Journal of Biomolecular NMR

, Volume 58, Issue 2, pp 123–128

The IR-15N-HSQC-AP experiment: a new tool for NMR spectroscopy of paramagnetic molecules

  • Simone Ciofi-Baffoni
  • Angelo Gallo
  • Riccardo Muzzioli
  • Mario Piccioli
Article

Abstract

A crucial factor for the understanding of structure-function relationships in metalloproteins is the identification of NMR signals from residues surrounding the metal cofactor. When the latter is paramagnetic, the NMR information in the proximity of the metal center may be scarce, because fast nuclear relaxation quenches signal intensity and coherence transfer efficiency. To identify residues at a short distance from a paramagnetic center, we developed a modified version of the 15N-HSQC experiment where (1) an inversion recovery filter is added prior to HSQC, (2) the INEPT period has been optimized according to fast relaxation of interested spins, (3) the inverse INEPT has been eliminated and signals acquired as antiphase doublets. The experiment has been successfully tested on a human [Fe2S2] protein which is involved in the biogenesis of iron-sulfur proteins. Thirteen HN resonances, unobserved with conventional HSQC experiments, could be identified. The structural arrangement of the protein scaffold in the proximity of the Fe/S cluster is fundamental to comprehend the molecular processes responsible for the transfer of Fe/S groups in the iron-sulfur protein assembly machineries.

Keywords

Iron-sulfur proteins Paramagnetic NMR 15N-HSQC Pulse sequences Paramagnetic relaxation Anamorsin 

