Journal of Biomolecular NMR

, Volume 61, Issue 3–4, pp 185–196 | Cite as

Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA)

  • Sang Ho Park
  • Vivian S. Wang
  • Jasmina Radoicic
  • Anna A. De Angelis
  • Sabrina Berkamp
  • Stanley J. Opella


The use of paramagnetic constraints in protein NMR is an active area of research because of the benefits of long-range distance measurements (>10 Å). One of the main issues in successful execution is the incorporation of a paramagnetic metal ion into diamagnetic proteins. The most common metal ion tags are relatively long aliphatic chains attached to the side chain of a selected cysteine residue with a chelating group at the end where it can undergo substantial internal motions, decreasing the accuracy of the method. An attractive alternative approach is to incorporate an unnatural amino acid that binds metal ions at a specific site on the protein using the methods of molecular biology. Here we describe the successful incorporation of the unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) into two different membrane proteins by heterologous expression in E. coli. Fluorescence and NMR experiments demonstrate complete replacement of the natural amino acid with HQA and stable metal chelation by the mutated proteins. Evidence of site-specific intra- and inter-molecular PREs by NMR in micelle solutions sets the stage for the use of HQA incorporation in solid-state NMR structure determinations of membrane proteins in phospholipid bilayers.


Membrane protein UAA PRE Protein structure CXCR1 p7 


  1. Abragam A, Bleaney B (2012) Electron paramagnetic resonance of transition ions. Oxford University Press, OxfordGoogle Scholar
  2. Balayssac S, Bertini I, Lelli M, Luchinat C, Maletta M (2007) Paramagnetic ions provide structural restraints in solid-state NMR of proteins. J Am Chem Soc 129:2218–2219. doi:10.1021/ja068105a CrossRefGoogle Scholar
  3. Barthelmes K et al (2011) Engineering encodable lanthanide-binding tags into loop regions of proteins. J Am Chem Soc 133:808–819. doi:10.1021/ja104983t CrossRefGoogle Scholar
  4. Bertini I, Luchinat C, Parigi G, Pierattelli R (2008) Perspectives in paramagnetic NMR of metalloproteins. Dalton Trans 3782–3790. doi:10.1039/b719526e
  5. Bodenhausen G, Ruben DJ (1980) Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy. Chem Phys Lett 69:185–189. doi:10.1016/0009-2614(80)80041-8
  6. Campbell ID, Dobson CM, Williams RJ, Xavier AV (1973) The determination of the structure of proteins in solution: lysozyme. Ann N Y Acad Sci 222:163–174CrossRefADSGoogle Scholar
  7. Casagrande F, Maier K, Kiefer H, Opella SJ, Park SH (2011) Expression and purification of G-protein-coupled receptors for nuclear magnetic resonance structural studies. In: Robinson AS (ed) production of membrane proteins: strategies for expression and isolation. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, pp 297–316. doi:10.1002/9783527634521.ch11
  8. Cellitti SE et al (2008) In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy. J Am Chem Soc 130:9268–9281. doi:10.1021/ja801602q CrossRefGoogle Scholar
  9. Chen H et al (2011) Optimal mutation sites for PRE data collection and membrane protein structure prediction. Structure 19:484–495. doi:10.1016/j.str.2011.02.002 CrossRefGoogle Scholar
  10. Clore G (2013) Seeing the invisible by paramagnetic and diamagnetic NMR. Biochem Soc Trans 41:1343–1354Google Scholar
  11. Cohen LS, Arshava B, Neumoin A, Becker JM, Guntert P, Zerbe O, Naider F (2011) Comparative NMR analysis of an 80-residue G protein-coupled receptor fragment in two membrane mimetic environments. Biochim Biophys Acta 1808:2674–2684. doi:10.1016/j.bbamem.2011.07.011 CrossRefGoogle Scholar
