Cellular solid-state nuclear magneticresonance spectroscopyMarie Renaulta, Ria Tommassen-van Boxtelb, Martine P. Bosb, Jan Andries Postc, Jan Tommassenb,1, and Marc Baldusa,1

aBijvoet Center for Biomolecular Research, bDepartment of Molecular Microbiology, Institute of Biomembranes, and cDepartment of BiomolecularImaging, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

Edited by Robert Tycko, National Institutes of Health, Bethesda, MD, and accepted by the Editorial Board January 6, 2012 (received for reviewOctober 11, 2011)

Decrypting the structure, function, and molecular interactions ofcomplex molecular machines in their cellular context and at atomicresolution is of prime importance for understanding fundamentalphysiological processes. Nuclear magnetic resonance is a well-established imaging method that can visualize cellular entities atthe micrometer scale and can be used to obtain 3D atomic struc-tures under in vitro conditions. Here, we introduce a solid-stateNMR approach that provides atomic level insights into cell-asso-ciated molecular components. By combining dedicated protein pro-duction and labeling schemes with tailored solid-state NMR pulsemethods, we obtained structural information of a recombinantintegral membrane protein and the major endogenous molecularcomponents in a bacterial environment. Our approach permitsstudying entire cellular compartments as well as cell-associatedproteins at the same time and at atomic resolution.

cellular envelope ∣ Escherichia coli ∣ lipoprotein ∣ PagL ∣ magic anglespinning

Physiological processes rely on the concerted action of mole-cular entities in and across different cellular compartments.

Whereas advancements in molecular imaging have providedunprecedented insights into the macromolecular organizationin the subnanometer range (1), studying atomic structure and mo-tion in situ has been challenging for structural biology. NMR hasprovided insight into cellular processes (2–4) and can determineentire 3Dmolecular structures inside living cells (5) provided thatmolecular entities tumble rapidly in a cellular setting. In princi-ple, solid-state NMR (ssNMR) spectroscopy offers a comple-mentary spectroscopic tool to monitor molecular structure anddynamics at atomic resolution in a complex setting (see ref. 6for a recent review). Indeed, ssNMR has already been used tostudy individual molecular components in the context of naturalbilayers (7, 8), bacterial cell walls (9), and cellular organelles (10).

Here, we introduce a general approach to investigate structureand dynamics of an arbitrary molecular target and its potentialmolecular partners in a cellular setting. Our studies focuses onthe Gram-negative bacterial cell that is characterized by a mole-cularly complex but architecturally unique envelope, consisting oftwo lipid bilayers, the inner and outer membrane (IM, OM), se-parated by the periplasm containing the peptidoglycan (PG) layer(Fig. 1A). The IM is a phospholipid bilayer and harbors α-helicalproteins, whereas the OM is asymmetrical and consists of phos-pholipids, lipopolysaccharides (LPS), lipoproteins, and β-barrel-fold integral membrane proteins. LPS forms the outermost layerof the OM and protects the cell against harmful compounds fromthe environment. PG is a large macromolecule that gives the cellits shape and rigidity.

Using uniformly 13C,15N-labeled cellular preparations ofEscherichia coli, we characterized the structure and dynamicsof a recombinant integral membrane protein (PagL) and othermajor endogenous molecular components of the cell envelopeincluding lipids, the peptidoglycan, and the lipoprotein Lpp (alsoknown as Braun’s lipoprotein). These studies highlight theinfluence of the surrounding compartment on molecular struc-ture and establish ssNMR under magic angle spinning (MAS)

conditions (11) as a high-resolution method to investigate atomicstructures of major cell-associated (macro)molecules.

