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

Abstract

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.

Figure 1

X-band field sweep spectrum of KcsA R64C-SL in detergent at 40 K, The pump (red) and probe (at 70 MHz offset; blue) excitation profiles are indicated. (Inset) The molecular axis system of the nitroxide spin label; the g- and A-tensors are collinear with this axis system.

Figure 2

(a) X-band PELDOR time traces of KcsA R64C-SL in detergent and (b) apparent distance distribution obtained by the Tikhonov regularization method of the DEER Analysis program (8). The frequency difference Δv between the constant pump and variable detection frequencies ranged from 38–80 MHz. Simulated PELDOR time traces are plotted above the experimental data in (a). Data for the different detection frequencies are shifted relative to each other for clarity.

Figure 3

Spin label orientations within the KcsA R64C-SL tetramer used for the simulations in Figure 2a. Viewed from the top (left) and side (right), the nitroxides are aligned with the N–O axis parallel to the ion conduction pore (K⁺ ions within the pore are indicated by green spheres) and at an angle β = 65° between the normal of the nitroxide plane and the direction to the channel center. The spin label side chains are shown using ball-and-stick representations while the rest of the protein in shown as a ribbon. This figure and Figure 5 were prepared using PyMol (DeLano Scientific LLC) and Protein Data Bank coordinates from KcsA entry 1K4C.

Figure 4

Q-band PELDOR time traces of KcsA R64C-SL in detergent at several probe frequencies. The frequency difference Δv between the constant pump and variable detection frequencies ranged from 40–70 MHz. Simulated PELDOR time traces are plotted above the experimental data. Data for the different detection frequencies are shifted relative to each other for clarity.

Figure 5

Structure of wild-type KcsA highlighting R64 and its local environment viewed from the top (a) and side (b). For comparison, our proposed structure of the spin label is also shown (c). R64 and spin label side chains are shown using ball-and-stick representations while the rest of the protein is shown as a surface representation. K⁺ ions in the central ion conduction pore are indicated as green spheres.

Figure 6

Room temperature cw-X-band EPR spectrum of KcsA R64C-SL solubilized in detergent. Lines marked with * are due to residual free spin labels (not bound to protein).

Figure 7

X-band PELDOR time traces of liposomal KcsA R64C-SL reconstituted with 3-fold excess wild-type KcsA into PE:PG at a 10:1 lipid to total protein mass ratio. Simulated time traces (shown above the experimental data) are based on the nitroxide positions in Figure 3.