  12. Cook GA, Stefer S, Opella SJ (2011) Expression and purification of the membrane protein p7 from hepatitis C virus. Biopolymers 96:32–40. doi:10.1002/bip.21453 CrossRefGoogle Scholar
  13. Cross TA, Opella SJ (1979) NMR of fd coat protein. J Supramol Struct 11:139–145. doi:10.1002/jss.400110204 CrossRefGoogle Scholar
  14. Cross TA, Opella SJ (1980) Structural properties of fd coat protein in sodium dodecyl sulfate micelles. Biochem Biophys Res Commun 92:478–484CrossRefGoogle Scholar
  15. Deiters A, Geierstanger BH, Schultz PG (2005) Site-specific in vivo labeling of proteins for NMR studies. Chembiochem Eur J Chem Biol 6:55–58. doi:10.1002/cbic.200400319 CrossRefGoogle Scholar
  16. Fleissner MR et al (2009) Site-directed spin labeling of a genetically encoded unnatural amino acid. Proc Natl Acad Sci USA 106:21637–21642. doi:10.1073/pnas.0912009106 CrossRefADSGoogle Scholar
  17. Ganguly S, Weiner BE, Meiler J (2011) Membrane protein structure determination using paramagnetic tags. Structure 19:441–443. doi:10.1016/j.str.2011.03.008 CrossRefGoogle Scholar
  18. Gaponenko V, Dvoretsky A, Walsby C, Hoffman BM, Rosevear PR (2000) Calculation of z-coordinates and orientational restraints using a metal binding tag. Biochemistry 39:15217–15224CrossRefGoogle Scholar
  19. Gottstein D, Reckel S, Dotsch V, Guntert P (2012) Requirements on paramagnetic relaxation enhancement data for membrane protein structure determination by NMR. Structure 20:1019–1027. doi:10.1016/j.str.2012.03.010 CrossRefGoogle Scholar
  20. Hagen DS, Weiner JH, Sykes BD (1978) Fluorotyrosine M13 coat protein: fluorine-19 nuclear magnetic resonance study of the motional properties of an integral membrane protein in phospholipid vesicles. Biochemistry 17:3860–3866Google Scholar
  21. Hagen DS, Weiner JH, Sykes BD (1979) Investigation of solvent accessibility of the fluorotyrosyl residues of M13 coat protein in deoxycholate micelles and phospholipid vesicles. Biochemistry 18:2007–2012CrossRefGoogle Scholar
  22. Hass MA, Ubbink M (2014) Structure determination of protein-protein complexes with long-range anisotropic paramagnetic NMR restraints. Curr Opin Struct Biol 24:45–53. doi:10.1016/ CrossRefGoogle Scholar
  23. Hilty C, Wider G, Fernandez C, Wuthrich K (2004) Membrane protein-lipid interactions in mixed micelles studied by NMR spectroscopy with the use of paramagnetic reagents. Chembiochem Eur J Chem Biol 5:467–473. doi:10.1002/cbic.200300815 CrossRefGoogle Scholar
  24. Inubushi T, Becker E (1983) Efficient detection of paramagnetically shifted NMR resonances by optimizing the WEFT pulse sequence. J Magn Reson 51:128–133ADSGoogle Scholar
  25. Jaroniec CP (2012) Solid-state nuclear magnetic resonance structural studies of proteins using paramagnetic probes. Solid State Nucl Magn Reson 43–44:1–13. doi:10.1016/j.ssnmr.2012.02.007 CrossRefGoogle Scholar
  26. Jones DH et al (2010) Site-specific labeling of proteins with NMR-active unnatural amino acids. J Biomol NMR 46:89–100. doi:10.1007/s10858-009-9365-4 CrossRefGoogle Scholar
  27. Keizers PH, Ubbink M (2011) Paramagnetic tagging for protein structure and dynamics analysis. Prog Nucl Magn Reson Spectrosc 58:88–96. doi:10.1016/j.pnmrs.2010.08.001 CrossRefGoogle Scholar
  28. Knight MJ et al (2012) Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR. Proc Natl Acad Sci USA 109:11095–11100. doi:10.1073/pnas.1204515109 CrossRefADSGoogle Scholar
  29. Knight MJ, Felli IC, Pierattelli R, Emsley L, Pintacuda G (2013) Magic angle spinning NMR of paramagnetic proteins. Acc Chem Res 46:2108–2116. doi:10.1021/ar300349y CrossRefGoogle Scholar
