Skip to main content
SpringerLink
Log in

The effects of cation adduction upon the conformation of three-helix bundle protein domains

  • Original Research
  • Published:
International Journal for Ion Mobility Spectrometry

Abstract

The ubiquitin-binding, three-helix bundle domains of the proteins ubiquilin 1 (UQ1) and hHR23A both exhibited remarkably high, but discrete, ammonium ion adduction when electrosprayed from aqueous ammonium acetate. The degree of adduction was highly charge state dependent with, unusually, the lowest charge states (+3 for UQ1 and +4 for hHR23A) showing almost no adducts and the highest charge states (+5 for UQ1 and +6 for hHR23A) exhibiting adduction with two ammonium cations as the most abundant form. As the charge state of protein ions produced by electrospray ionisation (ESI) is related to solvent-accessible surface area we inferred that the ammonium-carrying ions were of a more open conformation than their protonated counterparts. This was confirmed by ESI-travelling wave ion mobility spectrometry-mass spectrometry (TWIMS-MS), which showed that, although the purely protonated ions were compact, their equivalents bearing one or two ammonium adducts exhibited populations of significantly larger collisional cross section (CCS). We postulate that complexation with the ammonium cation may disrupt a key salt bridge(s) in the compact structure. A similar effect is observed with mono-sodium ion adduction, but this is diminished with each additional sodium ion in the complex to produce more compact structures.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Notes

  1. http://www.indiana.edu/~clemmer

  2. http://depts.washington.edu/bushlab

  3. http://www.vanderbilt.edu/AnS/Chemistry/groups/mcleanlab/ccs.html

References

  1. Kanu AB, Dwivedi P, Tam M, Matz L, Hill HH (2008) Ion mobility-mass spectrometry. J Mass Spectrom 43(1):1–22. doi:10.1002/jms.1383

    Article  CAS  Google Scholar 

  2. Dwivedi P, Wu C, Matz LM, Clowers BH, Siems WF, Hill HH (2006) Gas-phase chiral separations by ion mobility spectrometry. Anal Chem 78(24):8200–8206. doi:10.1021/ac0608772

    Article  CAS  Google Scholar 

  3. Tao L, McLean JR, McLean JA, Russell DH (2007) A collision cross-section database of singly-charged peptide ions. J Am Soc Mass Spectrom 18(7):1232–1238. doi:10.1016/j.jasms.2007.04.003

    Article  CAS  Google Scholar 

  4. Barran PE, Polfer NC, Campopiano DJ, Clarke DJ, Langridge-Smith PRR, Langley RJ, Govan JRW, Maxwell A, Dorin JR, Millar RP, Bowers MT (2005) Is it biologically relevant to measure the structures of small peptides in the gas-phase? Int J Mass Spectrom 240(3):273–284. doi:10.1016/j.ijms.2004.09.013

    Article  CAS  Google Scholar 

  5. Clemmer DE, Hudgins RR, Jarrold MF (1995) Naked protein conformations–cytochrome-C in the gas-phase. J Am Chem Soc 117(40):10141–10142. doi:10.1021/ja00145a037

    Article  CAS  Google Scholar 

  6. Clemmer DE, Jarrold MF (1997) Ion mobility measurements and their applications to clusters and biomolecules. J Mass Spectrom 32(6):577–592. doi:10.1002/(sici)1096-9888(199706)32:6<577::aid-jms530>3.3.co;2-w

    Article  CAS  Google Scholar 

  7. McLean JA, Ruotolo BT, Gillig KJ, Russell DH (2005) Ion mobility-mass spectrometry: a new paradigm for proteomics. Int J Mass Spectrom 240(3):301–315. doi:10.1016/j.ijms.2004.10.003

    Article  CAS  Google Scholar 

  8. Wyttenbach T, Bowers MT (2003) Gas-phase conformations: the ion mobility/ion chromatography method. Mod Mass Spectrom 225:207–232. doi:10.1007/b10470