References

  1. Abriata LA, Ledesma GN, Pierattelli R, Vila AJ (2009) Electronic structure of the ground and excited states of the Cua site by NMR spectroscopy. J Am Chem Soc 131:1939–1946CrossRefGoogle Scholar
  2. Arnesano F, Banci L, Piccioli M (2006) NMR structures of paramagnetic metalloproteins. Q Rev Biophys 38:167–219CrossRefGoogle Scholar
  3. Balayssac S, Bertini I, Luchinat C, Parigi G, Piccioli M (2006) 13C direct detected NMR increases the detectability of residual dipolar couplings. J Am Chem Soc 128:15042–15043CrossRefGoogle Scholar
  4. Banci L, Bertini I, Ciurli S, Ferretti S, Luchinat C, Piccioli M (1993) The electronic structure of (Fe4S4)3+ clusters in proteins. An investigation of the oxidized high-potential iron-sulfur protein II from Ectothiorhodospira vacuolata. Biochemistry 32:9387–9397CrossRefGoogle Scholar
  5. Banci L, Bertini I, Ciofi-Baffoni S, Kandias NG, Spyroulias GA, Su XC, Robinson NJ, Vanarotti M (2006) The delivery of copper for thylakoid import observed by NMR. Proc Natl Acad Sci USA 103:8325ADSGoogle Scholar
  6. Banci L, Bertini I, Cantini F, Ciofi-Baffoni S (2010) Cellular copper distribution: a mechanistic systems biology approach. Cell Mol Life Sci 67:2563–2589CrossRefGoogle Scholar
  7. Banci L, Bertini I, Ciofi-Baffoni S, Boscaro F, Chatzi A, Mikolajczyk M, Tokatlidis K, Winkelmann J (2011) Anamorsin is a 2Fe2S cluster-containing substrate of the Mia40-dependent mitochondrial protein trapping machinery. Chem Biol 18:794–804CrossRefGoogle Scholar
  8. Banci L, Bertini I, Calderone V, Ciofi-Baffoni S, Giachetti A, Jaiswal D, Mikolajczyk M, Piccioli M, Winkelmann J (2013a) Molecular view of an electron transfer process essential for iron-sulfur protein biogenesis. Proc Natl Acad Sci USA 110:7136–7141ADSCrossRefGoogle Scholar
  9. Banci L, Ciofi-Baffoni S, Mikolajczyk M, Winkelmann J, Bill E and Eirini Pandelia M (2013b) Human anamorsin binds (2Fe.2S) clusters with unique electronic properties. J Biol Inorg Chem 18:883–893Google Scholar
  10. Bentrop D, Bertini I, Luchinat C, Mendes J, Piccioli M, Teixeira M (1996) Paramagnetic NMR of the 7Fe ferredoxin from the hyperthermoacidophilic archaeon Desulfurolobus ambivalens reveals structural similarity to other dicluster ferredoxins. Eur J Biochem 236:92–99CrossRefGoogle Scholar
  11. Bermel W, Bertini I, Felli IC, Piccioli M, Pierattelli R (2006) 13C-detected protonless NMR spectroscopy of proteins in solution. Progr NMR Spectrosc 48:25–45CrossRefGoogle Scholar
  12. Bertini I, Capozzi F, Luchinat C, Piccioli M, Vicens Oliver M (1992) NMR is a unique and necessary step in the investigation of iron- sulfur proteins: the HiPIP from R. gelatinosus as an example. Inorg Chim Acta 198–200:483–491CrossRefGoogle Scholar
  13. Bertini I, Capozzi F, Luchinat C, Piccioli M, Vila AJ (1994a) The Fe4S4 centers in ferredoxins studied through proton and carbon hyperfine coupling. Sequence specific assignments of cysteines in ferredoxins from Clostridium acidi urici and Clostridium pasteurianum. J Am Chem Soc 116:651–660CrossRefGoogle Scholar
  14. Bertini I, Felli IC, Kastrau DHW, Luchinat C, Piccioli M, Viezzoli MS (1994b) Sequence-specific assignment of the 1H and 15N Nuclear Magnetic Resonance spectra of the reduced recombinant high potential iron sulfur protein (HiPIP) I from Ectothiorhodospira halophila. Eur J Biochem 225:703–714CrossRefGoogle Scholar
  15. Bertini I, Luchinat C, Piccioli M (1994c) Copper zinc superoxide dismutase a paramagnetic protein that provides a unique frame for the NMR investigations. Progr NMR Spectrosc 26:91–141CrossRefGoogle Scholar
  16. Bertini I, Eltis LD, Felli IC, Kastrau DHW, Luchinat C, Piccioli M (1995) The solution structure of oxidized HiPIP I from Ectothiorhodospira halophila, can NMR probe rearrangements associated to electron transfer processes? Chem Eur J 1:598–607CrossRefGoogle Scholar