  30. Lee HS, Spraggon G, Schultz PG, Wang F (2009) Genetic incorporation of a metal-ion chelating amino acid into proteins as a biophysical probe. J Am Chem Soc 131:2481–2483. doi:10.1021/ja808340b CrossRefGoogle Scholar
  31. Li J, Pilla K, Li Q, Zhanag A, Su XC, Huber T, Yang J (2013) Magic angle spinning NMR structure determination of proteins from pseudocontact shifts. J Am Chem Soc 135:8294–8303CrossRefGoogle Scholar
  32. Liang B, Bushweller JH, Tamm LK (2006) Site-directed parallel spin-labeling and paramagnetic relaxation enhancement in structure determination of membrane proteins by solution NMR spectroscopy. J Am Chem Soc 128:4389–4397. doi:10.1021/ja0574825 CrossRefGoogle Scholar
  33. Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79:413–444. doi:10.1146/annurev.biochem.052308.105824 CrossRefGoogle Scholar
  34. Liu W-M, Overhand M, Ubbink M (2014a) The application of paramagnetic lanthanoid ions in NMR spectroscopy on proteins. Coord Chem Rev 273–274:2–12. doi:10.1016/j.ccr.2013.10.018
  35. Liu WM et al (2014b) A two-armed lanthanoid-chelating paramagnetic NMR probe linked to proteins via thioether linkages. Chemistry 20:6256–6258. doi:10.1002/chem.201400257 CrossRefGoogle Scholar
  36. Liu Z, Gong Z, Guo DC, Zhang WP, Tang C (2014c) Subtle dynamics of holo glutamine binding protein revealed with a rigid paramagnetic probe. Biochemistry 53:1403–1409. doi:10.1021/bi4015715 CrossRefGoogle Scholar
  37. Loh CT, Ozawa K, Tuck KL, Barlow N, Huber T, Otting G, Graham B (2013) Lanthanide tags for site-specific ligation to an unnatural amino acid and generation of pseudocontact shifts in proteins. Bioconjug Chem 24:260–268. doi:10.1021/bc300631z CrossRefGoogle Scholar
  38. Ma C, Opella SJ (2000) Lanthanide ions bind specifically to an added “EF-hand” and orient a membrane protein in micelles for solution NMR spectroscopy. J Magn Reson 146:381–384. doi:10.1006/jmre.2000.2172 CrossRefADSGoogle Scholar
  39. Markley JL, Putter I, Jardetzky O (1968) High-resolution nuclear magnetic resonance spectra of selectively deuterated staphylococcal nuclease. Science 161:1249–1251CrossRefADSGoogle Scholar
  40. Marley J, Lu M, Bracken C (2001) A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20:71–75CrossRefGoogle Scholar
  41. McConnell HM, McFarland BG (1970) Physics and chemistry of spin labels. Q Rev Biophys 3:91–136CrossRefGoogle Scholar
  42. Meadows DH, Markley JL, Cohen JS, Jardetzky O (1967) Nuclear magnetic resonance studies of the structure and binding sites of enzymes. I. Histidine residues. Proc Natl Acad Sci USA 58:1307–1313CrossRefADSGoogle Scholar
  43. Mesleh MF, Lee S, Veglia G, Thiriot DS, Marassi FM, Opella SJ (2003) Dipolar waves map the structure and topology of helices in membrane proteins. J Am Chem Soc 125:8928–8935. doi:10.1021/ja034211q CrossRefGoogle Scholar
  44. Morrisett JD, Wien RW, McConnell HM (1973) The use of spin labels for measuring distances in biological systems. Ann N Y Acad Sci 222:149–162CrossRefADSGoogle Scholar
  45. Nadaud PS, Helmus JJ, Kall SL, Jaroniec CP (2009) Paramagnetic ions enable tuning of nuclear relaxation rates and provide long-range structural restraints in solid-state NMR of proteins. J Am Chem Soc 131:8108–8120. doi:10.1021/ja900224z CrossRefGoogle Scholar
  46. Nguyen TH, Ozawa K, Stanton-Cook M, Barrow R, Huber T, Otting G (2011) Generation of pseudocontact shifts in protein NMR spectra with a genetically encoded cobalt(II)-binding amino acid. Angew Chem 50:692–694. doi:10.1002/anie.201005672 CrossRefGoogle Scholar
