Chemical shift assignment of the transmembrane helices of DsbB, a 20-kDa integral membrane enzyme, by 3D magic-angle spinning NMR spectroscopy

Abstract

The Escherichia coli inner membrane enzyme DsbB catalyzes disulfide bond formation in periplasmic proteins, by transferring electrons to ubiquinone from DsbA, which in turn directly oxidizes cysteines in substrate proteins. We have previously shown that DsbB can be prepared in a state that gives highly resolved magic-angle spinning (MAS) NMR spectra. Here we report sequential 13C and 15N chemical shift assignments for the majority of the residues in the transmembrane helices, achieved by three-dimensional (3D) correlation experiments on a uniformly 13C, 15N-labeled sample at 750-MHz 1H frequency. We also present a four-dimensional (4D) correlation spectrum, which confirms assignments in some highly congested regions of the 3D spectra. Overall, our results show the potential to assign larger membrane proteins using 3D and 4D correlation experiments and form the basis of further structural and dynamical studies of DsbB by MAS NMR.

Figure 1.

Representative 2D planes from 3D (A) NCACX, (B) CAN(CO)CX, (C) NCOCX, and (D) CON(CA)CX chemical shift correlation spectra acquired on a [U-¹³C,¹⁵N] DsbB C41S sample (∼1 μmol) at 750-MHz ¹H frequency, 12.5-kHz MAS frequency, and 223 K; 75-kHz ¹H TPPM decoupling (6.5 μs, 9°) was used during indirect evolution periods and acquisition period, and 90-kHz CW decoupling was used during ¹⁵N-¹³C and ¹³C-¹⁵N SPECIFIC CP periods. Additional acquisition and all processing parameters for each spectrum are included in the Electronic supplementary material.

Figure 2.

A strip plot of residues A73–V75. The plot consists of strips from four 3D spectra: (A) CON(CA)CX, (B) NCACX, (C) NCOCX, and (D) CAN(CO)CX.

Figure 3.

Backbone torsion angles of assigned residues in DsbB C41S determined by TALOS analysis.

Figure 4.

Representative 2D planes from 4D CANCOCX spectrum acquired on a [U-¹³C, ¹⁵N] DsbB C41S sample (∼1 μmol) at 500-MHz ¹H frequency, 11.111-kHz MAS frequency, and 223 K; 80-kHz ¹H TPPM decoupling (6.1 μs, 17°) was used during indirect evolution periods and acquisition period; 90-kHz CW decoupling was used during ¹⁵N-¹³C and ¹³C-¹⁵N SPECIFIC CP periods. The data were acquired with 0.45-ms ¹H-¹⁵C CP, 40 points with a dwell time of 90 μs in t₁ (¹³C), 6.0 ms ¹³Cα-¹⁵N CP, 40 points with a dwell time of 180 μs in t₂ (¹⁵N), 6.0 ms ¹⁵N-¹³CO SPECIFIC CP, 20 points with a dwell time of 270 μs in t₃ (¹³C), 25 ms ¹³C-¹³C DARR mixing, 1024 points with a dwell time of 15 μs in t₄ (¹³C). Twelve scans were acquired for each row. The data were processed with 150-Hz, 90-Hz, 100-Hz, and 100-Hz net line broadening (Lorentzian-to-Gausssian apodization) in F1, F2, F3, and F4 dimensions, respectively, and zero filled to 128 (F1) × 80 (F2) × 128 (F3) × 2048 (F4) complex points prior to Fourier transformation.