A Two-Armed Probe for In-Cell DEER Measurements on Proteins*

Abstract

The application of double electron-electron resonance (DEER) with site-directed spin labeling (SDSL) to measure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin-label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances. Here, three two-armed Gd^III complexes, Gd^III -CLaNP13a/b/c were synthesized. Rather than the disulfide linkage of most other CLaNP molecules, a thioether linkage was used to avoid reductive dissociation of the linker. The doubly Gd^III labeled N55C/V57C/K147C/T151C variants of T4Lysozyme were measured by 95 GHz DEER. The constructs were measured in vitro, in cell lysate and in Dictyostelium discoideum cells. Measured distances were 4.5 nm, consistent with results from paramagnetic NMR. A narrow distance distribution and typical modulation depth, also in cell, indicate complete and durable labeling and probe rigidity due to the dual attachment sites.

Keywords: EPR spectroscopy; double electron-electron resonance (DEER); gadolinium; protein structures; spin labels.

Figure 1

CLaNP13 and T4Lys as model protein. a) Structures of Ln^III‐CLaNP5 and Ln^III‐CLaNP13. b) Model of the structure of T4Lys based on PDB entry 3dke ^[47] with two Cys pairs for the attachment of two probes. The positions of the metal ions are based on PCS analysis using Yb^III‐CLaNP5 as a paramagnetic probe. The backbone is drawn in ribbon representation. The Cys residues used for attachment have been modeled into the structure and are shown as sticks. The metal ions are shown as yellow spheres.

Figure 2

Details of overlaid ¹H‐¹⁵N HSQC spectra of Yb^III and Lu^II loaded CLaNP5 attached to T4Lys N55C/V57C (a) and T4Lys K147C/T151C (b). Several PCS are indicated with solid lines and residue numbers. The NMR spectra were recorded at 14.1 T (600 MHz). The full spectra are shown in Figure S3.

Figure 3

DEER data of Gd13aT4L, Gd13bT4L, Gd13cT4L. a) Background corrected DEER traces. Traces are shifted vertically for clarity. Measurements were performed at 10 K for 6 to 12 hours. Red lines: fits obtained with the distance distributions shown in (b) obtained after Tikhonov regularization (α=100). Peaks marked with an asterisk do not contribute significantly to the data, as determined by the DeerAnalysis suppression tool. ^[48] Inset: 95 GHz field‐swept electron‐spin echo spectrum (FSESE) of the central transition region, position of the pump and observer frequencies are shown.

Figure 4

The DEER trace of the protein in Dictyostelium discoideum (D. discoideum) cells for Gd‐CLaNP13bT4L (blue). Reference (black): in vitro trace of Gd‐CLaNP13bT4L, truncated to the total evolution time of the in cell data (1356 ns). a) Background corrected DEER traces. Red lines: fits obtained with the distance distributions shown in b) obtained by Tikhonov regularization (α=100). The difference in the distance distributions shown in (b) is not significant (see result of DEER validation Figure S11 d).