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. 2009 Sep 16;131(36):12970-8.
doi: 10.1021/ja902012x.

Coupled decomposition of four-dimensional NOESY spectra

Sebastian Hiller  1 , Ilghis Ibraghimov, Gerhard Wagner, Vladislav Y Orekhov
Affiliations

Coupled decomposition of four-dimensional NOESY spectra

Sebastian Hiller et al. J Am Chem Soc. .

Abstract

Four-dimensional (4D) NOESY spectra provide unambiguous distance information at a resolution that cannot be achieved in fewer dimensions and thus increase the quality of biomolecular structure determination substantially. Since the degree of chemical shift degeneracy increases with protein size, the use of 4D NOESY spectra is particularly important for large proteins. The potential high resolution in 4D spectra cannot be achieved in a reasonable time with conventional acquisition routines that sample the Nyquist grid uniformly. It can, however, be obtained with nonuniform sampling of the data grid, but optimal processing of such data has not yet been established. Here we describe a processing method for a pair of sparsely sampled 4D NOESY spectra, a methyl-methyl and an amide-methyl NOESY, recorded on a perdeuterated protein with protonated isoleucine, leucine, and valine methyl groups. The coupled multidimensional decomposition (Co-MDD) of these two spectra together with a 2D template spectrum results in a substantial increase in sensitivity, evidenced by 50-100% additional cross peaks, when compared to alternative processing schemes. At the same time, Co-MDD allows the use of low sparse levels of 10-15% of the full data grid for NOESY spectra. For the 283-residue integral human membrane protein VDAC-1, which has a rotational correlation time of about 70 ns in detergent micelles, the two 4D Co-MDD NOESYs yielded a total of 366 NOEs, resulting in 139 unambiguous upper limit distance constraints for the structure calculation.