ResultsSample Design for Cellular ssNMR Spectroscopy. Our goal was toestablish general expression and purification procedures that leadto uniformly 13C,15N-labeled preparations of whole cells (WC)and cell envelopes (CE) containing an arbitrary (membrane)protein target (Fig. 1B). As our model system, we selected the150-residue integral membrane-protein PagL from Pseudomonasaeruginosa, an OM enzyme that removes a fatty acyl chain fromLPS (12). We overexpressed pagL under control of the bacter-iophage T7 promoter, which is inducible with IPTG, in a mutantE. coli BL21Star(DE3) strain, lacking the two major OM proteins(OMPs) OmpF and OmpA. The suppression of these majorOMPs prevented to a large extent the accumulation of the unpro-cessed signal-peptide-bearing precursor of PagL and led to signif-icant amounts of mature protein in the host membrane when mildrecombinant-protein-expression conditions were used (Fig. S1).For optimal analysis of major cell-associated molecular compo-nents, E. coli cultures were switched from unlabeled to 15N,13C-isotope labeled growth conditions at the beginning of the expo-nential growth phase, when recombinant protein production wasinduced, leading to the incorporation of isotopes in PagL and co-expressed endogenous molecular components. WC and CE sam-ples were prepared from the same exponentially growing culture.As a reference, (U-13C,15N)-labeled PagL was produced in intra-cellular inclusion bodies, purified, and reconstituted in proteoli-posomes (PL, Fig. 1B). Before analysis, WC pellet was washedwith PBS, whereas CE and PL were resuspended in Hepes atpH 7.0 and harvested by ultracentrifugation using identical pro-cedures. Both in CE preparations and reconstituted in PL, PagLexhibited similar heat modifiability, a property typical of the well-folded protein (12, 13) (Fig. 1C). To verify that PagL was correctlyfolded in vivo and in vitro, we monitored its LPS 3-O-deacylaseactivity in CE and PL preparations as described previously (12,13). In both cases, LPS was converted into a form with higherelectrophoretic mobility (Fig. 1D), in agreement with the ex-pected hydrolysis of the primary acyl chain at position 3 of lipidA. Taken together, our data (heat modifiability and activity as-says) indicate that cellular and PL preparations contained well-folded and functional PagL.

NMR Spectra of E. coli Whole Cells and Cell Envelopes Versus Proteo-liposomes. To characterize rigid—presumably membrane-asso-

Author contributions: M.R., J.T., and M.B. designed research; M.R., R.T.-v.B. and M.P.B.performed research; J.-A.P. contributed new reagents/analytic tools; M.R., J.T., and M.B.analyzed data; and M.R., J.T., and M.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. R.T. is a guest editor invited by the Editorial Board.

See Commentary on page 4715.1To whom correspondence may be addressed. E-mail: j.p.m.tommassen@uu.nl orm.baldus@uu.nl.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1116478109/-/DCSupplemental.

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ciated—molecular components in WC and CE, we performed aset of 2D 13C-13C correlation experiments employing dipolar-based magnetization transfer steps. Overall, both preparationsyielded NMR spectra of astonishing quality considering samplecomplexity and noncrystallinity (Fig. 2 A and B), with well-dis-persed cross-peaks characteristic for protein and lipid signals.As anticipated, we observed an improvement in both sensitivityand spectral resolution for the CE preparation (Fig. S2A), poten-tially due to the single contribution of CE-associated compo-nents. These results were corroborated by SDS/PAGE analysis(Fig. 1C), which showed a significant decrease of the amount ofproteins after removal of the protoplasm by cell lysis and ultra-centrifugation. Over time, WC and CE preparations did not re-veal any marked spectroscopic changes at −2 °C, and the cellmorphology and the structural integrity of the CE were preservedafter extended NMR studies (Fig. S3). When comparing WC andCE spectra with the reference PL spectrum recorded under simi-lar measurement conditions (Fig. 2C), we found that a large set ofintraresidue correlations from PagL, notably cross-peaks of Ala,Thr, and Ser residues in β-sheet protein segments (see below), arewell preserved in WC, CE, and PL spectra.

Conformational Analysis of the PagL Protein in the E. coli Cell Envel-ope.To examine in further detail the conformation of PagL in CE,we performed 2D 15N-13C correlation experiments (14) in whichsignals arising from nonproteinaceous molecular components arelargely reduced. Comparison with the reference PL spectrum(Fig. 3A, red) revealed astonishing similarities. With average13C and 15N line widths of 0.6–0.8 and 1.5–1.6 ppm, respectively,ssNMR spectra of the CE preparation exhibited comparable, ifnot superior spectral resolution (Fig. S2B). Because of the favor-able spectroscopic dispersion among Thr, Ser, and Gly residues instandard 2D CC/NC correlation experiments, we could subse-quently perform a residue-specific analysis, which consisted ofthree stages. First we determined sequential resonance assign-ments in PagL-PL preparations in spectroscopically favorableregions. Second, comparison to the same spectral regions in da-tasets obtained for CE served to obtain tentative assignments forthe same PagL residues in the CE case. These were finally cross-validated using additional 2D and 3D datasets. With this strategy,we obtained 15N and 13C resonance assignments for 13 PagLresidues located in different topological regions of the proteinusing CE and PL preparations (Fig. S4 and Table S1). An analysisbased on 13C secondary chemical shifts of PagL in CE and in PLwas consistent with the presence of Ser33, Thr34, and Thr91 inunstructured protein regions and Ala9, Thr10, Thr16, Thr32,