    Article  CAS  Google Scholar 

  9. Wyttenbach T, vonHelden G, Bowers MT (1996) Gas-phase conformation of biological molecules: Bradykinin. J Am Chem Soc 118(35):8355–8364. doi:10.1021/ja9535928

    Article  CAS  Google Scholar 

  10. Ruotolo BT, Benesch JLP, Sandercock AM, Hyung SJ, Robinson CV (2008) Ion mobility-mass spectrometry analysis of large protein complexes. Nat Protoc 3(7):1139–1152. doi:10.1038/nprot.2008.78

    Article  CAS  Google Scholar 

  11. Uetrecht C, Rose RJ, van Duijn E, Lorenzen K, Heck AJR (2010) Ion mobility mass spectrometry of proteins and protein assemblies. Chem Soc Rev 39(5):1633–1655. doi:10.1039/b914002f

    Article  CAS  Google Scholar 

  12. Breuker K, McLafferty FW (2008) Stepwise evolution of protein native structure with electrospray into the gas phase, 10(−12) to 10(2) S. Proc Natl Acad Sci U S A 105(47):18145–18152. doi:10.1073/pnas.0807005105

    Article  CAS  Google Scholar 

  13. Giles K, Pringle SD, Worthington KR, Little D, Wildgoose JL, Bateman RH (2004) Applications of a travelling wave-based radio-frequency only stacked ring ion guide. Rapid Commun Mass Spectrom 18(20):2401–2414. doi:10.1002/rcm.1641

    Article  CAS  Google Scholar 

  14. Pringle SD, Giles K, Wildgoose JL, Williams JP, Slade SE, Thalassinos K, Bateman RH, Bowers MT, Scrivens JH (2007) An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/travelling wave IMS/oa-ToF instrument. Int J Mass Spectrom 261(1):1–12. doi:10.1016/j.ijms.2006.07.021

    Article  CAS  Google Scholar 

  15. Duijn EV, Barendregt A, Synowsky S, Versluis C, Heck AJR (2009) Chaperonin complexes monitored by ion mobility mass spectrometry. J Am Chem Soc 131(4):1452–1459. doi:10.1021/ja8055134

    Article  Google Scholar 

  16. Smith DP, Knapman TW, Campuzano I, Malham RW, Berryman JT, Radford SE, Ashcroft AE (2009) Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies. Eur J Mass Spectrom 15(2):113–130. doi:10.1255/ejms.947

    Article  CAS  Google Scholar 

  17. Hilton GR, Thalassinos K, Grabenauer M, Sanghera N, Slade SE, Wyttenbach T, Robinson PJ, Pinheiro TJT, Bowers MT, Scrivens JH (2010) Structural analysis of prion proteins by means of drift cell and traveling wave ion mobility mass spectrometry. J Am Soc Mass Spectrom 21(5):845–854. doi:10.1016/j.jasms.2010.01.017

    Article  CAS  Google Scholar 

  18. Smith DP, Radford SE, Ashcroft AE (2010) Elongated oligomers in beta(2)-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry. Proc Natl Acad Sci U S A 107(15):6794–6798. doi:10.1073/pnas.0913046107

    Article  CAS  Google Scholar 

  19. Wyttenbach T, Grabenauer M, Thalassinos K, Scrivens JH, Bowers MT (2010) The effect of calcium ions and peptide ligands on the relative stabilities of the calmodulin dumbbell and compact structures. J Phys Chem B 114(1):437–447. doi:10.1021/jp906242m

    Article  CAS  Google Scholar 

  20. Ruotolo BT, Giles K, Campuzano I, Sandercock AM, Bateman RH, Robinson CV (2005) Evidence for macromolecular protein rings in the absence of bulk water. Science 310(5754):1658–1661. doi:10.1126/science.1120177

