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. 2015 Apr;61(3-4):275-86.
doi: 10.1007/s10858-014-9893-4. Epub 2015 Jan 13.

Solid-state NMR of the Yersinia Pestis Outer Membrane Protein Ail in Lipid Bilayer Nanodiscs Sedimented by Ultracentrifugation

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Solid-state NMR of the Yersinia Pestis Outer Membrane Protein Ail in Lipid Bilayer Nanodiscs Sedimented by Ultracentrifugation

Yi Ding et al. J Biomol NMR. .
Free PMC article


Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396-10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure-activity correlation experiments across a wide range of timescales.


Figure 1
Figure 1. Characterization of Ail nanodiscs
(A) Size exclusion chromatography of Ail nanodiscs (solid line) or empty nanodiscs (dashed lines) with optimized molar ratios of Ail to MSP to phospholipid (Ail / MSP / PL). (B) TROSY 1H-15N correlation NMR spectrum of 15N labeled Ail in MSP1D1Δh5 nanodiscs in solution (0.5 mM Ail). (C) INEPT 13C NMR spectrum of 13C labeled Ail in MSP1E3D1 nanodiscs in solution (0.5 mM Ail). Assigned lipid signals were derived from published data (Oldfield and Chapman 1971; Birdsall et al. 1972; Levine et al. 1972; Haberkorn et al. 1978); asterisks indicate signals from protein aromatic sites. (D) Hahn echo 31P-detected 1H-decoupled spectrum of Ail-MSP1E3D1 nanodiscs in solution (0.5 mM Ail).
Figure 2
Figure 2. Fibronectin-bnding activity of Ail in nanodiscs and detergent
(A) Purified refolded Ail-His in DePC or in nanodiscswas added at increasing concentrations to fibronectin-coated plates and incubated overnight. Binding was detected by ELISA using a mouse anti-His antibody. Ail-His in MSP1D1Δh5 or MSP1E3D1 nanodiscs bind fibronectin (black solid lines). Ail-His in 170 mM DePC (green) does not bind fibronectin. Empty nanodiscs lacking Ail-His were probed with anti-ApoA1 antibody and do not bind fibronectin (black dotted line). Each point in each data set represents the average of three experiments. (B) TROSY 1H-15N correlation NMR spectrum of Ail in 170 mM DePC solution (0.5 mM Ail). (C) TROSY 1H-15N correlation NMR spectra of Ail in MSP1D1Δh5 nanodiscs in solution obtained in the absence of detergent (black), and after addition of 4 mM DePC (orange) or 11 mM DePC (green).
Figure 3
Figure 3. NMR spectra of Ail-MSP1D1Δh5 nanodiscs obtained after sedimentation (A, B) and subsequent redilution (C)
(A, B) Solid-state NMR 15N spectra of sedimented Ail nanodiscs obtained with CP (A) or INEPT (B) under 5 kHz MAS, at 25°C. (C) TROSY 1H-15N correlation solution NMR spectra obtained for Ail nanodiscs before sedimentation (black; 0.5 mM Ail) and after re-dilution of the sedimented sample (red; 0.5 mM Ail) at 45°C.
Figure 4
Figure 4. One-dimensional solid-state NMR spectra of sedimented Ail-MSP1E3D1 nanodiscs (5.5 mM Ail) obtained at 25°C (A-C) or 10°C (D-F)
(A, D) 13C spectra obtained with CP (black) or DP (red) and 10 kHz MAS. (B, E) 13C spectra obtained with INEPT at 10 kHz MAS. (C, F) 31P spectra obtained with DP for static sample. Isotropic signal intensity (~0 ppm) is attributed to the presence of Na phosphate in the NMR buffer.
Figure 5
Figure 5. Two-dimensional solid-state NMR 13C correlation spectra of Ail in sedimented MSP1E3D1 nanodiscs (black; 5.5 mM Ail) or proteoliposomes (red; 2.4 mM Ail)
Spectra were recorded at 27°C (for sedimented nanodiscs) or 0°C (for liposomes), with 10 kHz MAS, using PDSD with 32 scans for sedimented nanodiscs or 512 scans for liposomes. (A) Boxes highlight signals with chemical shifts characteristic for Ala, Ile, Pro and Thr residues. (B) Selected region of the spectra showing the Cα-Cβ correlations derived from the assigned spectrum of Ail in 170 mM DePC (green). (C, D) Expanded regions of the spectra.

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