In-Cell Protein Magnetic Resonance Spectroscopy

Abstract

Abstract:

The cellular interior contains macromolecules at concentrations exceeding 300 mg/mL and volume occupancies of 30%. Until recently, however, most protein studies were performed in vitro and in dilute solutions. In-cell NMR spectroscopy provides a means to gain atomiclevel data noninvasively in the cellular environment. Although in-cell NMR has recently undergone significant advances, it is still in its infancy. Here, we review the limitations and potential pitfalls of in-cell NMR and recent advances in the field.

Key words: in-cell NMR, protein, Escherichia coli, EPR

CLC Number:

O482.53

WANG Ya-Qiang, LI Cong-Hang, PIELAK Gary J. In-Cell Protein Magnetic Resonance Spectroscopy[J]. Chinese Journal of Magnetic Resonance, 2012, 29(4): 475-488.

References

[1] Zimmerman S B, Trach S O. Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli[J]. J Mol Biol, 1991, 222(3): 599-620.

[2] Serber Z, D tsch V. In-cell NMR spectroscopy[J]. Biochemistry, 2001, 40(48): 14 317-14 323.

[3] Llinas M, Wüthrich K, Schwotzer W, et al.¹⁵N nuclear magnetic resonance of living cells[J]. Nature, 1975, 257(5 529): 817-818.

[4] Reckel S, Hansel R, Lohr F, et al. In-cell NMR spectroscopy[J]. Prog Nucl Magn Reson Spectrosc, 2007, 51(2): 91-101.

[5] Sharaf N G, Barnes C O, Charlton L M, et al. A bioreactor for in-cell protein NMR[J]. J Magn Reson, 2010, 202(2): 140-146.

[6] Augustus A M, Reardon P N, Spicer L D. MetJ repressor interactions with DNA probed by in-cell NMR[J]. Proc Natl Acad Sci USA, 2009, 106(13): 5 065-5 069.

[7] Banci L, Barbieri L, Bertini I, et al. In-cell NMR in E.coli to monitor maturation steps of hSOD1[J]. PLoS ONE, 2011, 6(8): e23561.

[8] Barnes C O, Monteith W B, Pielak G J. Internal and global protein motion assessed with a fusion construct and in-cell NMR spectroscopy\
[J]. ChemBioChem, 2011, 12(3): 390-391.

[9] Bertini I, Felli I C, Gonnelli L, et al. ¹³C direct-detection biomolecular NMR spectroscopy in living cells[J]. Angew Chem Int Ed, 2011, 50(10): 2 339-2 341.

[10] Burz D S, Dutta K, Cowburn D, et al. In-cell NMR for protein-protein interactions (STINT-NMR)[J]. Nat Protoc, 2006, 1(1): 146-152.

[11] Burz D S, Dutta K, Cowburn D, et al. Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR)[J]. Nat Meth, 2006, 3(2): 91-93.

[12] Crowley P B, Chow E, Papkovskaia T. Protein interactions in the Escherichia coli cytosol: An impediment to in-cell NMR spectroscopy[J]. ChemBioChem, 2011, 12(7): 1 043-1 048

[13] Cruzeiro-Silva C, Albernaz F P, Valente A P, et al. In-cell NMR spectroscopy: Inhibition of autologous protein expression reduces Escherichia coli- lysis[J]. Cell Biochem Biophys, 2006, 44(3): 497-502.

[14] Dedmon M M, Patel C N, Young G B, et al. FlgM gains structure in living cells[J]. Proc Natl Acad Sci USA, 2002, 99(20): 12 681-12 684.

[15] Gochin M, James T L, Shafer R H. In vivo ¹⁹F NMR of 5-fluorouracil incorporation into RNA and metabolites in Escherichia coli cells[J]. Biochim Biophys Acta, 1984, 804(1): 118-124.

[16] Li C, Wang G F, Wang Y, et al. Protein ¹⁹F NMR in Escherichia coli [J]. J Am Chem Soc, 2010, 132(1): 321-327.

[17] Serber Z, Lai H C, Yang A, et al. A C-terminal inhibitory domain controls the activity of p63 by an intramolecular mechanism[J]. Mol Cell Biol, 2002, 22(24): 8 601-8 611.

[18] Serber Z, Richter C, Moskau D, et al. New carbon-detected protein NMR experiments using CryoProbes[J]. J Am Chem Soc, 2000, 122(14): 3 554-3 555.

[19] Serber Z, Straub W, Corsini L, et al. Methyl groups as probes for proteins and complexes in in-cell NMR experiments[J]. J Am Chem Soc, 2004, 126(22): 7 119-7 125.

