Pulsed Electron-Electron Double-Resonance Determination of Spin-Label Distances and Orientations on the Tetrameric Potassium Ion Channel KcsA

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