    Article  CAS  Google Scholar 

  21. Jenner M, Ellis J, Huang WC, Raven EL, Roberts GCK, Oldham NJ (2011) Detection of a protein conformational equilibrium by electrospray ionisation-ion mobility-mass spectrometry. Angew Chem Int Ed 50(36):8291–8294. doi:10.1002/anie.201101077

    Article  CAS  Google Scholar 

  22. Bleiholder C, Dupuis NF, Wyttenbach T, Bowers MT (2011) Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to beta-sheet in amyloid fibril formation. Nat Chem 3(2):172–177. doi:10.1038/nchem.945

    Article  CAS  Google Scholar 

  23. De Cecco M, Seo ES, Clarke DJ, McCullough BJ, Taylor K, Macmillan D, Dorin JR, Campopiano DJ, Barran PE (2010) Conformational preferences of linear beta-defensins are revealed by ion mobility-mass spectrometry. J Phys Chem B 114(6):2312–2318. doi:10.1021/jp9111662

    Article  Google Scholar 

  24. Loo JA, Berhane B, Kaddis CS, Wooding KM, Xie YM, Kaufman SL, Chernushevich IV (2005) Electrospray ionization mass spectrometry and ion mobility analysis of the 20S proteasome complex. J Am Soc Mass Spectrom 16(7):998–1008. doi:10.1016/j.jasms.2005.02.017

    Article  CAS  Google Scholar 

  25. Halgand F, Laprevote O (2001) Mean charge state and charge state distribution of proteins as structural probes. An electrospray ionisation mass spectrometry study of lysozyme and ribonuclease A. Eur J Mass Spectrom 7(6):433–439. doi:10.1255/ejms.458

    Article  CAS  Google Scholar 

  26. Lemaire D, Marie G, Serani L, Laprevote O (2001) Stabilization of gas-phase noncovalent macromolecular complexes in electrospray mass spectrometry using aqueous triethylammonium bicarbonate buffer. Anal Chem 73(8):1699–1706. doi:10.1021/ac001276s

    Article  CAS  Google Scholar 

  27. Pagel K, Hyung SJ, Ruotolo BT, Robinson CV (2010) Alternate dissociation pathways identified in charge-reduced protein complex ions. Anal Chem 82(12):5363–5372. doi:10.1021/ac101121r

    Article  CAS  Google Scholar 

  28. Touboul D, Jecklin MC, Zenobi R (2008) Investigation of deprotonation reactions on globular and denatured proteins at atmospheric pressure by ESSI-MS. J Am Soc Mass Spectrom 19(4):455–466. doi:10.1016/j.jasms.2007.12.011

    Article  CAS  Google Scholar 

  29. Winger BE, Lightwahl KJ, Smith RD (1992) Gas-phase proton-transfer reactions involving multiply charged cytochrome-C ions and water under thermal conditions. J Am Soc Mass Spectrom 3(6):624–630

    Article  CAS  Google Scholar 

  30. Loo RRO, Udseth HR, Smith RD (1991) Evidence of charge inversion in the reaction of singly charged anions with multiply charged macroions. J Phys Chem 95(17):6412–6415. doi:10.1021/j100170a006

    Article  CAS  Google Scholar 

  31. Zhao Q, Schieffer GM, Soyk MW, Anderson TJ, Houk RS, Badman ER (2010) Effects of ion/ion proton transfer reactions on conformation of gas-phase cytochrome C ions. J Am Soc Mass Spectrom 21(7):1208–1217. doi:10.1016/j.jasms.2010.03.032

    Article  CAS  Google Scholar 

  32. Zhao Q, Soyk MW, Schieffer GM, Fuhrer K, Gonin MM, Houk RS, Badman ER (2009) An ion trap-ion mobility-time of flight mass spectrometer with three ion sources for ion/ion reactions. J Am Soc Mass Spectrom 20(8):1549–1561. doi:10.1016/j.jasms.2009.04.014

    Article  CAS  Google Scholar 

  33. Hopper JTS, Sokratous K, Oldham NJ (2012) Charge state and adduct reduction in electrospray ionization-mass spectrometry using solvent vapor exposure. Anal Biochem 421(2):788–790. doi:10.1016/j.ab.2011.10.034