Fig. 1. Cellular ssNMR spectroscopy: overall strategy and sample prepara-tion, including inner and outer membrane proteins (IMP, OMP). (A) Schematicstructure of the E. coli K-12 cell envelope. (B) Overall scheme for the prepara-tion of WC and CE from strain CE1535 carrying plasmid pPagLðPaÞ, and of pur-ified PagL protein from strain BL21Star(DE3) carrying plasmid pPagLðPaÞ(-)reconstituted in PL. (C) Coomassie-stained SDS/PAGE analysis of WC, CE,and protoplasm (P) fractions obtained from exponentially growing E. coliCE1535 cells containing plasmid pPagLðPaÞ and comparison with the referencePL sample. Molecular-mass markers (MM) are indicated next to the gels. Sam-ples were denatured by boiling in SDS (d) or left on ice (n) before electrophor-esis. F and U denote the positions of folded and heat-denatured forms ofPagL, respectively. (D) In vitro and in vivo LPS deacylase activity of PagL.(Upper) PurifiedNeisseria meningitidis LPS was incubated in a detergent-con-taining buffer with (lane 3) or without (lane 1) PagL-containing PL and ana-lyzed by Tricine SDS/PAGE and staining with silver. Membranes from N.meningitidis harboring functional PagL were coanalyzed for reference (lane2). (Lower) Silver-stained Tricine SDS/PAGE analysis of CE isolated from plas-midless 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 fromIPTG-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 sinebell 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.

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Arg36-Thr38, and Thr46 situated in β-strands (Fig. S5A). Thesecorrelations as well as the comparison to the CC correlation pat-tern predicted on the basis of the X-ray structure suggested thatthe backbone fold of PagL seen in crystals is largely conserved inthe cellular envelope as well as in proteoliposomes (Fig. S5B). Inaddition to using 15N and 13C chemical shifts, we investigatedwhich protein regions were sensitive to the cellular environmentby analyzing cross-peak amplitudes as spectral parameters(Fig. 3B). Detailed ssNMR signal sets extracted from 2D correla-tion spectra of CE isolated from induced cells (black) or nonin-duced cells (green) and PL (red) at selected PagL 15N and 13Cresonance frequencies are shown in Fig. 3B, Left. The bar dia-gram (Fig. 3B, Right) displays chemical-shift differences in back-bone 15N,13Cα and side-chainCβ resonances between CE andPL preparations. Overall, most differences in the backbone che-mical shifts were small. Only for Ala9, Thr16, Thr32, and Arg36we observed 15N or 13Cα chemical-shift deviations of around 0.6

and 0.4 ppm, respectively (Fig. 3C, arrows). In addition, signifi-cant side-chain chemical-shift changes were observed for residueswithin transmembrane segments (Ala9, Thr10, Leu37), the firstperiplasmic turn (Ser33), and the third extracellular loop(Thr91). For several assigned correlations, we also observed asizable attenuation in NMR signal intensity (ranging between30% and 50%), notably for backbone resonances involving Ala9,Thr91, and Thr32 (Fig. S6A). Even in the absence of residue-specific assignments, we could analyze other amino acid types,including Ser, Ala, Gly, Val, Lys, Tyr, and Trp, based on theircharacteristic intraresidue correlation pattern and peak positions.Besides subtle backbone chemical-shift changes, we found signif-icant alterations in side-chain resonances of Val, Lys, and Tyr