[20] Wang G F, Li C, Pielak G J. ¹⁹F NMR studies of alpha-synuclein-membrane interactions[J]. Protein Sci, 2010, 19(9): 1 686-1 691.

[21] Sakakibara D, Sasaki A, Ikeya T, et al. Protein structure determination in living cells by in-cell NMR spectroscopy[J]. Nature, 2009, 458(7 234): 102-105.

[22] Inomata K, Ohno A, Tochio H, et al. High-resolution multi-dimensional NMR spectroscopy of proteins in human cells[J]. Nature, 2009, 458(7 234): 106-109.

[23] Selenko P, Serber Z, Gadea B, et al. Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes[J]. Proc Natl Acad Sci USA, 2006, 103(32): 11 904-11 909.

[24] Bodart J F, Wieruszeski J M, Amniai L, et al. NMR observation of Tau in Xenopus oocytes[J]. J Magn Reson, 2008, 192(2): 252-257.

[25] Schnizler K, Kuster M, Methfessel C, et al. The roboocyte: Automated cDNA/mRNA injection and subsequent TEVC recording on Xenopus oocytes- in 96-well microtiter plates[J]. Receptors Channels, 2003, 9(1): 41-48.

[26] Gard D L, Kirschner M W. Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs[J]. J Cell Biol, 1987, 105(5): 2 191-2 201.

[27] Smet C, Leroy A, Sillen A, et al. Accepting its random coil nature allows a partial NMR assignment of the neuronal Tau protein[J]. Chembiochem, 2004, 5(12): 1 639-1 646.

[28] Lippens G, Wieruszeski J M, Leroy A, et al. Proline directed random coil chemical shift values as a tool for the NMR assignment of the tau phosphorylation sites[J]. ChemBioChem, 2004, 5(1): 73-78.

[29] Sillen A, Barbier P, Landrieu I, et al. NMR investigation of the interaction between the neuronal protein tau and the microtubules[J]. Biochemistry, 2007, 46(11): 3 055-3 064.

[30] Sakai T, Tochio H, Inomata K, et al. Fluoroscopic assessment of protein leakage during Xenopus oocytes in-cell NMR experiment by co injected EGFP[J]. Anal Biochem, 2007, 371(2): 247-249.

[31] Sakai T, Tochio H, Tenno T, et al. In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes[J]. J Biomol NMR, 2006, 36(3): 179-188.

[32] Charlton L M, Pielak G J. Peeking into living eukaryotic cells with high-resolution NMR[J]. Proc Natl Acad Sci USA, 2006, 103(32): 11 817-11 818.

[33] Schlesinger A P, Wang Y, Tadeo X, et al. Macromolecular crowding fails to fold a globular protein in cells[J]. J Am Chem Soc, 2011, 133(21): 8 082-8 085.

[34] Xie J, Thapa R, Reverdatto S, et al. Screening of small molecule interactor library by using in-cell NMR spectroscopy (SMILI-NMR)[J]. J Med Chem, 2009, 52(11): 3 516-3 522.

[35] Arnesano F, Banci L, Bertini I, et al. Probing the interaction of Cisplatin with the human copper chaperone atox1 by solution and in-cell NMR spectroscopy[J]. J Am Chem Soc, 2011, 133(45): 18 361-18 369.

[36] Vogt P K. PI 3-kinase, mTOR, protein synthesis and cancer[J]. Trends Mol Med, 2001, 7(11): 482-484.

[37] Selenko P, Frueh D P, Elsaesser S J, et al. In situ observation of protein phosphorylation by high-resolution NMR spectroscopy[J]. Nat Struct Mol Biol, 2008, 15(3): 321-329.

[38] Burz D S, Shekhtman A. In-cell biochemistry using NMR spectroscopy[J]. PLoS ONE, 2008, 3(7): e2 571.

[39] H-nsel R, Foldynov Trantírkov S, L hr F, et al. Evaluation of parameters critical for observing nucleic acids inside living Xenopus laevis oocytes by in-cell NMR spectroscopy[J]. J Am Chem Soc, 2009, 131(43): 15 761-15 768.

[40] Burz D S, Shekhtman A. The STINT-NMR method for studying in cell proteinprotein interactions[J]. Curr Protoc Protein Sci, 2010, 61: 17.11.11-17.11.15.

[41] Maldonado A Y, Burz D S, Shekhtman A. In-cell NMR spectroscopy[J]. Prog Nucl Magn Reson Spectrosc, 2011, 59(3): 197-212.