    Article  CAS  Google Scholar 

  34. Sokratous K, Roach LV, Channing D, Strachan J, Long J, Searle MS, Layfield R, Oldham NJ (2012) Probing affinity and ubiquitin linkage selectivity of ubiquitin-binding domains using mass spectrometry. J Am Chem Soc 134(14):6416–6424. doi:10.1021/ja300749d

    Article  CAS  Google Scholar 

  35. Kaltashov IA, Abzalimov RR (2008) Do ionic charges in ESI MS provide useful information on macromolecular structure? J Am Soc Mass Spectrom 19(9):1239–1246. doi:10.1016/j.jasms.2008.05.018

    Article  CAS  Google Scholar 

  36. Benesch JLP (2009) Collisional activation of protein complexes: picking up the pieces. J Am Soc Mass Spectrom 20(3):341–348. doi:10.1016/j.jasms.2008.11.014

    Article  CAS  Google Scholar 

  37. Hopper JTS, Oldham NJ (2011) Alkali metal cation-induced destabilization of gas-phase protein-ligand complexes: consequences and prevention. Anal Chem 83(19):7472–7479. doi:10.1021/ac201686f

    Article  CAS  Google Scholar 

  38. Rozman M, Gaskell SJ (2010) Non-covalent interactions of alkali metal cations with singly charged tryptic peptides. J Mass Spectrom 45(12):1409–1415. doi:10.1002/jms.1856

    Article  CAS  Google Scholar 

  39. Wu C, Klasmeier J, Hill HH (1999) Atmospheric pressure ion mobility spectrometry of protonated and sodiated peptides. Rapid Commun Mass Spectrom 13(12):1138–1142. doi:10.1002/(sici)1097-0231(19990630)13:12<1138::aid-rcm625>3.0.co;2-8

    Article  CAS  Google Scholar 

  40. Counterman AE, Valentine SJ, Srebalus CA, Henderson SC, Hoaglund CS, Clemmer DE (1998) High-order structure and dissociation of gaseous peptide aggregates that are hidden in mass spectra. J Am Soc Mass Spectrom 9(8):743–759. doi:10.1016/s1044-0305(98)00052-x

    Article  CAS  Google Scholar 

  41. Salbo R, Bush MF, Naver H, Campuzano I, Robinson CV, Pettersson I, Jorgensen TJD, Haselmann KF (2012) Traveling-wave ion mobility mass spectrometry of protein complexes: accurate calibrated collision cross-sections of human insulin oligomers. Rapid Commun Mass Spectrom 26(10):1181–1193. doi:10.1002/rcm.6211

    Article  CAS  Google Scholar 

  42. Mesleh MF, Hunter JM, Shvartsburg AA, Schatz GC, Jarrold MF (1996) Structural information from ion mobility measurements: effects of the long-range potential. J Phys Chem 100(40):16082–16086. doi:10.1021/jp961623v

    Article  CAS  Google Scholar 

  43. Shvartsburg AA, Jarrold MF (1996) An exact hard-spheres scattering model for the mobilities of polyatomic ions. Chem Phys Lett 261(1–2):86–91. doi:10.1016/0009-2614(96)00941-4

    Article  CAS  Google Scholar 

  44. Williams JP, Lough JA, Campuzano I, Richardson K, Sadler PJ (2009) Use of ion mobility mass spectrometry and a collision cross-section algorithm to study an organometallic ruthenium anticancer complex and its adducts with a DNA oligonucleotide. Rapid Commun Mass Spectrom 23(22):3563–3569. doi:10.1002/rcm.4285

    Article  CAS  Google Scholar 

  45. Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D: Biol Crystallogr 66:12–21. doi:10.1107/s0907444909042073

    Article  Google Scholar 

  46. Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral GJ, Wang X, Murray LW, Arendall WB, Snoeyink J, Richardson JS, Richardson DC (2007) MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res 35:W375–W383. doi:10.1093/nar/gkm216

