Dipolar Assisted Assignment Protocol (DAAP) for MAS solid-state NMR of rotationally aligned membrane proteins in phospholipid bilayers

Abstract

A method for making resonance assignments in magic angle spinning solid-state NMR spectra of membrane proteins that utilizes the range of heteronuclear dipolar coupling frequencies in combination with conventional chemical shift based assignment methods is demonstrated. The Dipolar Assisted Assignment Protocol (DAAP) takes advantage of the rotational alignment of the membrane proteins in liquid crystalline phospholipid bilayers. Improved resolution is obtained by combining the magnetically inequivalent heteronuclear dipolar frequencies with isotropic chemical shift frequencies. Spectra with both dipolar and chemical shift frequency axes assist with resonance assignments. DAAP can be readily extended to three- and four-dimensional experiments and to include both backbone and side chain sites in proteins.

Keywords: MAS; Membrane protein; Solid state NMR; Vpu.

Figure 1

Multidimensional pulse schemes for dipolar assisted assignment protocol experiments. (A) Intra or inter residue correlation of ¹H-¹⁵N DC/¹⁵N(Cα)¹³CX or ¹H-¹⁵N DC/¹⁵N(C)¹³CX. (B) ¹H-¹⁵N DC/¹³Cα¹⁵N¹³CO. (C) ¹Hα-¹³Cα DC/¹³C CS/¹³C CS. (D) ¹H-¹⁵N DC/¹Hα-¹³Cα DC/¹³Cα CS. (E) ¹Hα-¹³Cα DC/¹⁵N CS/¹³Cα CS. Thin and wide solid lines are for 90° and 180° pulses. Delta symbol represents the delay for z-filter. CP and DCP represents cross polarization and double cross polarization under SPECIFIC CP condition. CS stands for chemical shift ϕ represents the phase change under States mode.

Figure 2

Rotational alignment of uniformly ¹³C,¹⁵N labeled single trans-membrane helix from Vpu in DMPC proteoliposome. (A) ¹³C detected one-dimensional spectra recorded at various temperatures ranging from 5 °C to 25 °C. Data were collected using ¹H to ¹³C cross-polarization under 5 kHz magic angle sample spinning. (B) Graphical plot showing the variation in intensity ratios (central peak to the first sideband on the left) as a function of temperature. Central peak is marked with an arrow in A and asterisks denote spinning side bands. One-dimensional data were acquired with 100 kHz spectral width, 15 ms acquisition time, and 2 s recycle delay for 16 scans (5 °C), 32 scans (10 °C, 15 °C, 20 °C) and 64 scans (25 °C).

Figure 3

¹³C detected two-dimensional correlation spectra of uniformly ¹³C, ¹⁵N labeled Vpu-TM. (A) ¹³C/¹³C correlation spectrum obtained from 50 ms proton driven spin diffusion (PDSD) mixing. Single resonance assignment for ¹³C shifts in P3, L11and S23 are labeled. Chemical shift dispersion for other residues such as Ala, Val, Ile, Glu, Gln, Trp, and Lys are also been labeled. (B) Two-dimensional N(CA)CX correlation spectra. 20 ms DARR mixing was used for carbon spin exchange in B. Residue numbers are marked following the sequential resonance assignment. Homo- and hetero- nuclear correlation two-dimensional spectra were acquired with 128 scans (¹³C/¹³C) and 512 scans (¹³C/¹⁵N) with a 2 s recycle delay. Experiments were carried out with 242 ppm (¹³C), 80 ppm (¹³C) and 32 ppm (¹⁵N) spectral widths for direct and indirect acquisition. The acquisition periods were 12 ms for direct and 4 ms (¹³C)/6 ms (¹⁵N) for indirect detection. 100 μs, 500 μs and 3000 μs contact times were used for ¹H to ¹³C, ¹H to ¹⁵N and ¹⁵N to ¹³C cross-polarization, respectively.

Figure 4

Strip-plots for resonance assignment for residues V9–V12 in uniformly¹³C,¹⁵N labeled Vpu TM. Two-dimensional ¹³C/¹³C correlation plot from NCACX (red) and NCOCX (blue) data extracted at ¹⁵N shifts. ¹³C shifts are marked according to their positions in backbone and side chains. Three-dimensional data were acquired with 256 scans (NCACX) and 512 scans (NCOCX). The experiments were carried out under similar conditions to those described in Figure 3 except that the homonuclear spin diffusion was obtained with 40 ms DARR mixing, and 40 ppm and 20 ppm ¹³C spectral widths for NCACX and NCOCX, respectively.

