Advertisement

Size Determination of Protein Oligomers/Aggregates Using Diffusion NMR Spectroscopy

  • Pancham S. Kandiyal
  • Ji Yoon Kim
  • Daniel L. Fortunati
  • K. H. MokEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2039)

Abstract

Diffusion-ordered spectroscopy (DOSY) is a widely used NMR technique for the identification of different chemical moieties/compounds contained in mixtures and has been successfully employed for the separation of small molecules based on hydrodynamic radii. Herein we show that DOSY can also be applied for the size determination of larger biomolecules such as proteins and protein oligomers/aggregates. Proof-of-principle is first shown with a cross-linked oligomeric protein mixture where the hydrodynamic volumes of each component are estimated and subsequently verified with size-exclusion HPLC and SDS polyacrylamide gel electrophoresis. We then determine the sizes of protein oligomers contained in a protein solution subjected under amyloid fibrillogenesis conditions. These studies aim to provide insight into the kinetics behind protein aggregation involved in amyloidosis as well as to determine the hydrodynamic radii of proteins within the mixture.

Key words

DOSY Hydrodynamic radii Protein Oligomers Aggregation Diffusion coefficient Pulsed-field gradient (PFG) Stokes–Einstein equation 

References

  1. 1.
    Cavanagh J, Fairbrother WJ, Palmer AG III, Rance M, Skelton NJ (2006) Protein NMR spectroscopy (2nd Ed): principles and practice. Academic, New York, NYGoogle Scholar
  2. 2.
    Li D, Keresztes I, Hopson R, Williard PG (2009) Characterization of reactive intermediates by multinuclear diffusion-ordered NMR spectroscopy (DOSY). Acc Chem Res 42(2):270–280CrossRefGoogle Scholar
  3. 3.
    Stejskal EO, Tanner JE (1965) Spin diffusion measurements: spin echoes in the presence of a time-dependent field gradient. J Chem Phys 42:288–292CrossRefGoogle Scholar
  4. 4.
    Dehner A, Kessler H (2005) Diffusion NMR spectroscopy: folding and aggregation of domains in p53. Chembiochem 6:1550–1565CrossRefGoogle Scholar
  5. 5.
    Pagès G, Gilard V, Martinob R, Malet-Martino M (2017) Pulsed-field gradient nuclear magnetic resonance measurements (PFG NMR) for diffusion ordered spectroscopy (DOSY) mapping. Analyst 142:3771CrossRefGoogle Scholar
  6. 6.
    Morris KF, Johnson CS Jr (1992) Diffusion-ordered two-dimensional nuclear magnetic resonance spectroscopy. JACS 114:3139–3141CrossRefGoogle Scholar
  7. 7.
    Barjat H, Morris GA, Smart SC, Swanson AG, Williams SCR (1995) High resolution diffusion ordered 2D spectroscopy (HR-DOSY)—a new tool for the analysis of complex mixtures. J Magn Reson B 108:170–171CrossRefGoogle Scholar
  8. 8.
    Balayssac S, Delsuc M-A, Gilard V, Prigent Y, Malet-Martino M (2009) Two-dimensional DOSY experiment with excitation sculpting water suppression for the analysis of natural and biological media. J Magn Reson 196:78–83CrossRefGoogle Scholar
  9. 9.
    Arora B, Tandon R, Attri P, Bhatia R (2017) Chemical crosslinking: role in protein and peptide science. Curr Protein Pept Sci 18(9):946–955CrossRefGoogle Scholar
  10. 10.
    Wu D, Chen A, Johnson CS Jr (1995) An improved diffusion-ordered spectroscopy experiment incorporating bipolar-gradient pulses. J Magn Reson A 115(2):260–226CrossRefGoogle Scholar
  11. 11.
    Jones JA, Wilkins DK, Smith LJ (1997) Characterization of protein unfolding by NMR diffusion measurement. J Biomol NMR 10:199–203CrossRefGoogle Scholar
  12. 12.
    Pelta MD, Barjat H, Morris GA, Davis AL, Hammond SJ (1998) Pulse sequences for high-resolution diffusion-ordered spectroscopy (HR-DOSY). Magn Reson Chem 36:706–714CrossRefGoogle Scholar
  13. 13.
    Shimizu A, Ikeguchi M, Sugai S (1994) Appropriateness of DSS and TSP as internal references for (1)H NMR studies of molten globule proteins in aqueous media. J Biomol NMR 4:859–862CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Pancham S. Kandiyal
    • 1
  • Ji Yoon Kim
    • 1
  • Daniel L. Fortunati
    • 1
  • K. H. Mok
    • 1
    Email author
  1. 1.Trinity Biomedical Sciences Institute (TBSI), School of Biochemistry & ImmunologyTrinity College DublinDublinIreland

Personalised recommendations