Fig. 3. Conformational analysis of PagL. (A) Overlay of 2D NCA correlationspectra of CE isolated from IPTG-induced WC (black) and PL (red), recordedwith identical acquisition and processing parameters—i.e., with a sine bellfunction (SSB ¼ 4.5) and using linear prediction in indirect dimension. (B)Comparison of PagL in CE and PL environments. (Left) Selected spectral re-gions from 2D homonuclear and heteronuclear spectra of CE isolated fromIPTG-induced WC (black) showing isolated PagL resonances, and overlaidwith spectra obtained on CE isolated from noninduced WC (green) andPL (red). Assignments are indicated where available. (Right) Backbone N,Cα, and Cβ chemical-shift changes observed for PagL embedded in E. coliCE and in PL. Horizontal lines indicate the threshold for significant chemi-cal-shift changes. The threshold was set to 2 times ΔðδCE − δPLÞ ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

½ΔðδCEÞ�2 þ ½ΔðδPLÞ�2p

. Residues with a chemical-shift deviation larger thanthe threshold (+ 2 SD) are labeled. (C) Summary of PagL ssNMR spectralchanges between CE and PL preparations plotted onto the topological repre-sentation of PagL according to the crystal structure (β-sheet protein segmentsare represented by open rectangles and transmembrane segments TM1–8are labeled). Arrows point to residues that experienced significant backbone(solid lines) and side-chain (dashed lines) chemical-shift changes, whereasorange filled bars indicate major alterations in signal intensities (>50%) be-tween 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 CEisolated from noninduced WCs overlaid with backbone Cα-Cβ and N-Cα (redcrosses) intraresidue correlations predicted from the crystal structure of Lpp(Protein Data Bank ID code 1EQ7) using SPARTA+ (33). For other carbon posi-tions (black crosses), average 13C chemical-shift values given in the BiologicalMagnetic Resonance Data Bank (http://www.bmrb.wisc.edu/ref_info/statsel.htm) were used. Characteristic correlations are labeled and color coded: or-ange for correlations absent in the experimental data and green for correla-tions that are in agreement with chemical-shift predictions. (Inset, UpperRight) Spectral region characteristic of Gly residues. (Inset, Lower Left) An over-lay of 2D NCA spectra for α-helical Thr, Val, and Ile N-Cα correlations in CE iso-lated from non- (black) and IPTG-induced (blue) cells. (C) Summary of thestructural analysis of Lpp based on ssNMR spectra obtained on CE isolated fromnoninduced cells. Residues for which backbone correlations significantly devi-ate 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 cor-relation patterns for the free (Upper) and the bound (Lower) forms of Lpp.Meso-diaminopimelic acid, m-DAP.

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residues, whereas Thr residues are not affected (Fig. 3 B, Left andC, orange bars; see also Fig. S6B). Taken together, these findingssuggest an overall conservation of the backbone structure ofPagL in the cellular envelope. Larger changes observed for somebackbone residues and, in particular, side-chain conformations(Fig. 3C) may reflect structural and/or dynamical alterations thatare potentially related to the presence of endogenous membrane-associated molecular components in the CE environment.

Characterization of the Endogenous E. coli Lipoprotein Lpp. With upto about 7.2 × 105 copies per cell, the lipoprotein Lpp, or Braun’slipoprotein (15), belongs to the most abundant CE proteins inexponentially growing E. coli cells. Lpp is found in both “free”and “bound” forms, the latter being covalently attached to thePG network (16, 17). In solution, the 56-residue polypeptide moi-ety, called Lpp-56, associates to form a hydrophilic homotrimercomposed of a three-stranded coiled-coil domain and two helix-capping motifs (18), but a model for a lipophilic superhelical as-sembly containing six subunits has also been proposed (16). Wefirst monitored the presence of free Lpp by SDS/PAGE analysisof CE preparations followed by immunoblotting (Fig. S7A). Wenext performed a series of 2D 15N-13C and 13C-13C correlationexperiments on CE isolated from noninduced WC and comparedresults with predictions based on the available high-resolution 3Dstructure of Lpp-56. We found good agreement between our dataand predicted intraresidue backbone C–C (Fig. 4A) as well asN–Cα (Fig. 4B) correlation patterns, suggesting the predomi-nance of well-folded Lpp in the CE preparations. These resultswere further supported by weak signal intensities in spectral re-gions characteristic for glycine (Fig. 4B, Upper Right Inset), whichis missing in the amino acid composition of Lpp. In addition, iso-lated backbone and side-chain resonances corresponding to Ala,Val, and Ile residues were readily observed around peak positionspredicted from the crystal structure (18) (Fig. 4 A and B, redcrosses). In the X-ray structure, these residues pack against eachother to form the hydrophobic interface between the three he-lices (Fig. 4C, green). Interestingly, both peak positions and spec-