Figure 5

¹³C-detected two-dimensional separated local field NMR spectra. (A) ¹⁵N edited spectrum correlating ¹³Cα chemical shifts and ¹H-¹⁵N dipolar couplings. The spectrum was obtained using the pulse scheme shown in Figure 1A without the ¹⁵N chemical shift evolution and spin diffusion mixing periods. (B) ¹³C CS/¹H-¹³C DC correlation spectrum. All spectra were recorded under 11.111 kHz MAS at 25 °C. Two-dimensional data were acquired with 512 scans (A) and 128 scans (B) using a 2 s recycle delay. Dwell times of 90 μs for A and 30 μs for B were used in the indirect dimensions to record the spectra. A 12 ms acquisition time was used for direct ¹³C detection.

Figure 6

¹³C detected three-dimensional separated local field spectra of uniformly ¹³C,¹⁵N labeled Vpu TM. (A) and (B) Two-dimensional SLF planes correlating ¹³Cα CS/¹H-¹⁵N DC extracted from a three-dimensional SLF data set for ¹⁵N shifts at 121.6 ppm and 122.8 ppm, respectively. The spectrum was recorded using the pulse scheme shown in Figure 1A without incorporating the spin diffusion mixing. (C) and (D) Two-dimensional SLF planes correlating ¹³Cα CS/¹Hα-¹³Cα DC for ¹⁵N shifts at 121.6 ppm and 122.8 ppm, respectively. The experiment correlating ¹³Cα CS/¹⁵N CS/¹Hα-¹³Cα DC was acquired using the pulse scheme in Figure 1E. (E) and (F) Two-dimensional SLF planes correlating ¹H-¹⁵N/¹Hα-¹³Cα dipolar frequencies extracted from a three-dimensional SLF data set for ¹³C shifts at 65.7 ppm and 53.5 ppm, respectively. The three-dimensional data was collected using the pulse scheme in Figure 1D. (G) ¹³C/¹³C two-dimensional plane obtained at 9.9 kHz ¹Hα-¹³Cα DC. Three-dimensional SLF data correlating ¹Hα-¹³Cα DC/¹³C CS/¹³C CS recorded using the pulse scheme shown in Figure 1C. All spectra were collected at a spinning frequency of 11.111 kHz and 25 °C sample temperature. t1 noise from lipid signals is denoted with asterisks. The three-dimensional data in A–F were collected with 512 scans (A–F) or128 scans (G). A 2 s recycle delay and 90 μs dwell time were used in the experiments.

Figure 7

¹³C detected three-dimensional SLF data correlating ¹³C and ¹⁵N isotropic chemical shifts with ¹H-¹⁵N dipolar frequencies. (A) N(CA)CX two-dimensional plane obtained at 2.4 kHz ¹H-¹⁵N dipolar coupling (DC) frequency. The three-dimensional data were collected using the pulse scheme shown in Figure 1A with a 20 ms DARR (dipolar assisted rotational resonance) mixing. (B) N(CO)CX 2D-plane at 2.55 kHz ¹H-¹⁵N DC. The three-dimensional data were collected using the pulse scheme shown in Figure 1A with 40 ms DARR mixing. (C) CONCA two-dimensional correlation plane obtained for 2.55 kHz ¹H-¹⁵N DC. The three-dimensional data were collected using the pulse scheme shown in Figure 1B with 1.44 ms TEDOR mixing. Dashed lines illustrate the connectivity for residues A14 and V13 for ¹⁵N and ¹³C shifts. (D) ¹³C/¹³C two-dimensional plane obtained at 7.4 kHz ¹Hα-¹³Cα DC. Three-dimensional SLF data correlating ¹Hα-¹³Cα DC/¹³C CS/¹³C CS recorded using the pulse scheme shown in Figure 1C. The three-dimensional data were collected with 1024 scans (A–C), 128 scans (D), 2 s recycle delay and 90 μs dwell time.

Figure 8

Dipolar wave plot for ¹H-¹⁵N dipolar frequencies as a function of residue type. Comparison between the dipolar waves of normalized dipolar couplings (x) with order parameter S = 0.8 obtained from 14-O-PC/6-O-PC bicelles (q = 3.2) at 42°C and those of dipolar couplings (o) obtained from DMPC liposomes at a spinning rate of 11.111 kHz and 25°C.