Characterizing proteins in a native bacterial environment using solid-state NMR spectroscopy.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
02 2021
02 2021
Historique:
received:
07
07
2020
accepted:
09
10
2020
pubmed:
15
1
2021
medline:
9
3
2021
entrez:
14
1
2021
Statut:
ppublish
Résumé
For a long time, solid-state nuclear magnetic resonance (ssNMR) has been employed to study complex biomolecular systems at the detailed chemical, structural, or dynamic level. Recent progress in high-resolution and high-sensitivity ssNMR, in combination with innovative sample preparation and labeling schemes, offers novel opportunities to study proteins in their native setting irrespective of the molecular tumbling rate. This protocol describes biochemical preparation schemes to obtain cellular samples of both soluble as well as insoluble or membrane-associated proteins in bacteria. To this end, the protocol is suitable for studying a protein of interest in both whole cells and in cell envelope or isolated membrane preparations. In the first stage of the procedure, an appropriate strain of Escherichia coli (DE3) is transformed with a plasmid of interest harboring the protein of interest under the control of an inducible T7 promoter. Next, the cells are adapted to grow in minimal (M9) medium. Before the growth enters stationary phase, protein expression is induced, and shortly thereafter, the native E. coli RNA polymerase is inhibited using rifampicin for targeted labeling of the protein of interest. The cells are harvested after expression and prepared for ssNMR rotor filling. In addition to conventional
Identifiants
pubmed: 33442051
doi: 10.1038/s41596-020-00439-4
pii: 10.1038/s41596-020-00439-4
doi:
Substances chimiques
Membrane Proteins
0
Proteins
0
Protons
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
893-918Subventions
Organisme : Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organisation for Scientific Research)
ID : 700.10.443
Références
Griffin, R. G. Solid state nuclear magnetic resonance of lipid bilayers. Methods Enzymol. 72, 108–174 (1981).
pubmed: 7311829
doi: 10.1016/S0076-6879(81)72010-X
Seelig, J. Deuterium magnetic resonance: theory and application to lipid membranes. Q. Rev. Biophys. 10, 353–418 (1977).
pubmed: 335428
doi: 10.1017/S0033583500002948
Brown, L. S. & Ladizhansky, V. Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy. Prot. Sci. 24, 1333–1346 (2015).
doi: 10.1002/pro.2700
Hong, M., Zhang, Y. & Hu, F.H. Membrane protein structure and dynamics from NMR spectroscopy. Annu. Rev. Phys. Chen., 63, (eds. Johnson, M.A. & Martinez, T.J.) 1-24 (2012).
Kaplan, M., Pinto, C., Houben, K. & Baldus, M. Nuclear magnetic resonance (NMR) applied to membrane–protein complexes. Q. Rev. Biophys. 49, e15 (2016).
pubmed: 27659286
doi: 10.1017/S003358351600010X
Herzfeld, J. & Lansing, J. C. Magnetic resonance studies of the bacteriorhodopsin pump cycle. Annu. Rev. Biophys. Biomol. Struct. 31, 73–95 (2002).
pubmed: 11988463
doi: 10.1146/annurev.biophys.31.082901.134233
Ketchem, R., Hu, W. & Cross, T. High-resolution conformation of gramicidin A in a lipid bilayer by solid-state NMR. Science 261, 1457–1460 (1993).
pubmed: 7690158
doi: 10.1126/science.7690158
Lange, A. et al. Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR. Nature 440, 959–962 (2006).
pubmed: 16612389
doi: 10.1038/nature04649
Cady, S. D. et al. Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers. Nature 463, 689–U127 (2010).
pubmed: 20130653
pmcid: 2818718
doi: 10.1038/nature08722
Luca, S. et al. The conformation of neurotensin bound to its G protein-coupled receptor. Proc. Natl Acad. Sci. USA 100, 10706–10711 (2003).
pubmed: 12960362
doi: 10.1073/pnas.1834523100
Goncalves, J. A., Ahuja, S., Erfani, S., Eilers, M. & Smith, S. O. Structure and function of G protein-coupled receptors using NMR spectroscopy. Prog. Nucl. Magn. Reson. Spectrosc. 57, 159–180 (2010).
pubmed: 20633362
pmcid: 2907352
doi: 10.1016/j.pnmrs.2010.04.004
Loquet, A., Habenstein, B. & Lange, A. Structural investigations of molecular machines by solid-state NMR. Acc. Chem. Res. 46, 2070–2079 (2013).
pubmed: 23496894
doi: 10.1021/ar300320p
Tycko, R. Solid-state NMR studies of amyloid fibril structure. Annu. Rev. Phys. Chem., 62 (eds. Leone, S.R., Cremer, P.S., Groves, J.T. & Johnson, M.A.) 279-299 (2011).