tral resolution are well preserved in the presence of recombinantprotein PagL in the OM (Fig. 4B, Lower Left Inset, blue spec-trum). In contrast, we observed a systematic deviation betweenour data and predicted (Fig. 4 A and B, orange) 13C and 15N re-sonances for N- and C-terminal residues (Ser3, Asn4, Met52,Thr54), which exceeded the standard deviation. We thus specu-late that potential conformational and/or dynamical changes oc-cur between crystalline Lpp and native Lpp around theseresidues. In vivo, Lpp has characteristic covalent modificationsat its N and C termini (19). About one-third of Lpp moleculesis covalently bound to the PG layer. Such a modification involvesthe formation of a peptide bond between the free ϵ-amino groupof the C-terminal Lys of Lpp and the free carboxylate group ofmeso-diaminopimelic acid residues in PG (Fig. 4C). We thus re-corded a 2D (15N- 13Ca) correlation spectrum using a larger spec-tral window that includes the side-chain region (Fig. 4D). Weidentified two intense cross-peaks in the amide region at the ex-pected Cϵ carbon frequency of free Lys. These signals were cor-related to a set of additional signals at 32, 27, and 29.5 ppm, ingood agreement with averaged 13C chemical shifts of Lys Cβ, Cγ ,and Cδ side-chain resonances and strongly suggesting that abound Lpp contribution is detected in the CE spectrum.

Resonance Assignment of Mobile LPS and PG.We could readily char-acterize highly flexible PG, located in the periplasm, and LPS inthe OM in 2D (1H,13C) as well as (13C-13C) through-bond corre-lation spectra obtained on CE preparations. LPS consists of a1,4′-bisphosphorylated β-1,6-linked glucosamine disaccharide(α- and β-GlcN), substituted with fatty acid chains at positions2, 3, 2′, and 3′ and with an oligosaccharide core at position 6′.PG is a giant heteropolymer made of linear glycan strands ofalternating β-1,4-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (NAM) residues, which are cross-linked byshort peptides. First, we recorded a 2D (1H-13C) insensitivenuclei enhanced by polarization transfer (INEPT) spectrum toexamine the sugar heterogeneity/composition in our sample.Intense and very well-resolved cross-peaks were found at peak

Fig. 5. Resonance assignment of flexible LPS and PG using through-bond ssNMR correlation spectroscopy. (A) Expansion of the 2D (1H-13C) insensitive nucleienhanced by polarization transfer (INEPT) correlation spectrum obtained on CE isolated from IPTG-induced WC, showing dispersion and resolution of indi-vidual 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 variedbetween 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 thesame spin system are connected by dashed lines. (C) Residue-specific ssNMR assignment of LPS and PG glucosamine units based on 13C-13C connectivities withinthe sugar rings and their substituents (see also Fig. S7A). Assigned resonances from LPS and PG glucosamine units are highlighted by filled circles onto chemicalstructures with the same color coding as in A.

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positions corresponding to anomeric H1/C1 atoms from carbohy-drate units (Fig. 5A). The 13C dispersion between 90 and 104 ppmwas consistent with the presence of nine carbohydrate species, inα- and β-configuration. Glucosamine units, constituting exclu-sively the PG backbone (GlcNAc and NAM) and the lipid A moi-ety of the LPS (β-D-GlcN and α-D-GlcN) can be distinguishedfrom sugars of the LPS core (labeled A–E) based on a character-istic one-bond CC connectivity between anomeric carbon andnitrogen-substituted C2 in a 2D (13C,13C) correlation spectrum(Fig. 5B). Next, we identified carbohydrate units by tracing CCand HC connectivities within the sugar rings and their substitu-ents (20, 21) (Fig. 5C and Fig. S7B). Using this strategy, we ob-tained de novo ssNMR assignments of LPS and PG glucosamineunits and some of their substituents (Table S1). Solid-state NMRchemical shifts were compatible with the presence of polysubsti-tuted glucosamine units within the lipid A of LPS and backbonePG moieties, as previously deduced from the analysis of purifiedmolecules (21–23), even when part of cell envelope preparations.However, carbohydrates from the core of the LPS could not beidentified unambiguously due to the large overlap of 13C and 1Hresonances and the inherent structural heterogeneity of the LPScore region (Fig. S7C).