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Figures

Figure 1
Figure 1
Pulse sequence for the 4D NUS-Co-MDD-15N-HMQC-[1H,1H]-NOESY-13C-HMQC experiment. Radio-frequency pulses were applied at 4.7 ppm for 1H, 118.0 ppm for 15N and 17.3 ppm for 13C. Narrow and wide bars represent 90° and 180° pulses with rectangular shape and high power at maximal power, respectively. Pulses with a bell shape on 1H are water flip-back pulses, applied as Gaussian shapes, with 1 ms duration. The last three 1H pulses, a WATERGATE element, are centered with respect to the sequence Δ2−90°−t3−90°−Δ2 on the 13C channel, so that its water flip pulses overlap with the delays Δ2 for short t3 times. Decoupling using GARP on 13C is indicated by a rectangle. t4 represents the acquisition period. τm is the NOESY mixing time. On the line marked PFG, curved shapes indicate sine bell-shaped pulsed magnetic field gradients applied along the z-axis with the following durations and strengths: G1, 1000 μs, 35 G/cm; G2, 200 μs, 6 G/cm; G3, 300 μs, 9 G/cm; G4, 800 μs, 30 G/cm; G5, 600 μs, 20 G/cm. Phase cycling: φ1 = x, φ2 = {x, −x}, φ3 = {x, x, −x, −x}, φrec = {−x, x, x, −x}, ψ1 = x + 45°, all other pulses = x. Fixed delays were Δ1 = 3.8 ms and Δ2 = 3.5 ms. Quadrature detection with States-TPPI for the indirect dimensions was achieved with the angles φ1,φ2 and φ3 for t1, t2 and t3, respectively.
Figure 2
Figure 2
Data flow and data generation in the Co-MDD processing scheme. (A) Data flowchart. The conventional Fourier transform steps are not shown. See the Methods section for details. (B) Generation of the dimensions α and ζ in the 4D data set COS from the dimensions t1 and t2 in the 4D data set CCS and the 2D data set HMQCS. Squares correspond to complex points in the data grids. Corresponding points are marked by index numbers or the letter “A”. Grey points have zero intensity. Dimensions t3 and ω4 have been omitted in this drawing.
Figure 3
Figure 3
3D and 4D NOESY spectra of the integral membrane protein VDAC-1 in LDAO micelles. (A) Plane from the NUS-Co-MDD-13C-HMQC-[1H,1H]-NOESY-13C-HMQC at the position of I123 CδHδ, (B) Plane from the NUS-Co-MDD-15N-HMQC-[1H,1H]-NOESY-13C-HMQC at the position of V184 Hδ1/Cδ1. Resonance assignments are indicated. (C) Strip from the 3D-13C-HMQC-[1H,1H]-NOESY at the position of V184 Hδ1/Cδ1. Corresponding peaks in (B) and (C) are connected by dashed lines.
Figure 4
Figure 4
Display of all methyl–methyl and methyl–amide NOEs observed in the two 4D Co-MDD-NOESYs for the integral membrane protein VDAC-1 in LDAO micelles. Boxes are amino acid residues in β-sheet secondary structure and circles denote residues in loops or helical secondary structure. Selected residue numbers, the strand numbers β1–β19 and all residues of type I, L and V are indicated. Grey are residues with an unassigned amide moieties, white and yellow are residues with an assigned amide moieties. The side chains of yellow and white I, L or V residues point to the outside and inside of the barrel, respectively. Blue lines are NOEs observed in the 4D 15N-HMQC-[1H,1H]-NOESY-13C-HMQC. A blue letter shows an NOE in this experiment from an I, L or V methyl group to its own amide moiety. Red lines denote NOE contacts observed in the 4D 13C-HMQC-[1H,1H]-NOESY-13C-HMQC. Note that a red line can represent up to 8 actually observed NOEs. Residues L150 and V143, which have NOE contacts to residue L10, are plotted twice. Strand β19, which closed the β-barrel by parallel pairing to β1, is plotted twice.
Figure 5
Figure 5
Comparison of different processing schemes for the 4D NUS 13C-HMQC-[1H,1H]-NOESY-13C-HMQC data. A: 2D planes obtained by the three methods Co-MDD, DFT and MDD for I194 HδCδ, a representative from group M1, which contains 85% of all methyls (see text), and for L10 Hδ1Cδ1, a representative from group M2. Potential peak positions are indicated by orange circles. All spectral planes are plotted with a base level of 3.0 times their spectral noise. B: 1D cross sections along the dashed lines in A, as indicated by corresponding roman numbers. I–III and IV–VI were scaled according to the noise level of the 2D planes.
Figure 6
Figure 6
Comparison of different processing schemes for the 4D NUS 15N-HMQC-[1H,1H]-NOESY-13C-HMQC. A: 2D planes obtained by the three methods Co-MDD, DFT and MDD for L39 Hδ1Cδ1 and L208 Hδ1Cδ1. Potential peak positions are indicated by orange circles. All spectral planes are plotted with a base level of 3.0 times their spectral noise. B: 1D cross sections along the dashed lines in A, as indicated by corresponding roman numbers. I–III and IV–VI were scaled according to the noise level of the 2D planes.
Figure 7
Figure 7
Peak statistics of the two Co-MDD 4D NOESYs. The fraction f is the number of observed peaks divided by the number of expected peaks from the 2.3 Å crystal structure, plotted as a histogram of the 1H–1H distance d. The absolute number of observed peaks in each distance category is indicated above each bar. (A) 4D 13C-HMQC-[1H,1H] -NOESY-13C-HMQC, (B) 4D 15N-HMQC-[1H,1H] -NOESY-13C-HMQC.
Figure 8
Figure 8
Correlations of peak intensities I in non-uniformly sampled 4D NOESY spectra processed with either discrete Fourier transformation (DFT) and Co-MDD (see text). Unobserved peaks (intensities below noise level) were assigned a value of 0. (A) Cross and diagonal peaks in the 4D 13C-HMQC-[1H,1H] -NOESY-13C-HMQC, the region in the dashed box is shown enlarged in (B).(C) cross peaks in the 4D 15N-HMQC-[1H,1H] -NOESY-13C-HMQC.
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