Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins

Abstract

We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water, K_a = 1.9 × 10⁶ M^-1, is at least 1 order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase in the rotational correlation time, τ_rot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in τ_rot and electron spin coherence time, T_m, along with the narrowing of interspin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4-9, compared to the common spin label such as MTSL, which is not affected by CB-7. Interspin distances of 3 nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 Å resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating an undisturbed structure and conformation of the protein.

Figure 1.

Structures of spin labels, model nitroxide ClA-DZD, and dicationic nitroxide bPTO.

Figure 2.

X-ray crystallography of ClA-DZD@CB-7 at 123 K: (A) top view, Ortep plot with carbon, nitrogen, oxygen, and chlorine atoms depicted with thermal ellipsoids set at the 50% probability level and (B) side view, the guest and host are shown in ball-and-stick and in wire plots. Molecules of water are omitted for clarity. (C) CW EPR spectra of polycrystalline ClA-DZD@CB-7 and ClA-DZD at room temperature. For more details, see: SI, Tables S2 and S3, and Figs. S2–S6.

Figure 3.

EPR spectroscopy in fluid Tris-MOPS-1 buffer (Table 1) at 295 K: rotational correlation times, τ_cor, and ¹⁴N hyperfine tensor components, A_zz, and isotropic ¹⁴N hyperfine couplings, A_iso. (A) 0.28 mM ClA-DZD (B) 0.28 mM ClA-DZD with 2 equiv of CB-7; (C) 0.25 mM model pyrroline nitroxide 1; (D) 0.25 mM 1 with 2 equiv of CB-7. Enlarged version of this figure and a summary of fitting parameters may be found in the SI: Fig. S14 and Table S5.

Figure 4.

DZD-T4L 65: (A) The ribbon diagram of T4L showing the DZD label at C65 near the N-terminal region of helix C. (B) Minimally biased omit mF_o-DF_c electron density contoured at 3.0 σ (green) for the DZD label, which was calculated prior to the introduction of DZD into the structural model. Hydrogen bonds are shown as dotted lines with distances. R137 is from a neighboring molecule in the crystal lattice (blue, primed label) and makes a bifurcated hydrogen bond with the oxygen atom of the nitroxide label. (C) The CW EPR spectrum of polycrystalline DZD-T4L in reservoir solution prior to X-ray data collection confirms the presence of the radical on the spin label (Fig. S1, SI). (D) A comparison of the DZD label (this work, gray) and the MTN spin label from PDB 6PGY, showing similar overall orientations in the crystal.

Figure 5.

DEER distance measurements on doubly spin labeled DZD-T4L and MTSL-T4L in 2:1 w/g matrix without CB-7 (gly) and with 1 mM CB-7 (gly/CB-7) at 83 K. The water phase is Tris-MOPS-1 buffer (Table 1), i.e., 6 mM Tris, 9 mM MOPS, 0.01 mM EDTA, and 50 mM NaCl (pH 7.2). Main plots: normalized echo vs. time with fits, using a sum of Gaussians as the distance distribution. Inset plots: distance distributions with 95% confidence bands (orange lines). For additional details, including alternative distance distributions for DZD-T4L 65/135, see: SI, Tables S7 and S8, and Fig. S30 and S31.

Figure 6.

DEER distance measurements on doubly spin labeled DZD-T4L 65/80 in 2:1 w/g matrix with 1 mM CB-7 (gly/CB-7) at T = 83–200 K: normalized echo vs. time with fits, using a sum of Gaussians, and distance distributions, P(r), with 95% confidence bands (orange lines). The water phase is Tris-MOPS-1 buffer (Table 1). Additional information may be found in Table 3 and the SI: Table S9 and Fig. S32.