Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR

Abstract

We present a new method that combines carbonyl-selective labeling with frequency-selective heteronuclear recoupling to resolve the spectral overlap of magic angle spinning (MAS) NMR spectra of membrane proteins in fluid lipid membranes with broad lines and high redundancy in the primary sequence. We implemented this approach in both heteronuclear (15)N-(13)C(α) and homonuclear (13)C-(13)C dipolar assisted rotational resonance (DARR) correlation experiments. We demonstrate its efficacy for the membrane protein phospholamban reconstituted in fluid PC/PE/PA lipid bilayers. The main advantage of this method is to discriminate overlapped (13)C(α) resonances by strategically labeling the preceding residue. This method is highly complementary to (13)C(i-1)(')-(15)N(i)-(13)C(i)(α) and (13)C(i-1)(α)-(15)N(i-1)-(13)C(i)(') experiments to distinguish inter-residue spin systems at a minimal cost to signal-to-noise.

Figure 1

Pulse sequences used to obtain (A) 2D FDR-¹⁵N-¹³C^α and (B) 3D FDR-¹⁵N-¹³C^α-¹³C^X-DARR spectra. The 90° pulses on ¹³C are phase cycled with XY-4 [62] and applied at the end of each rotor cycle, while those 180° pulses applied to ¹⁵N are applied in the middle of the rotor cycle with MLEV-4 phase cycling [61]. Phase cycling is as follows: ₁ {x,-x}, ₂ {y}, ₃ {x,x,y,y,-x,-x,-y,-y}, ₄ {y,y,-x,-x,-y,-y,x,x}, ₅ {-y,-y,x,x,y,y,-x,-x}, _rec {x,-x,y,-y,-x,x,-y,y}. Phases ₂ and ₃ are adjusted by 90° to generate phase-sensitive data in t₁ and t₂, respectively.

Figure 2

FDR dephasing ¹⁵N{¹³C} curves for one-bond (1.3 Å) and two-bond (2.5 Å) ¹³C′-¹⁵N distances. The dotted line indicates the maximum dephasing for one-bond transfer, showing less than 0.1 dephasing for the two-bond curve. The FDR dephasing data is scaled by $1/\sqrt{2}$ with respect to the REDOR experiment [44, 58].

Figure 3

FDR-¹⁵N-¹³C^α spectra for NAVL. Spectra were acquired with (FDR, panel A) and without ¹³C 90° pulses (reference, panel B). A total dephasing time of 2 msec was used with an MAS rate of 8 kHz. (C) Subtraction of the FDR spectrum from that of the reference. Note that in the subtracted spectrum, the noise increases by a factor of $\sqrt{2}$.

Figure 4

Comparison of ¹H-¹³C cross-polarization, ¹⁵N-¹³C^α SPECIFIC-CP, ¹³C′-¹⁵N-¹³C^α double SPECIFIC-CP, and FDR-¹⁵N-¹³C^α (2.0 msec dephasing time) experiments. The integrated intensities from these spectra are shown in Table 1.

Figure 5

FDR-¹⁵N-¹³C^α spectra for AFA-PLN labeled [U-¹³C,¹⁵N] at residues 30–33. 1D spectra were acquired with (FDR, panel A) and without ¹³C 90° pulses (reference, panel B). A total dephasing time of 2 msec was used with an MAS rate of 8 kHz. (C) Subtraction of the FDR spectrum from the reference.

Figure 6

2D FDR-¹⁵N-¹³C^α spectra for AFA-PLN labeled [U-¹³C,¹⁵N] at residues 30–33. (A) Difference spectrum for AFA-PLN^30-33 using similar parameters as that in Figure 5. (B) ¹⁵N-¹³C^α spectrum of AFA-PLN^30-33.

Figure 7

FDR-¹³C^α-¹³C^X-DARR spectra for NAVL. (A) Spectra were acquired with (FDR, panel i) and without ¹³C 90° pulses (reference, panel ii) using the pulse sequence in Figure 1B. A total dephasing time of 2 msec was used with an MAS rate of 8 kHz. (iii) Subtraction of the FDR spectrum from that of the reference. A 100 msec DARR mixing time was used. Panels B and C show the 1D traces from the 2D spectra in panel A.