DiscussionOur results demonstrate that cellular ssNMR spectroscopy can beused to probe atomic details of integral membrane proteins andendogenous membrane-associated molecular components in abacterial cellular setting. We showed that 15N-edited datasetsas well as the combined use of through-space and through-bondssNMR experiments reduces spectral complexity and facilitatesthe discrimination between proteinaceous and nonproteinaceousmolecular components at different levels of molecular mobility.

By analyzing (U-13C,15N)-labeled E. coli cell envelopes, wefound that LPS and PG moieties can exhibit a remarkable degreeof molecular motion (Fig. 6, red), while the protein componentsinvestigated were well folded in the cellular membrane context(Fig. 6, blue). In the case of Lpp, our results strongly supportthe anchoring of the protein to the underlying PG layer by virtueof the side chain of the C-terminal lysine. Although our data areconsistent with the overall 3D model for the isolated Lpp proteinmoiety, substantial conformational changes are predicted for re-sidues located at the N- and C-terminal extremities of the protein,which are covalently substituted in vivo. Further cellular ssNMRstudies may reveal the conformation of the free form of Lpp thathas been postulated to cross the OM (17). In the case of PagL, forwhich high-resolution ssNMR spectra were obtained for all stu-died preparations, we demonstrated that the global fold of theprotein as seen in the crystal structure was largely preserved inboth PL and CE environments, including the presence of β-sheetstructure in the first periplasmic turn, which involved Thr32. Onthe other hand, several protein backbone resonances related toresidues located in the first periplasmic turn, the transmembranesegments 1–3, and in the third extracellular loop were sensitive tothe molecular environment. Alterations in ssNMR signal inten-sity were detected predominantly for aromatic, hydrophobic, andcharged residues. Interestingly, these PagL residues are located inprotein segments potentially exposed to other major membrane-associated cellular components, including LPS and the PG (Fig. 6,orange). Similar to the case of Lpp, further ssNMR studies willhelp to establish the structural details of the molecular interac-tion of PagL and its substrate LPS, to dissect active and latentPagL conformations that depend on its molecular environment(24), and to establish the physiological relevance of PagL dimers(13) in a native-like environment.

With increasing levels of molecular complexity, spectroscopicsensitivity becomes a critical factor. Nevertheless, one- and two-dimensional ssNMR spectroscopy of whole cell preparationssuch as shown in this study is possible (Fig. 2A). In addition,

WC preparations can be readily combined with state-of-the-artssNMR signal enhancement methods operating at low tempera-ture, thereby reducing acquisition times significantly (25). Suchtechnologies are likely to reduce the expression levels neededto perform cellular ssNMR experiments. In the present study,the expression levels of PagL were comparable to those of theendogenous protein Lpp (Fig. S2B). Dedicated preparationmethods (see, e.g., ref. 6) including the single-protein productionmethod (26) are available to further reduce unwanted back-ground contributions. Cellular ssNMR spectroscopy as describedherein opens opportunities for the structural investigation oflarge and/or membrane-associated macromolecules. Already, thecell envelope of Gram-negative bacteria, including the IM, theperiplasm, and the OM, epitomizes a model organelle involvedin a large number of functions critical for cellular physiology.With recent advances in using insect and mammalian cells forproducing recombinant eukaryotic proteins, cellular ssNMR asshown here could also be applicable to recombinant eukaryoticproteins in specific compartments—i.e., after isolation of the cel-lular membrane or intracellular organelles. Using cellular ssNMR,the structural investigation of fundamental processes, such asligand/drug binding or membrane-protein folding mediated by

Fig. 6. Atomic-level insights of E. coli CE-associated macromolecules revealedby cellular ssNMR spectroscopy. Schematic representation of the CE fromE. coli showing rigid (blue) and flexible (red) (non)proteinaceous molecularcomponents characterized by through-bond and through-space ssNMR experi-ments, respectively. The topological representations of PagL and Lpp as seen inthe available 3Dmodels of isolated molecules are indicated. For PagL, residueslocated in β-strand and random-coil protein segments are represented bysquares and circles, respectively. Residues that were not included in the analysisare colored in gray. Amino acids are given in single-letter notation. Spectro-scopic changes of large (chemical-shift deviation >0.4 ppm and/or signal inten-sity variations >50%) and small magnitude that are potentially related toassociation betweenmembrane protein and OM (or PG) are indicated for bothproteins in orange and blue, respectively.