  17. Bertini I, Dalvit C, Huber JG, Luchinat C, Piccioli M (1997) ePHOGSY experiment on a paramagnetic protein: location of the catalytic water molecule in the heme crevice of the oxidized form of horse heart Cytochrome c. FEBS Lett 415:45–48CrossRefGoogle Scholar
  18. Bertini I, Donaire A, Jiménez B, Luchinat C, Parigi G, Piccioli M, Poggi L (2001a) Paramagnetism-based versus classical constraints: an analysis of the solution structure of Ca Ln Calbindin D9k. J Biomol NMR 21:85–98CrossRefGoogle Scholar
  19. Bertini I, Luchinat C, Parigi G (2001b) Solution NMR of paramagnetic molecules. Elsevier, AmsterdamGoogle Scholar
  20. Bertini I, Cavallaro G, Cosenza M, Kümmerle R, Luchinat C, Piccioli M, Poggi L (2002) Cross correlation rates between curie spin and dipole-dipole relaxation in paramagnetic proteins: the case of cerium substituted Calbindin D9k. J Biomol NMR 23:115–125CrossRefGoogle Scholar
  21. Bertini I, Jiménez B, Piccioli M (2005) 13C direct detected experiments: optimisation to paramagnetic signals. J Magn Reson 174:125–132ADSCrossRefGoogle Scholar
  22. Bertini I, Gupta YK, Luchinat C, Parigi G, Peana M, Sgheri L, Yuan J (2007) Paramagnetism-based NMR restraints provide maximum allowed probabilities for the different conformations of partially independent protein domains. J Am Chem Soc 129:12786–12794CrossRefGoogle Scholar
  23. Boal AK, Rosenzweig AC (2009) Structural biology of copper trafficking. Chem Rev 109:4760–4779CrossRefGoogle Scholar
  24. Caillet-Saguy C, Piccioli M, Turano P, Lukat-Rodgers G, Wolff N, Rodgers K, Izadi-Pruneyre N, Delepierre M, Lecroisey A (2012) Heme carrier HasA: learning about the role of the iron axial ligands in the heme uptake and release processes. J Biol Chem 287:26932–26943CrossRefGoogle Scholar
  25. Clore GM, Iwahara J (2009) Theory, practice, and applications of paramagnetic relaxation enhancement for the characterization of transient low-population states of biological macromolecules and their complexes. Chem Rev 109:4108–4139CrossRefGoogle Scholar
  26. Fetrow JS, Baxter SM (1999) Assignment of 15N chemical shifts and 15N relaxation measurements for oxidized and reduced iso-1-cytochrome c. Biochemistry 38:4480–4492CrossRefGoogle Scholar
  27. Finney LA, O’Halloran TV (2003) Transition metal speciation in the cell: insights from the chemistry of metal ion receptors. Science 300:931–936ADSCrossRefGoogle Scholar
  28. Gaponenko V, Sarma SP, Altieri AS, Horita DA, Li J, Byrd RA (2004) Improving the accuracy of NMR structures of large proteins using pseudocontact shifts as long/range restraints. J Biomol NMR 28:205–212CrossRefGoogle Scholar
  29. Gelis I, Katsaros N, Luchinat C, Piccioli M, Poggi L (2003) A simple protocol to study blue copper proteins by NMR. Eur J Biochem 270:600–609CrossRefGoogle Scholar
  30. Hsueh KL, Westler WM, Markley JL (2010) NMR investigations of the Rieske protein from thermus thermophilus support a coupled proton and electron transfer mechanism. J Am Chem Soc 132:7908–7918CrossRefGoogle Scholar
  31. Im S-C, Liu G, Luchinat C, Sykes AG, Bertini I (1998) The solution structure of parsley [2Fe-2S] ferredoxin. Eur J Biochem 258:465–477CrossRefGoogle Scholar
  32. Iwahara J, Schwieters CD, Clore GM (2004) Characterization of nonspecific protein-DNA interactions by H-1 paramagnetic relaxation enhancement. J Am Chem Soc 126:12800–12808CrossRefGoogle Scholar
  33. Keizers PHJ, Ubbink M (2011) Paramagnetic tagging for protein structure and dynamics analysis. Prog Nucl Magn Reson Spectrosc 58:88–96CrossRefGoogle Scholar
  34. Knight MJ, Felli IC, Pierattelli R, Emsley L, Pintacuda G (2013) Magic angle spinning NMR of paramagnetic proteins. Acc Chem Res 46:2108–2116CrossRefGoogle Scholar
  35. Leary SC, Winge DR, Cobine PA (2009) “Pulling the plug” on cellular copper: the role of mitochondria in copper export. Biochim Biophys Acta 1793:146–153CrossRefGoogle Scholar
