Cellular solid-state nuclear magnetic resonance spectroscopy

Abstract

Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a well-established imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution.

Fig. 1.

Cellular ssNMR spectroscopy: overall strategy and sample preparation, including inner and outer membrane proteins (IMP, OMP). (A) Schematic structure of the E. coli K-12 cell envelope. (B) Overall scheme for the preparation of WC and CE from strain CE1535 carrying plasmid pPagL_(Pa), and of purified PagL protein from strain BL21Star(DE3) carrying plasmid pPagL_(Pa)(-) reconstituted in PL. (C) Coomassie-stained SDS/PAGE analysis of WC, CE, and protoplasm (P) fractions obtained from exponentially growing E. coli CE1535 cells containing plasmid pPagL_(Pa) and comparison with the reference PL sample. Molecular-mass markers (MM) are indicated next to the gels. Samples were denatured by boiling in SDS (d) or left on ice (n) before electrophoresis. F and U denote the positions of folded and heat-denatured forms of PagL, respectively. (D) In vitro and in vivo LPS deacylase activity of PagL. (Upper) Purified Neisseria meningitidis LPS was incubated in a detergent-containing buffer with (lane 3) or without (lane 1) PagL-containing PL and analyzed by Tricine SDS/PAGE and staining with silver. Membranes from N. meningitidis harboring functional PagL were coanalyzed for reference (lane 2). (Lower) Silver-stained Tricine SDS/PAGE analysis of CE isolated from plasmidless CE1535 cells (lane 1) or noninduced (lane 2) and IPTG-induced (lane 3) CE1535 cells carrying the PagL-encoding plasmid.

Fig. 2.

NMR spectra of whole cells, cell envelopes, and proteoliposomes. ¹³C-¹³C correlation spectra of fully hydrated IPTG-induced WC (A), CE isolated from IPTG-induced WC (B), and (U-¹³C, ¹⁵N)-labeled PagL-containing PL (C) recorded using respectively 224, 336, and 192 scans and processed identically using a sine bell function (SSB of 3.5) and linear prediction in the indirect dimension. Contour plots were adjusted to the same noise level. Characteristic cross-peaks of Ala, Ser, and Thr residues located within PagL β-sheet protein segments and of endogenous E. coli lipids (Lip) are indicated in red and green, respectively.

Fig. 3.

Conformational analysis of PagL. (A) Overlay of 2D NCA correlation spectra of CE isolated from IPTG-induced WC (black) and PL (red), recorded with identical acquisition and processing parameters—i.e., with a sine bell function (SSB = 4.5) and using linear prediction in indirect dimension. (B) Comparison of PagL in CE and PL environments. (Left) Selected spectral regions from 2D homonuclear and heteronuclear spectra of CE isolated from IPTG-induced WC (black) showing isolated PagL resonances, and overlaid with spectra obtained on CE isolated from noninduced WC (green) and PL (red). Assignments are indicated where available. (Right) Backbone N, C^α, and C^β chemical-shift changes observed for PagL embedded in E. coli CE and in PL. Horizontal lines indicate the threshold for significant chemical-shift changes. The threshold was set to 2 times . Residues with a chemical-shift deviation larger than the threshold (+ 2 SD) are labeled. (C) Summary of PagL ssNMR spectral changes between CE and PL preparations plotted onto the topological representation of PagL according to the crystal structure (β-sheet protein segments are represented by open rectangles and transmembrane segments TM1–8 are labeled). Arrows point to residues that experienced significant backbone (solid lines) and side-chain (dashed lines) chemical-shift changes, whereas orange filled bars indicate major alterations in signal intensities (> 50%) between CE and PL.

Fig. 4.

Identification and characterization of the lipoprotein Lpp. (A) Two-dimensional (¹³C-¹³C) and (B) 2D NCA correlation spectra obtained on the CE isolated from noninduced WCs overlaid with backbone C^α-C^β and N-C^α (red crosses) intraresidue correlations predicted from the crystal structure of Lpp (Protein Data Bank ID code 1EQ7) using SPARTA+ (33). For other carbon positions (black crosses), average ¹³C chemical-shift values given in the Biological Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu/ref_info/statsel.htm) were used. Characteristic correlations are labeled and color coded: orange for correlations absent in the experimental data and green for correlations that are in agreement with chemical-shift predictions. (Inset, Upper Right) Spectral region characteristic of Gly residues. (Inset, Lower Left) An overlay of 2D NCA spectra for α-helical Thr, Val, and Ile N-C^α correlations in CE isolated from non- (black) and IPTG-induced (blue) cells. (C) Summary of the structural analysis of Lpp based on ssNMR spectra obtained on CE isolated from noninduced cells. Residues for which backbone correlations significantly deviate or not from predictions are labeled and highlighted in orange or green, respectively. (D) Two-dimensional NCA correlation spectrum including side-chain amide ¹⁵Nζ resonances of Lys revealing distinct and characteristic NC correlation patterns for the free (Upper) and the bound (Lower) forms of Lpp. Meso-diaminopimelic acid, m-DAP.

Fig. 5.

Resonance assignment of flexible LPS and PG using through-bond ssNMR correlation spectroscopy. (A) Expansion of the 2D (¹H-¹³C) insensitive nuclei enhanced by polarization transfer (INEPT) correlation spectrum obtained on CE isolated from IPTG-induced WC, showing dispersion and resolution of individual α- and β-anomeric resonances of LPS and PG sugar moieties. Splitting due to the C1-C2 scalar coupling is visible for all anomeric resonances and varied between 47 and 53 Hz. Color coding: β-GlcN, dark blue; α-GlcN, light blue; PG GlcNAc, orange; NAM, red; LPS core, black. (B) Sections from the 2D (¹³C-¹³C) INEPT-total through-bond correlation spectroscopy showing characteristic correlations between anomeric C1 and one-bond nitrogen-substituted C2 carbons (Upper) or one-to-three bonded non nitrogen-substituted C2-C4 carbons (Lower) of LPS and PG sugar moieties. The CC and HC correlations that belong to the same spin system are connected by dashed lines. (C) Residue-specific ssNMR assignment of LPS and PG glucosamine units based on ¹³C-¹³C connectivities within the sugar rings and their substituents (see also Fig. S7A). Assigned resonances from LPS and PG glucosamine units are highlighted by filled circles onto chemical structures with the same color coding as in A.

Fig. 6.

Atomic-level insights of E. coli CE-associated macromolecules revealed by cellular ssNMR spectroscopy. Schematic representation of the CE from E. coli showing rigid (blue) and flexible (red) (non)proteinaceous molecular components characterized by through-bond and through-space ssNMR experiments, respectively. The topological representations of PagL and Lpp as seen in the available 3D models of isolated molecules are indicated. For PagL, residues located in β-strand and random-coil protein segments are represented by squares and circles, respectively. Residues that were not included in the analysis are colored in gray. Amino acids are given in single-letter notation. Spectroscopic changes of large (chemical-shift deviation > 0.4 ppm and/or signal intensity variations > 50%) and small magnitude that are potentially related to association between membrane protein and OM (or PG) are indicated for both proteins in orange and blue, respectively.