  47. Noren CJ, Anthony-Cahill SJ, Griffith MC, Schultz PG (1989) A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244:182–188CrossRefADSGoogle Scholar
  48. Opella SJ (2013) Structure determination of membrane proteins in their native phospholipid bilayer environment by rotationally aligned solid-state NMR spectroscopy. Acc Chem Res 46:2145–2153. doi:10.1021/ar400067z CrossRefGoogle Scholar
  49. Otting G (2010) Protein NMR using paramagnetic ions. Annu Rev Biophys 39:387–405. doi:10.1146/annurev.biophys.093008.131321 CrossRefGoogle Scholar
  50. Page RC, Lee S, Moore JD, Opella SJ, Cross TA (2009) Backbone structure of a small helical integral membrane protein: a unique structural characterization. Protein sci Publ Protein Soc 18:134–146. doi:10.1002/pro.24 Google Scholar
  51. Park SH, Berkamp S, Cook GA, Chan MK, Viadiu H, Opella SJ (2011a) Nanodiscs versus macrodiscs for NMR of membrane proteins. Biochemistry 50:8983–8985. doi:10.1021/bi201289c CrossRefGoogle Scholar
  52. Park SH, Casagrande F, Cho L, Albrecht L, Opella SJ (2011b) Interactions of interleukin-8 with the human chemokine receptor CXCR1 in phospholipid bilayers by NMR spectroscopy. J Mol Biol 414:194–203. doi:10.1016/j.jmb.2011.08.025 CrossRefGoogle Scholar
  53. Park SH, Casagrande F, Das BB, Albrecht L, Chu M, Opella SJ (2011c) Local and global dynamics of the G protein-coupled receptor CXCR1. Biochemistry 50:2371–2380. doi:10.1021/bi101568j CrossRefGoogle Scholar
  54. Parthasarathy S, Nishiyama Y, Ishii Y (2013) Sensitivity and resolution enhanced solid-state NMR for paramagnetic systems and biomolecules under very fast magic angle spinning. Acc Chem Res 46:2127–2135. doi:10.1021/ar4000482 CrossRefGoogle Scholar
  55. Prestegard JH, al-Hashimi HM, Tolman JR (2000) NMR structures of biomolecules using field oriented media and residual dipolar couplings. Q Rev Biophys 33:371–424CrossRefGoogle Scholar
  56. Radoicic J, Lu GJ, Opella SJ (2014) NMR structures of membrane proteins in phospholipid bilayers. Quart Rev Biophys 47:249–283. doi:10.1017/S0033583514000080 CrossRefGoogle Scholar
  57. Rajarathnam K, Kay CM, Clark-Lewis I, Sykes BD (1997) Characterization of quaternary structure of interleukin-8 and functional implications. Methods Enzym 287:89–105CrossRefGoogle Scholar
  58. Saunders M, Wishnia A, Kirkwood JG (1957) The nuclear magnetic resonance spectrum of ribonuclease. J Am Chem Soc 79:3289–3290. doi:10.1021/ja01569a083 CrossRefGoogle Scholar
  59. Schmidt M, Borbas J, Drescher J, Summerer D (2014) A genetically encoded spin label for electron paramagnetic resonance distance measurements. J Am Chem Soc 136:138–141Google Scholar
  60. Sengupta I, Nadaud PS, Jaroniec CP (2013) Protein structure determination with paramagnetic solid-state NMR spectroscopy. Acc Chem Res 46:2117–2126. doi:10.1021/ar300360q CrossRefGoogle Scholar
  61. Son WS et al (2012) ‘q-Titration’ of long-chain and short-chain lipids differentiates between structured and mobile residues of membrane proteins studied in bicelles by solution NMR spectroscopy. J Magn Reson 214:111–118. doi:10.1016/j.jmr.2011.10.011 CrossRefADSGoogle Scholar
  62. Soroka K, Vithanage RS, Phillips DA, Walker B, Dasgupta PK (1987) Fluorescence properties of metal complexes of 8-hydroxyquinoline-5-sulfonic acid and chromatographic applications. Anal Chem 59:629–636. doi:10.1021/ac00131a019 CrossRefGoogle Scholar
  63. Su XC et al (2008) A dipicolinic acid tag for rigid lanthanide tagging of proteins and paramagnetic NMR spectroscopy. J Am Chem Soc 130:10486–10487. doi:10.1021/ja803741f CrossRefGoogle Scholar