  36. Li J, Ding S, Cowan JA (2013) Thermodynamic and structural analysis of human NFU conformational chemistry. Biochemistry 52(29):4904–4913CrossRefGoogle Scholar
  37. Lin IJ, Xia B, King DS, Machonkin TE, Westler WM, Markley JL (2009) Hyperfine-Shifted 13C and 15 N NMR signals from clostridium pasteurianum rubredoxin: extensive assignments and quantum chemical verification. J Am Chem Soc 131:15555–15563CrossRefGoogle Scholar
  38. Lutsenko S (2010) Human copper homeostasis: a network of interconnected pathways. Curr Opin Chem Biol 14:211–217CrossRefGoogle Scholar
  39. Lyons TA, Ratnaswamy G, Pochapsky TC (1996) Redox-dependent dynamics of putidaredoxin characterized by amide proton exchange. Protein Sci 5:627–639CrossRefGoogle Scholar
  40. Machonkin TE, Westler WM, Markley JL (2002) 13C-13C 2D NMR: a novel strategy for the study of paramagnetic proteins with slow electronic relaxation times. J Am Chem Soc 124:3204–3205CrossRefGoogle Scholar
  41. Machonkin TE, Westler WM, Markley JL (2004) Strategy for the study of paramagnetic proteins with slow electronic relaxation rates by NMR spectroscopy application to oxidized human [2Fe-2S] ferredoxin. J Am Chem Soc 126:5413–5426CrossRefGoogle Scholar
  42. Markley JL, Kim JH, Dai Z, Bothe JR, Cai K, Frederick RO, Tonelli M (2013) Metamorphic protein IscU alternates conformations in the course of its role as the scaffold protein for iron-sulfur cluster biosynthesis and delivery. FEBS Lett 587:1172–1179CrossRefGoogle Scholar
  43. Mo H, Pochapsky SS, Pochapsky TC (1999) A model for the solution structure of oxidized terpredoxin, a Fe2S2 ferredoxin from Pseudomonas. Biochemistry 38:5666CrossRefGoogle Scholar
  44. Otting G (2010) Protein NMR using paramagnetic ions. Annu Rev Biophys 39:387–405CrossRefGoogle Scholar
  45. Piccioli M, Poggi L (2002) Tailored HCCH-TOCSY experiment for resonance assignment in the proximity of a paramagnetic center. J Magn Reson 155:236–243ADSCrossRefGoogle Scholar
  46. Russo L, Maestre-Martinéz M, Wolff S, Becker S and Griesinger C (2013) Inter-domain dynamics explored by paramagnetic NMR. J Am Chem Soc 135:17111–17120Google Scholar
  47. Skjeldal L, Markley JL, Coghlan VM, Vickery LE (1991) 1H-NMR spectra of vertebrate (2Fe-2S) ferredoxins. Hyperfine resonances suggest different electron delocalization patterns from plant ferredoxins. Biochemistry 30:9078–9083CrossRefGoogle Scholar
  48. Ubbink M (2012) Dynamics in transient complexes of redox proteins. Biochem Soc Trans 40:415–418CrossRefGoogle Scholar
  49. Volkman BF, Wilkens SJ, Lee AL, Xia B, Westler WM, Berger R, Markley JL (1999) Redox-depedendent magnetic alignment of Clostridium pasteurianum rubredoxin: measurement of magnetic susceptibility anisotropy and prediction of pseudocontact shift contributions. J Am Chem Soc 121:4677–4683CrossRefGoogle Scholar
  50. Volkov AN, Ubbink M, Van Nuland NAJ (2010) Mapping the encounter state of a transient protein complex by PRE NMR spectroscopy. J Biomol NMR 48:225–236CrossRefGoogle Scholar
  51. Wilkens SJ, Xia B, Weinhold F, Markley JL, Westler WM (1998) NMR investigations of clostridium pasteurianum rubredoxin. Origin of hyperfine 1H, 2H, 13C and 15 N NM chemical shfits in iron-sulfur proteins as determined by comparison of experimental data with hybrid density functional calculations. J Am Chem Soc 120:4806–4814CrossRefGoogle Scholar
  52. Yagi H, Pilla KB, Maleckis A, Graham B, Huber T and Otting G (2013) Three-dimensional protein fold determination from backbone amide pseudocontact shifts generated by lanthanide tags at multiple sites. Structure 21:883–890Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Simone Ciofi-Baffoni
    • 1
  • Angelo Gallo
    • 1
  • Riccardo Muzzioli
    • 1
  • Mario Piccioli
    • 1
  1. 1.Magnetic Resonance Center and Department of ChemistryUniversity of FlorenceSesto FiorentinoItaly

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