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complex proteinaceous machineries, should be possible, therebybridging the gap between structural and cellular biology.

Materials and MethodsExpression Vectors and Bacterial Strains. The pET11a-derived plasmidspPagLðPaÞ and pPagLðPaÞ(-) encoding P. aeruginosa PagL with and without sig-nal sequence, respectively, have been described (12). Mutant derivatives of E.coli BL21Star(DE3) lacking the OmpF and/or OmpA proteins were isolated byselection for resistance to the OmpF- and OmpA-specific bacteriophages TuIa(27) and K3 (28), respectively. Mutants lacking OmpF, OmpA, or both weredesignated CE1536, CE1537, and CE1535, respectively.

Sample Preparation. WC and CE NMR samples were prepared from 50 and200 mL of the cultures, respectively, following the procedure described inSI Materials and Methods. For CE NMR samples, cells were disrupted in pre-sence of EDTA-free protease inhibitor cocktail (Sigma-Aldrich) using a pre-cooled French pressure cell (8,000 psi). Unbroken cells were removed bymultiple centrifugation steps (1;000 × g, 10 min, 4 °C) until a pellet wasno longer detectable. The CE fraction was isolated by ultracentrifugationof the cell lysate for 8 min at 150;000 × g and at 4 °C. The correct localizationand membrane insertion of PagL was confirmed on Coomassie-stained SDS/PAGE gels after extracting the CE with 1% N-lauroylsarcosine or 6 M urea asdescribed previously (29, 30). To obtain the reference PL ssNMR spectra, PagLwas expressed as intracellular inclusion bodies in E. coli BL21Star(DE3) harbor-ing the plasmid pPagLðPaÞ(-), then purified, and reconstituted in dimyristoyl-phosphatidylcholine vesicles at a final protein-to-lipid molar ratio of 1∶50 asdescribed in SI Materials and Methods. Before packing, the CE and PL pelletswere resuspended in 500 μL of 10 mM Hepes (pH 7.0) and harvested by ultra-

centrifugation (90;000 × g for 45 min at 4 °C). Freshly prepared and fullyhydrated WC, CE, and PL samples were transferred into 3.2-mm MAS rotorsby low-speed centrifugation, packed with bottom and top spacers, and sub-sequently analyzed by ssNMR spectroscopy. In the PagL-PL case, we estimate8 mg of protein present in the NMR rotor.

NMR Spectroscopy. The ssNMR analysis of cellular and proteoliposome pre-parations was based on a set of multidimensional homonuclear and hetero-nuclear ssNMR experiments employing dipolar- or scalar-based magneti-zation transfer steps as detailed in SI Materials and Methods. Solid-stateNMR experiments were performed at a regulated sample temperature of−2 °C on a narrow-bore Bruker Avance 700 spectrometer equipped with a3.2-mm triple-resonance (1H,13C,15N) Efree MAS probe. 13C and 1H resonanceswere calibrated using adamantane as an external reference. The upfield 13Cresonance and isotropic 1H resonance of adamantane were set to 31.47 and1.7 ppm, respectively, to allow for a direct comparison of the solid-state che-mical shifts to solution-state NMR data. Accordingly, 15N resonances were ca-librated using the tripeptide AGG (31) as an external reference. A summary ofacquisition and process parameters is given in Table S2. NMR spectra wereprocessed using Topspin 3.0 (Bruker Biospin) and analyzed with Sparky (32).

ACKNOWLEDGMENTS. Technical assistance by Deepak Nand, Christian Ader,and Hans Meeldijk is gratefully acknowledged. This work was supportedby Nederlandse organisatie voor wetenschappelijk onderzoek (700.26.121,700.10.433, and 815.02.012) and received funding from the European Com-munity’s Seventh Framework Program (FP7/2007-2013) under Grant Agree-ment 211800.

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