Room-temperature distance measurements of immobilized spin-labeled protein by DEER/PELDOR

Abstract

Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (Tm) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer Tm between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the Tm for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-(methyl)methanethio-sulfonate label.

Figure 1

Pulse sequence for 4-pulse DEER. The spin echo is formed with pulses at the probe frequency, ν₁, with constant τ₁ and τ₂, whereas the pump pulse at ν₂ is stepped by varying T. The resulting signal is a measure of the modulation of the amplitude of the spin echo from spins excited at ν₁ by the pump pulse at ν₂.

Figure 2

Nitroxide radicals.

Figure 3

Temperature dependence of log(1/T_m) at X-band for 1, 2, and 3 in glassy mixtures. Data for 1 and 2 in water/glycerol are reproduced from (11). To see this figure in color, go online.

Figure 4

Background-subtracted DEER data at 80 K (A, black) and 160 K (B, black) for T4L labeled with 3 in 24% glycerol. Tikhonov fitting is shown in red. The distance distributions (C) were similar at 80 K (black) and 160 K (gray) for each double mutant. To see this figure in color, go online.

Figure 5

Room temperature DEER of T4L 65/80 labeled with 3 in a glassy trehalose matrix: raw data (A, black) with background subtraction (A, red); Pake pattern (B, black) with Tikhonov regularization fitting, α = 100 (B, red); background subtracted raw data (C, black) with Tikhonov fitting (C, red); distance distribution (D). To see this figure in color, go online.