[42] Pielak G J, Li C, Miklos A C, et al. Protein nuclear magnetic resonance under physiological conditions[J]. Biochemistry, 2009, 48(2): 226-234.

[43] Robinson K E, Reardon P N, Spicer L D. In-cell NMR spectroscopy in Escherichia coli[J]. Methods Mol Biol, 2012, 831: 261-277.

[44] Wang Q, Zhuravleva A, Gierasch L M. Exploring weak, transient protein-protein interactions in crowded in vivo environments by in cell nuclear magnetic resonance spectroscopy[J]. Biochemistry, 2011, 50(43): 9 225-9 236.

[45] Pielak G J. Retraction[J]. Biochemistry, 2007, 46(27): 8 206.

[46] Serber Z, Corsini L, Durst F, et al. In-cell NMR spectroscopy[J]. Methods Enzymol, 2005, 394: 17-41.

[47] Spitzer J, Poolman B. The role of biomacromolecular crowding, ionic strength, and physicochemical gradients in the complexities of life's emergence[J]. Microbiol Mol Biol Rev, 2009, 73(2): 371-388.

[48] Spitzer J J, Poolman B. Electrochemical structure of the crowded cytoplasm[J]. Trends Biochem Sci, 2005, 30(10): 536-541.

[49] Palmer A G, 3rd, Kroenke C D, Loria J P. Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules[J]. Methods Enzymol, 2001, 339: 204-238.

[50] Cavanagh J, Fairbrother W J, Palmer A G, et al. Protein NMR Spectroscopy: Principles and Practice (2nd ed)[M]. London: Academic Press, 2006.

[51] Crowley P B, Brett K, Muldoon J. NMR spectroscopy reveals cytochrome c-poly(ethylene glycol) interactions[J]. ChemBioChem, 2008, 9(5): 685-688.

[52] Li C, Pielak G J. Using NMR to distinguish viscosity effects from nonspecific protein binding under crowded conditions[J]. J Am Chem Soc, 2009, 131(4): 1 368-1 369.

[53] Li C, Wang Y, Pielak G J. Translational and rotational diffusion of a small globular protein under crowded conditions[J]. J Phys Chem B, 2009, 113(40): 13 390-13 392.

[54] Wang Y, Li C, Pielak G J. Effects of proteins on protein diffusion[J]. J Am Chem Soc, 2010, 132(27): 9 392-9 397.

[55] Li C, Charlton L M, Lakkavaram A, et al. Differential dynamical effects of macromolecular crowding on an intrinsically disordered protein and a globular protein: implications for in-cell NMR spectroscopy[J]. J Am Chem Soc, 2008, 130(20): 6 310-6 311.

[56] Ikeya T, Sasaki A, Sakakibara D, et al. NMR protein structure determination in living E.coli cells using nonlinear sampling[J]. Nat Protoc, 2010, 5(6): 1 051-1 060.

[57] Reardon P N, Spicer L D. Multidimensional NMR spectroscopy for protein characterization and assignment inside cells[J]. J Am Chem Soc, 2005, 127(31): 10 848-10 849.

[58] Slade K M, Baker R, Chua M, et al. Effects of recombinant protein expression on green fluorescent protein diffusion in Escherichia coli[J]. Biochemistry, 2009, 48(23): 5 083-5 089.

[59] Hedrick W R, Mathew A, Zimbrick J D. Intracellular viscosity of lymphocytes determined by a N-15 spin label probe[J]. J Magn Reson, 1979, 36(2): 207-214.

[60] Kneller J M, Lu M, Bracken C. An effective method for the discrimination of motional anisotropy and chemical exchange[J]. J Am Chem Soc, 2002, 124(9): 1 852-1 853.

[61] Feig M, Sugita Y. Variable interactions between protein crowders and biomolecular solutes are important in understanding cellular crowding[J]. J Phys Chem B, 2012, 116(1): 599-605.

[62] McGuffee S R, Elcock A H. Diffusion, crowding & protein stability in a dynamic molecular model of the bacterial cytoplasm[J]. PLoS Comput Biol, 2010, 6(3): e1000694.

[63] Navon G, Ogawa S, Shulman R G, et al. High-resolution ³¹P nuclear magnetic resonance studies of metabolism in aerobic Escherichia coli cells[J]. Proc Natl Acad Sci USA, 1977, 74(3): 888-891.

[64] Bryant J E, Lecomte J T, Lee A L, et al. Protein dynamics in living cells[J]. Biochemistry, 2005, 44(26): 9 275-9 279.

[65] Bryant J E, Lecomte J T, Lee A L, et al. Cytosol has a small effect on protein backbone dynamics[J]. Biochemistry, 2006, 45(33): 10 085-10 091.