Das, N., Murray, D. T. & Cross, T. A. Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples. Nat. Protoc. 8, 2256 (2013).
pubmed: 24157546
pmcid: 4107459
doi: 10.1038/nprot.2013.129
Fricke, P. et al. Backbone assignment of perdeuterated proteins by solid-state NMR using proton detection and ultrafast magic-angle spinning. Nat. Protoc. 12, 764–782 (2017).
pubmed: 28277547
doi: 10.1038/nprot.2016.190
Ni, Q. Z. et al. High frequency dynamic nuclear polarization. Acc. Chem. Res. 46, 1933–1941 (2013).
pubmed: 23597038
pmcid: 3778063
doi: 10.1021/ar300348n
Ishii, Y. & Tycko, R. Sensitivity enhancement in solid state N-15 NMR by indirect detection with high-speed magic angle spinning. J. Magn. Reson. 142, 199–204 (2000).
pubmed: 10617453
doi: 10.1006/jmre.1999.1976
Chevelkov, V., Rehbein, K., Diehl, A. & Reif, B. Ultrahigh resolution in proton solid-state NMR spectroscopy at high levels of deuteration. Angew. Chem. Int. Ed. 45, 3878–3881 (2006).
doi: 10.1002/anie.200600328
Plitzko, J. M., Schuler, B. & Selenko, P. Structural biology outside the box—inside the cell. Curr. Opin. Struct. Biol. 46, 110–121 (2017).
pubmed: 28735108
doi: 10.1016/j.sbi.2017.06.007
Baker, L. A. et al. Combined H-1-detected solid-state NMR spectroscopy and electron cryotomography to study membrane proteins across resolutions in native environments. Structure 26, 161–170 (2018).
pubmed: 29249608
pmcid: 5758107
doi: 10.1016/j.str.2017.11.011
Narasimhan, S. et al. DNP-supported solid-state NMR spectroscopy of proteins inside mammalian cells. Angew. Chem. Int. Ed. 58, 12969–12973 (2019).
doi: 10.1002/anie.201903246
Damman, R. et al. Development of in vitro-grown spheroids as a 3D tumor model system for solid-state NMR spectroscopy. J. Biomol. NMR 74, 401–412 (2020).
pubmed: 32562030
pmcid: 7508937
doi: 10.1007/s10858-020-00328-8
Thongsomboon, W. et al. Phosphoethanolamine cellulose: a naturally produced chemically modified cellulose. Science 359, 334–338 (2018).
pubmed: 29348238
doi: 10.1126/science.aao4096
Renault, M. et al. Solid-state NMR spectroscopy on cellular preparations enhanced by dynamic nuclear polarization. Angew. Chem. Int. Ed. 51, 2998–3001 (2012).
doi: 10.1002/anie.201105984
Renault, M. et al. Cellular solid-state nuclear magnetic resonance spectroscopy. Proc. Natl Acad. Sci. USA 109, 4863–4868 (2012).
pubmed: 22331896
doi: 10.1073/pnas.1116478109
Gronenborn, A. M. & Clore, G. M. Rapid screening for structural integrity of expressed proteins by heteronuclear NMR spectroscopy. Prot. Sci. 5, 174–177 (1996).
doi: 10.1002/pro.5560050123
Qing, G. et al. Cold-shock induced high-yield protein production in Escherichia coli. Nat. Biotechnol. 22, 877–882 (2004).
pubmed: 15195104
doi: 10.1038/nbt984
Frederick, K. K. et al. Sensitivity-enhanced NMR reveals alterations in protein Sstructure by cellular milieus. Cell 163, 620–628 (2015).
