be sensitive to environmental mobility at the same time as to environmental polarity.9 In distinct, the fluorescence emission spectrum of the 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 with the emission spectrum to longer wavelengths with escalating occasions after excitation.10 The extent to which solvent can unwind around a dansyl group during the time it remains within the excited state depends upon the mobility of your solvent; big shifts inside the fluorescence emission spectrum to extended wavelengths are expected when the solvent is mobile, but only tiny shifts are anticipated for a rigid solvent. The atmosphere of a dansyl group bound to a website on a protein will consist of, at the least in aspect, amino acid residues whose mobility is most likely to become limited on the nanosecond fluorescence time scale; in contrast, a dansyl group embedded inside a lipid bilayer will experience an atmosphere with a lot greater mobility. This suggests that the fluorescence emission spectrum to get a dansyl-containing probe bound to a reconstituted membrane protein could include separate components because of protein-bound and lipid-bound probe. We show here that this can be the case for 11-dansylaminoundecanoic acid (Dauda) bound to KcsA and that Dauda is often used to characterize the fatty acid binding site within 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, as well as a set of correction components was generated by comparing the measured fluorescence intensity inside the presence of a provided concentration of KcsA to that in the absence of KcsA. It was also necessary to correct for the inner filter effect9,12 observed at high Dauda concentrations. Fluorescence intensities were measured for Dauda solutions in Tazobactam (sodium) sodium 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 growing Dauda concentration, but at higher concentrations, the fluorescence intensity was reduced because of the inner filter effect; comparison from the observed fluorescence intensities at high concentrations with those anticipated by extrapolation of the values observed at low concentrations gave the essential set of correction components. 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 had been fit to the sum of a saturable as well as a nonsaturable element, corresponding to binding to the cavity of K.