Cellular Solid-State Nuclear Magnetic Resonance Spectroscopy

Cellular solid-state nuclear magnetic SEE COMMENTARY resonance spectroscopy Marie 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 Biomolecular Imaging, 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 review October 11, 2011) Decrypting the structure, function, and molecular interactions of conditions (11) as a high-resolution method to investigate atomic complex molecular machines in their cellular context and at atomic structures of major cell-associated (macro)molecules. resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a well- Results established imaging method that can visualize cellular entities at Sample Design for Cellular ssNMR Spectroscopy. Our goal was to the micrometer scale and can be used to obtain 3D atomic struc- establish general expression and purification procedures that lead 13 15 tures under in vitro conditions. Here, we introduce a solid-state to uniformly C, N-labeled preparations of whole cells (WC) NMR approach that provides atomic level insights into cell-asso- and cell envelopes (CE) containing an arbitrary (membrane) ciated molecular components. By combining dedicated protein pro- protein target (Fig. 1B). As our model system, we selected the duction and labeling schemes with tailored solid-state NMR pulse 150-residue integral membrane-protein PagL from Pseudomonas methods, we obtained structural information of a recombinant aeruginosa, an OM enzyme that removes a fatty acyl chain from integral membrane protein and the major endogenous molecular LPS (12). We overexpressed pagL under control of the bacter- components in a bacterial environment. Our approach permits iophage T7 promoter, which is inducible with IPTG, in a mutant studying entire cellular compartments as well as cell-associated E. coli BL21Star(DE3) strain, lacking the two major OM proteins proteins at the same time and at atomic resolution. (OMPs) OmpF and OmpA. The suppression of these major OMPs prevented to a large extent the accumulation of the unpro- cellular envelope ∣ Escherichia coli ∣ lipoprotein ∣ PagL ∣ magic angle cessed signal-peptide-bearing precursor of PagL and led to signif- spinning icant amounts of mature protein in the host membrane when mild recombinant-protein-expression conditions were used (Fig. S1). hysiological processes rely on the concerted action of mole- For optimal analysis of major cell-associated molecular compo- 15 13 Pcular entities in and across different cellular compartments. nents, E. coli cultures were switched from unlabeled to N, C- Whereas advancements in molecular imaging have provided isotope labeled growth conditions at the beginning of the expo- unprecedented insights into the macromolecular organization nential growth phase, when recombinant protein production was in the subnanometer range (1), studying atomic structure and mo- induced, leading to the incorporation of isotopes in PagL and co- tion in situ has been challenging for structural biology. NMR has expressed endogenous molecular components. WC and CE sam- provided insight into cellular processes (2–4) and can determine ples were prepared from the same exponentially growing culture. 13 15 entire 3D molecular structures inside living cells (5) provided that As a reference, (U- C, N)-labeled PagL was produced in intra- molecular entities tumble rapidly in a cellular setting. In princi- cellular inclusion bodies, purified, and reconstituted in proteoli- ple, solid-state NMR (ssNMR) spectroscopy offers a comple- posomes (PL, Fig. 1B). Before analysis, WC pellet was washed mentary spectroscopic tool to monitor molecular structure and with PBS, whereas CE and PL were resuspended in Hepes at dynamics at atomic resolution in a complex setting (see ref. 6 pH 7.0 and harvested by ultracentrifugation using identical pro- for a recent review). Indeed, ssNMR has already been used to cedures. Both in CE preparations and reconstituted in PL, PagL BIOPHYSICS AND study individual molecular components in the context of natural exhibited similar heat modifiability, a property typical of the well- COMPUTATIONAL BIOLOGY bilayers (7, 8), bacterial cell walls (9), and cellular organelles (10). folded protein (12, 13) (Fig. 1C). Toverify that PagL was correctly Here, we introduce a general approach to investigate structure folded in vivo and in vitro, we monitored its LPS 3-O-deacylase and dynamics of an arbitrary molecular target and its potential activity in CE and