    Article  Google Scholar 

  47. Bertini I, Case DA, Ferella L, Giachetti A, Rosato A (2011) A grid-enabled web portal for NMR structure refinement with AMBER. Bioinformatics 27(17):2384–2390. doi:10.1093/bioinformatics/btr415

    Article  CAS  Google Scholar 

  48. Case DA, Cheatham TE, Darden T, Gohlke H, Luo R, Merz KM, Onufriev A, Simmerling C, Wang B, Woods RJ (2005) The Amber biomolecular simulation programs. J Comput Chem 26(16):1668–1688. doi:10.1002/jcc.20290

    Article  CAS  Google Scholar 

  49. Zhang D, Raasi S, Fushman D (2008) Affinity makes the difference: nonselective interaction of the UBA domain of ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains. J Mol Biol 377(1):162–180. doi:10.1016/j.jmb.2007.12.029

    Article  CAS  Google Scholar 

  50. Benesch JLP, Ruotolo BT (2011) Mass spectrometry: come of age for structural and dynamical biology. Curr Opin Struct Biol 21(5):641–649. doi:10.1016/j.sbi.2011.08.002

    Article  CAS  Google Scholar 

  51. Julian RR, Beauchamp JL (2002) The unusually high proton affinity of aza-18-crown-6 ether: implications for the molecular recognition of lysine in peptides by lariat crown ethers. J Am Soc Mass Spectrom 13(5):493–498. doi:10.1016/s1044-0305(02)00368-9

    Article  CAS  Google Scholar 

  52. Lomeli SH, Peng IX, Yin S, Loo RRO, Loo JA (2010) New reagents for increasing ESI multiple charging of proteins and protein complexes. J Am Soc Mass Spectrom 21(1):127–131. doi:10.1016/j.jasms.2009.09.014

    Article  CAS  Google Scholar 

  53. Verkerk UH, Kebarle P (2005) Ion-ion and ion-molecule reactions at the surface of proteins produced by nanospray. Information on the number of acidic residues and control of the number of ionized acidic and basic residues. J Am Soc Mass Spectrom 16(8):1325–1341. doi:10.1016/j.jasms.2005.03.018

    Article  CAS  Google Scholar 

  54. Steinberg MZ, Elber R, McLafferty FW, Gerber RB, Breuker K (2008) Early structural evolution of native cytochrome C after solvent removal. ChemBioChem 9(15):2417–2423. doi:10.1002/cbic.200800167

    Article  CAS  Google Scholar 

  55. Wyttenbach T, Bushnell JE, Bowers MT (1998) Salt bridge structures in the absence of solvent? the case for the oligoglycines. J Am Chem Soc 120(20). doi:10.1021/ja9801238

  56. Felitsyn N, Peschke M, Kebarle P (2002) Origin and number of charges observed on multiply-protonated native proteins produced by ESI. Int J Mass Spectrom 219(1):39–62. doi:10.1016/s1387-3806(02)00588-2

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to the Biotechnology and Biological Sciences Research Council (Grants BB/FQ19297/1 and BB/1006052/1) and the University of Nottingham for funding

Author information

Authors and Affiliations

  1. School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, UK

    Kleitos Sokratous & Neil J. Oldham

  2. School of Biomedical Sciences, University of Nottingham, Medical School, Queens Medical Centre, Nottingham, NG7 2UH, UK

    Robert Layfield

Authors
  1. Kleitos Sokratous
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert Layfield
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Neil J. Oldham
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Neil J. Oldham.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOCX 1151 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sokratous, K., Layfield, R. & Oldham, N.J. The effects of cation adduction upon the conformation of three-helix bundle protein domains. Int. J. Ion Mobil. Spec. 16, 19–27 (2013). https://doi.org/10.1007/s12127-012-0114-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12127-012-0114-0

Keywords

Search

Navigation