  64. Su Y, Hu F, Hong M (2012) Paramagnetic Cu(II) for probing membrane protein structure and function: inhibition mechanism of the influenza M2 proton channel. J Am Chem Soc 134:8693–8702. doi:10.1021/ja3026328 CrossRefGoogle Scholar
  65. Tang M, Berthold DA, Rienstra CM (2011) Solid-State NMR of a Large Membrane Protein by Paramagnetic Relaxation Enhancement. J Phys Chem Lett 2:1836–1841. doi:10.1021/jz200768r CrossRefGoogle Scholar
  66. Tolman JR, Flanagan J, Kennedy M, Prestegard JH (1995) Nuclear magnetic dipole interactions in filed-oriented proteins: information for structure determination in solution. Proc Natl Acad Sci USA 92:9279–9283CrossRefADSGoogle Scholar
  67. Ullrich SJ, Holper S, Glaubitz C (2014) Paramagnetic doping of a 7TM membrane protein in lipid bilayers by Gd(3)(+)-complexes for solid-state NMR spectroscopy. J Biomol NMR 58:27–35. doi:10.1007/s10858-013-9800-4 CrossRefGoogle Scholar
  68. Venditti V, Fawzi N, Clore G (2012) An efficient protocol for incorporation of an unnatural amino acid in perdeuterated recombinant proteins using glucose-based media. J Biomol NMR 52:191–195CrossRefGoogle Scholar
  69. Wang S, Munro RA, Kim SY, Jung KH, Brown LS, Ladizhansky V (2012) Paramagnetic relaxation enhancement reveals oligomerization interface of a membrane protein. J Am Chem Soc 134:16995–16998. doi:10.1021/ja308310z CrossRefGoogle Scholar
  70. Wang S et al (2013) Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein. Nat Methods 10:1007–1012. doi:10.1038/nmeth.2635 CrossRefGoogle Scholar
  71. Ward ME, Wang S, Krishnamurthy S, Hutchins H, Fey M, Brown LS, Ladizhansky V (2014) High-resolution paramagnetically enhanced solid-state NMR spectroscopy of membrane proteins at fast magic angle spinning. J Biomol NMR 58:37–47. doi:10.1007/s10858-013-9802-2 CrossRefGoogle Scholar
  72. Wien RW, Morrisett JD, McConnell HM (1972) Spin-label-induced nuclear relaxation. Distances between bound saccharides, histidine-15, and tryptophan-123 on lysozyme in solution. Biochemistry 11:3707–3716. doi:10.1021/bi00770a008 CrossRefGoogle Scholar
  73. Wohnert J, Franz KJ, Nitz M, Imperiali B, Schwalbe H (2003) Protein alignment by a coexpressed lanthanide-binding tag for the measurement of residual dipolar couplings. J Am Chem Soc 125:13338–13339. doi:10.1021/ja036022d CrossRefGoogle Scholar
  74. Yagi H, Pilla KB, Maleckis A, Graham B, Huber T, Otting G (2013) Three-dimensional protein fold determination from backbone amide pseudocontact shifts generated by lanthanide tags at multiple sites. Structure 21:883–890. doi:10.1016/j.str.2013.04.001 CrossRefGoogle Scholar
  75. Yeo KJ et al (2010) Rapid exploration of the folding topology of helical membrane proteins using paramagnetic perturbation. Protein Sci Publ Protein Soc 19:2409–2417. doi:10.1002/pro.521 CrossRefGoogle Scholar
  76. Young TS, Ahmad I, Yin JA, Schultz PG (2010) An enhanced system for unnatural amino acid mutagenesis in E. coli. J Mol Biol 395:361–374. doi:10.1016/j.jmb.2009.10.030 CrossRefGoogle Scholar
  77. Zhang WH, Otting G, Jackson CJ (2013) Protein engineering with unnatural amino acids. Curr Opin Struct Biol 23:581–587. doi:10.1016/ CrossRefGoogle Scholar
  78. Zhuang T, Lee H-S, Imperiali B, Prestegard JH (2008) Structure determination of a galectin-3-carbohydrate complex using paramagnetism-based NMR constraints. Protein Sci 17:1220–11231CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Sang Ho Park
    • 1
  • Vivian S. Wang
    • 1
  • Jasmina Radoicic
    • 1
  • Anna A. De Angelis
    • 1
  • Sabrina Berkamp
    • 1
  • Stanley J. Opella
    • 1
  1. 1.Department of Chemistry and BiochemistryUniversity of California, San DiegoLa JollaUSA

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