PL preparations as described previously (12, molecular partners in a cellular setting. Our studies focuses on 13). In both cases, LPS was converted into a form with higher the Gram-negative bacterial cell that is characterized by a mole- electrophoretic mobility (Fig. 1D), in agreement with the ex- cularly complex but architecturally unique envelope, consisting of pected hydrolysis of the primary acyl chain at position 3 of lipid two lipid bilayers, the inner and outer membrane (IM, OM), se- A. Taken together, our data (heat modifiability and activity as- parated by the periplasm containing the peptidoglycan (PG) layer says) indicate that cellular and PL preparations contained well- (Fig. 1A). The IM is a phospholipid bilayer and harbors α-helical folded and functional PagL. proteins, whereas the OM is asymmetrical and consists of phos- pholipids, lipopolysaccharides (LPS), lipoproteins, and β-barrel- NMR Spectra of E. coli Whole Cells and Cell Envelopes Versus Proteo- fold integral membrane proteins. LPS forms the outermost layer liposomes. To characterize rigid—presumably membrane-asso- of the OM and protects the cell against harmful compounds from the environment. PG is a large macromolecule that gives the cell Author contributions: M.R., J.T., and M.B. designed research; M.R., R.T.-v.B. and M.P.B. its shape and rigidity. performed research; J.-A.P. contributed new reagents/analytic tools; M.R., J.T., and M.B. 13 15 Using uniformly C, N-labeled cellular preparations of analyzed data; and M.R., J.T., and M.B. wrote the paper. Escherichia coli, we characterized the structure and dynamics The authors declare no conflict of interest. of a recombinant integral membrane protein (PagL) and other This article is a PNAS Direct Submission. R.T. is a guest editor invited by the Editorial Board. major endogenous molecular components of the cell envelope See Commentary on page 4715. including lipids, the peptidoglycan, and the lipoprotein Lpp (also 1To whom correspondence may be addressed. E-mail: [email protected] or known as Braun’s lipoprotein). These studies highlight the [email protected]. influence of the surrounding compartment on molecular struc- This article contains supporting information online at www.pnas.org/lookup/suppl/ ture and establish ssNMR under magic angle spinning (MAS) doi:10.1073/pnas.1116478109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1116478109 PNAS ∣ March 27, 2012 ∣ vol. 109 ∣ no. 13 ∣ 4863–4868 Downloaded by guest on September 30, 2021 ciated—molecular components in WC and CE, we performed a set of 2D 13C-13C correlation experiments employing dipolar- based magnetization transfer steps. Overall, both preparations yielded NMR spectra of astonishing quality considering sample complexity 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 sensitivity and spectral resolution for the CE preparation (Fig. S2A), poten- tially due to the single contribution of CE-associated components. These results were corroborated by SDS/PAGE analysis (Fig. 1C), which showed a significant decrease of the amount of proteins after removal of the protoplasm by cell lysis and ultracentrifugation. Over time, WC and CE preparations did not re- veal any marked spectroscopic changes at −2 °C, and the cell morphology and the structural integrity of the CE were preserved after extended NMR studies (Fig. S3). When comparing WC and CE spectra with the reference PL spectrum recorded under similar measurement conditions (Fig. 2C), we found that a large set of intraresidue correlations from PagL, notably cross-peaks of Ala, Thr, and Ser residues in β-sheet protein segments (see below), are well 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 which signals arising from nonproteinaceous molecular components are largely reduced. Comparison with the reference PL spectrum (Fig. 3A, red) revealed astonishing similarities. With average 13C and 15N line widths of 0.6–0.8 and 1.5–1.6 ppm, respectively, Fig. 1. Cellular ssNMR spectroscopy: overall strategy and sample prepara- ssNMR spectra of the CE preparation exhibited comparable, if tion, including inner and outer membrane proteins (IMP, OMP). (A) Schematic not superior spectral resolution (Fig. S2B). Because of the favor- structure of the E. coli K-12 cell envelope. (B) Overall scheme for the prepara- able spectroscopic dispersion among Thr, Ser, and Gly residues in tion of WC and CE from strain CE1535 carrying plasmid

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