77Se NMR Probes the Protein Environment of Selenomethionine

Abstract

Sulfur is critical for the correct structure and proper function of proteins. Yet, lacking a sensitive enough isotope, nuclear magnetic resonance (NMR) experiments are unable to deliver for sulfur in proteins the usual wealth of chemical, dynamic, and structural information. This limitation can be circumvented by substituting sulfur with selenium, which has similar physicochemical properties and minimal impact on protein structures but possesses an NMR compatible isotope (⁷⁷Se). Here we exploit the sensitivity of ⁷⁷Se NMR to the nucleus' chemical milieu and use selenomethionine as a probe for its proteinaceous environment. However, such selenium NMR spectra of proteins currently resist a reliable interpretation because systematic connections between variations of system variables and changes in ⁷⁷Se NMR parameters are still lacking. To start narrowing this knowledge gap, we report here on a biological ⁷⁷Se magnetic resonance data bank based on a systematically designed library of GB1 variants in which a single selenomethionine was introduced at different locations within the protein. We recorded the resulting isotropic ⁷⁷Se chemical shifts and relaxation times for six GB1 variants by solution-state ⁷⁷Se NMR. For four of the GB1 variants we were also able to determine the chemical shift anisotropy tensor of SeM by solid-state ⁷⁷Se NMR. To enable interpretation of the NMR data, the structures of five of the GB1 variants were solved by X-ray crystallography to a resolution of 1.2 Å, allowing us to unambiguously determine the conformation of the selenomethionine. Finally, we combine our solution- and solid-state NMR data with the structural information to arrive at general insights regarding the execution and interpretation of ⁷⁷Se NMR experiments that exploit selenomethionine to probe proteins.

Figure 1.

Structures of GB1 variants carrying SeM at different positions (top row), close-up of the SeM (middle row), and details of SeM’s local environment within 4.2 Å. (A) GB1 L5SeM PDB entry 6CNE; (B) GB1 I6SeM PDB entry 6CPZ; (C) GB1 V29SeM PDB entry 6C9O; (D) GB1 A34SeM PDB entry 6CHE; (E) GB1 V39SeM PDB entry 6CTE; (F) GB1 V54SeM low-resolution model. An imidazole ligand is displayed for GB1 A34SeM, and an MPD ligand is shown for GB1 I6SeM chain B. Hydrogen atoms were omitted for clarity. The selenium atom is colored orange, carbons are depicted in green, oxygens are depicted in red, and nitrogens are depicted in blue.

Figure 2.

Electron density of each SeM side chain found in the five crystal structures of GB1 variants. (A) GB1 L5SeM; (B) GB1 I6SeM; (C) GB1 V29SeM; (D) GB1A34SeM; (E) GB1 V39SeM. For variants with two copies of the protein in the asymmetric unit, the SeM in chain A is shown on the left and that of chain B is shown on the right. A34SeM has only one GB1 in the asymmetric unit. The selenium atom is shown in orange, carbons are shown in green, oxygens are shown in red, and nitrogens are shown in blue. Hydrogens are omitted for clarity. The 2F₀–F_c electron density map is shown in blue. The anomalous diffraction map is shown in magenta.

Figure 3.

⁷⁷Se solution NMR spectra of the six GB1 variants acquired at 11.74 T at 293 K. From the top to bottom: GB1 L5SeM, GB1 I6SeM, GB1 V29SeM, GB1 A34SeM, GB1 V39SeM, and GB1 V54SeM.

Figure 4.

⁷⁷Se solution NMR spectra of five GB1 variants acquired at 11.74 T at different temperatures.

Figure 5.

Two-dimensional ¹H–⁷⁷Se HMBC spectrum of five GB1 variants. (A) GB1 L5SeM; (B) GB1 I6SeM; (C) GB1 V29SeM; (D) GB1 V39SeM; (E) GB1 V54SeM.

Figure 6.

Solid-state ⁷⁷Se CP/MAS NMR spectra of [⁷⁷Se]-SeM incorporated in the protein GB1 L5SeM (left, panels A–C), GB1 I6SeM (middle panels D–F), GB1 V29SeM (middle, panels G–I) and GB1 V39SeM (right, panels J–L) at different spinning speeds, acquired at 14.1 T at 293 K. (A) GB1 L5SeM at 8 kHz; (B) GB1 L5SeM at 5 kHz; (C) GB1 L5SeM at 3 kHz; (D) GB1 I6SeM at 8 kHz; (E) GB1 I6SeM at 5 kHz; (F) GB1 I6SeM at 3 kHz; (G) GB1 V29SeM at 8 kHz; (H) GB1 V29SeM at 5 kHz; (I) GB1 V29SeM at 3 kHz; (J) GB1 V39SeM at 8 kHz; (K) GB1 V39SeM at 5 kHz; (L) GB1 V39SeM at 3 kHz.

Figure 7.

Solid-state ⁷⁷Se CP/MAS NMR spectra of GB1 variants recorded at different temperatures. All spectra were acquired at 14.1 T and a spinning speed of 5 kHz. Each column corresponds to one GB1 variant, in the order of (from left to right): GB1 L5SeM, GB1 I6SeM, GB1 V29SeM, and GB1 V39SeM. From top to the bottom: ⁷⁷Se CP/MAS NMR spectra acquired at 249, 260, 270, 279, and 293 K. Last row: superimposition of spectra acquired at 293 K (blue) and a lower temperature (red).

Figure 8.

Superimposition of ⁷⁷Se CP/MAS NMR spectra at 293 (blue), 279 (red), 270 (green), 260 (purple), and 249 K (yellow) for (A) GB1 L5SeM, (B) GB1 I6SeM, (C) GB1 V29SeM, and (D) GB1 V39SeM. The strongest spinning sideband is shown for all but the GB1 I6SeM spectrum, which has comparatively low sensitivity, and thus several spinning sidebands are shown to demonstrate the temperature dependence. All spectra are acquired at a field strength of 14.1 T NMR and a spinning speed of 5 kHz.