pubmed: 26456111
pmcid: 4621972
doi: 10.1016/j.cell.2015.09.024
Serber, Z. & Dötsch, V. In-cell NMR spectroscopy. Biochemistry 40, 14317–14323 (2001).
pubmed: 11724542
doi: 10.1021/bi011751w
Serber, Z. et al. Methyl groups as probes for proteins and complexes in in-cell NMR experiments. J. Am. Chem. Soc. 126, 7119–7125 (2004).
pubmed: 15174883
doi: 10.1021/ja049977k
Binolfi, A., Theillet, F.-X. & Selenko, P. Bacterial in-cell NMR of human α-synuclein: a disordered monomer by nature? Biochem. Soc. Trans. 40, 950–954 (2012).
pubmed: 22988846
doi: 10.1042/BST20120096
Barbieri, L., Luchinat, E. & Banci, L. Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells. Nat. Protoc. 11, 1101 (2016).
pubmed: 27196722
doi: 10.1038/nprot.2016.061
Tabaka, M., Sun, L., Kalwarczyk, T. & Hołyst, R. Implications of macromolecular crowding for protein–protein association kinetics in the cytoplasm of living cells. Soft Matter 9, 4386–4386 (2013).
doi: 10.1039/c3sm00013c
Siegal, G. & Selenko, P. Cells, drugs and NMR. J. Magn. Reson. 306, 202–212 (2019).
pubmed: 31358370
doi: 10.1016/j.jmr.2019.07.018
Lee, K. M., Androphy, E. J. & Baleja, J. D. A novel method for selective isotope labeling of bacterially expressed proteins. J. Biomol. NMR 5, 93–96 (1995).
pubmed: 7881274
doi: 10.1007/BF00227474
Pinto, C. et al. Studying assembly of the BAM complex in native membranes by cellular solid-state NMR spectroscopy. J. Struct. Biol. 206, 1–11 (2019).
pubmed: 29197585
doi: 10.1016/j.jsb.2017.11.015
Miao, Y. et al. M2 proton channel structural validation from full-length protein samples in synthetic bilayers and E. coli membranes. Angew. Chem. Int. Ed. 51, 8383–8386 (2012).
doi: 10.1002/anie.201204666
Medeiros-Silva, J. et al. 1 H-detected solid-state NMR studies of water-inaccessible proteins in vitro and in situ. Angew. Chem. Int. Ed. 55, 13606–13610 (2016).
doi: 10.1002/anie.201606594
Jacso, T. et al. Characterization of membrane proteins in isolated native cellular membranes by dynamic nuclear polarization solid-state NMR spectroscopy without purification and reconstitution. Angew. Chem. Int. Ed. 51, 432–435 (2012).
doi: 10.1002/anie.201104987
Ward, M. E. et al. In situ structural studies of anabaena sensory rhodopsin in the E. coli membrane. Biophys. J. 108, 1683–1696 (2015).
pubmed: 25863060
pmcid: 4390784
doi: 10.1016/j.bpj.2015.02.018
Etzkorn, M. et al. Complex formation and light activation in membrane-embedded sensory rhodopsin II as seen by solid-state NMR spectroscopy. Structure 18, 293–300 (2010).
pubmed: 20223212
doi: 10.1016/j.str.2010.01.011
Yamamoto, K., Caporini, M. A., Im, S.-C., Waskell, L. & Ramamoorthy, A. Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization. Biochim. Biophys. Acta 1848, 342–349 (2015).
pubmed: 25017802
doi: 10.1016/j.bbamem.2014.07.008
Schanda, P. et al. Atomic model of a cell-wall cross-linking enzyme in complex with an intact bacterial peptidoglycan. J. Am. Chem. Soc. 136, 17852–17860 (2014).
pubmed: 25429710
pmcid: 4544747
doi: 10.1021/ja5105987
Reckel, S., Lopez, J. J., Loehr, F., Glaubitz, C. & Doetsch, V. In-cell solid-state NMR as a tool to study proteins in large complexes. ChemBioChem 13, 534–537 (2012).
pubmed: 22298299
doi: 10.1002/cbic.201100721
Medeiros-Silva, J. et al. High-resolution NMR studies of antibiotics in cellular membranes. Nat. Commun. 9, 3963 (2018).