[66] Barnes C O, Pielak G J. In-cell protein NMR and protein leakage[J]. Proteins, 2011, 79(2): 347-351.

[67] Schanda P, Brutscher B. Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds[J]. J Am Chem Soc, 2005, 127(22): 8 014-8 015.

[68] Luby-Phelps K. Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area[J]. Int Rev Cytol, 2000, 192: 189-221.

[69] Jackson J C, Hammill J T, Mehl R A. Site-specific incorporation of a (19)F-amino acid into proteins as an NMR probe for characterizing protein structure and reactivity[J]. J Am Chem Soc, 2007, 129(5): 1 160-1 166.

[70] Haggie P M, Brindle K M. Mitochondrial citrate synthase is immobilized in vivo[J]. J Biol Chem, 1999, 274(7): 3 941-3 945.

[71] Williams S P, Fulton A M, Brindle K M. Estimation of the intracellular free ADP concentration by ¹⁹F NMR studies of fluorine-labeled yeast phosphoglycerate kinase in vivo[J]. Biochemistry, 1993, 32(18): 4 895-4 902.

[72] Williams S P, Haggie P M, Brindle K M. ¹⁹F NMR measurements of the rotational mobility of proteins in vivo[J]. Biophys J, 1997, 72(1): 490-498.

[73] Fu R, Wang X, Li C, et al. In situ structural characterization of a recombinant protein in native-Escherichia coli- membranes with solidstate magic angle spinning NMR[J]. J Am Chem Soc, 2011, 133(32): 12 370-12 373.

[74] Vogel E P, Curtis-Fisk J, Young K M, et al. Solid-state nuclear magnetic resonance (NMR) spectroscopy of human immunodeficiency virus gp41 protein that includes the fusion peptide: NMR detection of recombinant fgp41 in inclusion bodies in whole bacterial cells and structural characterization of purified and membrane-associated fgp41[J]. Biochemistry, 2011, 50(46): 10 013-10 026.

[75] Zavoisky Y. Spin-magnetic resonance in paramagnetics[J]. J Phys USSR, 1945, 9: 245-249.

[76] Schiemann O, Piton N, Plackmeyer J, et al. Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances[J]. Nat Protoc, 2007, 2(4): 904-923.

[77] Kohler S D, Weber A, Howard S P, et al. The proline-rich domain of TonB possesses an extended polyproline II-like conformation of sufficient length to span the periplasm of Gram-negative bacteria[J]. Protein Sci, 2010, 19(4): 625-630.

[78] Robotta M, Braun P, van Rooijen B, et al. Direct evidence of coexisting horseshoe and extended helix conformations of membranebound alpha-synuclein[J]. ChemPhysChem, 2011, 12(2): 267-269.

[79] Usselman R J, Walter E D, Willits D, et al. Monitoring structural transitions in icosahedral virus protein cages by site-directed spin labeling[J]. J Am Chem Soc, 2011, 133(12): 4 156-4 159.

[80] Azarkh M, Okle O, Eyring P, et al. Evaluation of spin labels for in-cell EPR by analysis of nitroxide reduction in cell extract of Xenopus laevis oocytes[J]. J Magn Reson, 2011, 212(2): 450-454.

[81] Azarkh M, Okle O, Singh V, et al. Long-range distance determination in a DNA model system inside Xenopus laevis oocytes by in cell spin label EPR[J]. ChemBioChem, 2011, 12(13): 1 992-1 995.

[82] Igarashi R, Sakai T, Hara H, et al. Distance determination in proteins inside Xenopus laevis oocytes by double electron electron resonance experiments[J]. J Am Chem Soc, 2010, 132(24): 8 228-8 829.

[83] Krstic I, Hansel R, Romainczyk O, et al. Long-range distance measurements on nucleic acids in cells by pulsed EPR spectroscopy[J]. Angew Chem Int Ed, 2011, 50(22): 5 070-5 074.

[84] Borbat P P, McHaourab H S, Freed J H. Protein structure determination using long-distance constraints from double quantum coherence ESR: study of T4 lysozyme[J]. J Am Chem Soc, 2002, 124(19): 5 304-5 314.

[85] Altenbach C, Kusnetzow A K, Ernst O P, et al. High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation[J]. Proc Natl Acad Sci USA, 2008, 105(21): 7 439-7 444.

[86] Gierasch L M, Gershenson A. Post-reductionist protein science, or putting Humpty Dumpty back together again[J]. Nat Chem Biol, 2009, 5(11): 774-777.