Pulsed electron-electron double-resonance determination of spin-label distances and orientations on the tetrameric potassium ion channel KcsA.

摘要: Pulsed electron-electron double-resonance (PELDOR) measurements are presented from the potassium ion channel KcsA both solubilized in detergent and reconstituted lipids. Site-directed spin-labeling using (1-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methyl methanethiosulfonate was performed with a R64C mutant of protein. The orientations spin-labels tetramer were determined by PELDOR experiments at two magnetic field strengths (0.3 T/X-band 1.2 T/Q-band) variable probe frequency. Quantitative simulation data supports strongly restricted nitroxide, oriented an angle 65 degrees relative to central axis. In general, poorer quality obtained membrane-reconstituted preparations compared soluble proteins or detergent-solubilized samples. One reason for this is reduced transverse spin relaxation time T(2) nitroxides due crowding tetramers within membrane that occurs even low protein lipid ratios. This can be overcome reconstituting mixtures unlabeled labeled proteins, yielding high-quality data. Identical oscillation frequencies their dependencies on frequency observed preparations, indicating position orientation same environments.