pubmed: 30262913
pmcid: 6160437
doi: 10.1038/s41467-018-06314-x
Shukla, R. et al. Mode of action of teixobactins in cellular membranes. Nat. Commun. 11, 2848 (2020).
pubmed: 32503964
pmcid: 7275090
doi: 10.1038/s41467-020-16600-2
Kaplan, M. et al. Probing a cell-embedded megadalton protein complex by DNP-supported solid-state NMR. Nat. Methods 12, 649–652 (2015).
pubmed: 25984698
doi: 10.1038/nmeth.3406
Almeida, F. C. L. et al. Selectively labeling the heterologous protein in Escherichia coli for NMR studies: A strategy to speed up NMR spectroscopy. J. Magn. Reson. 148, 142–146 (2001).
pubmed: 11133287
doi: 10.1006/jmre.2000.2213
Galvão-Botton, L. M. P. et al. High-throughput screening of structural proteomics targets using NMR. FEBS Lett. 552, 207–213 (2003).
pubmed: 14527688
doi: 10.1016/S0014-5793(03)00926-8
Baker, L. A., Daniëls, M., van der Cruijsen, E. A. W., Folkers, G. E. & Baldus, M. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling. J. Biomol. NMR 62, 199–208 (2015).
pubmed: 25956570
pmcid: 4451474
doi: 10.1007/s10858-015-9936-5
Serber, Z., Ledwidge, R., Miller, S. M. & Dötsch, V. Evaluation of parameters critical to observing proteins inside living Escherichia coli by in-cell NMR spectroscopy. J. Am. Chem. Soc. 123, 8895–8901 (2001).
pubmed: 11552796
doi: 10.1021/ja0112846
Chordia, S., Narasimhan, S., Lucini Paioni, A., Baldus, M. & Roelfes, G. In vivo assembly of artificial metalloenzymes and application in whole‐cell biocatalysis (ChemRxiv, 2020).
White, S. W., Zheng, J., Zhang, Y. M. & Rock, C. O. The structural biology of type II fatty acid biosynthesis. Annu. Rev. Biochem. 74,, 791–831 (2005).
doi: 10.1146/annurev.biochem.74.082803.133524
Roelfes, G. LmrR: a privileged scaffold for artificial metalloenzymes. Acc. Chem. Res. 52, 545–556 (2019).
pubmed: 30794372
pmcid: 6427492
doi: 10.1021/acs.accounts.9b00004
Mitchell, A. M. & Silhavy, T. J. Envelope stress responses: balancing damage repair and toxicity. Nat. Rev. Microbiol. 17, 417–428 (2019).
pubmed: 31150012
pmcid: 6596312
doi: 10.1038/s41579-019-0199-0
Fowler, D. M., Koulov, A. V., Balch, W. E. & Kelly, J. W. Functional amyloid – from bacteria to humans. Trends Biochem. Sci. 32, 217–224 (2007).
pubmed: 17412596
doi: 10.1016/j.tibs.2007.03.003
Otzen, D. & Riek, R. Functional amyloids. Cold Spring Harb. Perspect. Biol. 11, a:033860 (2019).
pubmed: 31088827
doi: 10.1101/cshperspect.a033860
Kim, K. W. Prokaryotic cytoskeletons: in situ and ex situ structures and cellular locations. Antonie van Leeuwenhoek 112, 145–157 (2019).
pubmed: 30128891
doi: 10.1007/s10482-018-1142-5
Albert, B. J. et al. Dynamic nuclear polarization nuclear magnetic resonance in human cells using fluorescent polarizing agents. Biochemistry 57, 4741–4746 (2018).
pubmed: 29924582
pmcid: 6842659
doi: 10.1021/acs.biochem.8b00257
Ghosh, R., Kragelj, J., Xiao, Y. & Frederick, K. K. Cryogenic sample loading into a magic angle spinning nuclear magnetic resonance spectrometer that preserves cellular viability. JoVE e61733 (2020).
Noinaj, N., Gumbart, J. C. & Buchanan, S. K. The β-barrel assembly machinery in motion. Nat. Rev. Microbiol. 15, 197–204 (2017).
pubmed: 28216659
pmcid: 5455337
doi: 10.1038/nrmicro.2016.191
Pinto, C. et al. Formation of the β-barrel assembly machinery complex in lipid bilayers as seen by solid-state NMR. Nat. Commun. 9, 4135–4145 (2018).
pubmed: 30297837
pmcid: 6175958
doi: 10.1038/s41467-018-06466-w
Koers, E. J. et al. NMR-based structural biology enhanced by dynamic nuclear polarization at high magnetic field. J. Biomol. NMR 60, 157–168 (2014).
pubmed: 25284462
doi: 10.1007/s10858-014-9865-8
Corzilius, B., Andreas, L. B., Smith, A. A., Ni, Q. Z. & Griffin, R. G. Paramagnet induced signal quenching in MAS–DNP experiments in frozen homogeneous solutions. J. Magn. Reson. 240, 113–123 (2014).
pubmed: 24394190
doi: 10.1016/j.jmr.2013.11.013
Narasimhan, S., Folkers, G. E. & Baldus, M. When small becomes too big: expanding the use of in-cell solid-state NMR spectroscopy. ChemPlusChem 85, 760–768 (2020).
pubmed: 32297474
doi: 10.1002/cplu.202000167
Zhai, W. et al. Postmodification via thiol-click chemistry yields hydrophilic trityl-nitroxide biradicals for biomolecular high-field dynamic nuclear polarization. J. Phys. Chem. B 124, 9047–9060 (2020).
pubmed: 32961049
doi: 10.1021/acs.jpcb.0c08321
T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco.
Paioni, A. L., Renault, M. A. M. & Baldus, M. DNP and cellular solid-state NMR. eMagRes 7, 51–61 (2018).
Weingarth, M., Bodenhausen, G. & Tekely, P. Broadband magnetization transfer using moderate radio-frequency fields for NMR with very high static fields and spinning speeds. Chem. Phys. Lett. 488, 10–16 (2010).
doi: 10.1016/j.cplett.2010.01.072
Gradmann, S. et al. Rapid prediction of multi-dimensional NMR data sets. J. Biomol. NMR 54, 377–387 (2012).
pubmed: 23143278
doi: 10.1007/s10858-012-9681-y
Narasimhan, S. et al. Rapid prediction of multi-dimensional NMR data sets using FANDAS. Protein NMR Methods Protoc. (ed. Ghose, R.) 111-132 (Springer, 2018).
Schägger, H. Tricine–SDS-PAGE. Nat. Protoc. 1, 16–22 (2006).
pubmed: 17406207
doi: 10.1038/nprot.2006.4
Heise, H., Seidel, K., Etzkorn, M., Becker, S. & Baldus, M. 3D NMR spectroscopy for resonance assignment and structure elucidation of proteins under MAS: novel pulse schemes and sensitivity considerations. J. Magn. Reson. 173, 64–74 (2005).
pubmed: 15705514
doi: 10.1016/j.jmr.2004.11.020
Lacabanne, D., Meier, B. H. & Böckmann, A. Selective labeling and unlabeling strategies in protein solid-state NMR spectroscopy. J. Biomol. NMR 71, 141–150 (2018).
pubmed: 29197975
doi: 10.1007/s10858-017-0156-z
Mandal, A., Boatz, J. C., Wheeler, T. & van der Wel, P. C. A. On the use of ultracentrifugal devices for routine sample preparation in biomolecular magic-angle-spinning NMR. J. Biomol. NMR 67, 165–178 (2017).
pubmed: 28229262
pmcid: 5445385
doi: 10.1007/s10858-017-0089-6
Knowles, T. J., McClelland, D. M., Rajesh, S., Henderson, I. R. & Overduin, M. Secondary structure and (1)H, (13)C and (15)N backbone resonance assignments of BamC, a component of the outer membrane protein assembly machinery in Escherichia coli. Biomol. NMR Assign. 3, 203–206 (2009).
pubmed: 19888691
doi: 10.1007/s12104-009-9175-3
Kim, K. H. et al. Structural characterization of Escherichia coli BamE, a lipoprotein component of the β-barrel assembly machinery complex. Biochemistry 50, 1081–1090 (2011).
pubmed: 21207987
doi: 10.1021/bi101659u