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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Cavanagh J, Fairbrother WJ, Palmer AG III, Rance M, Skelton NJ (2006) Protein NMR spectroscopy (2nd Ed): principles and practice. Academic, New York, NY
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):270–280
Stejskal EO, Tanner JE (1965) Spin diffusion measurements: spin echoes in the presence of a time-dependent field gradient. J Chem Phys 42:288–292
Dehner A, Kessler H (2005) Diffusion NMR spectroscopy: folding and aggregation of domains in p53. Chembiochem 6:1550–1565
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 142:3771
Morris KF, Johnson CS Jr (1992) Diffusion-ordered two-dimensional nuclear magnetic resonance spectroscopy. JACS 114:3139–3141
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 108:170–171
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 196:78–83
Arora B, Tandon R, Attri P, Bhatia R (2017) Chemical crosslinking: role in protein and peptide science. Curr Protein Pept Sci 18(9):946–955
Wu D, Chen A, Johnson CS Jr (1995) An improved diffusion-ordered spectroscopy experiment incorporating bipolar-gradient pulses. J Magn Reson A 115(2):260–226
Jones JA, Wilkins DK, Smith LJ (1997) Characterization of protein unfolding by NMR diffusion measurement. J Biomol NMR 10:199–203
Pelta MD, Barjat H, Morris GA, Davis AL, Hammond SJ (1998) Pulse sequences for high-resolution diffusion-ordered spectroscopy (HR-DOSY). Magn Reson Chem 36:706–714
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 4:859–862
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kandiyal, P.S., Kim, J.Y., Fortunati, D.L., Mok, K.H. (2019). Size Determination of Protein Oligomers/Aggregates Using Diffusion NMR Spectroscopy. In: McManus, J. (eds) Protein Self-Assembly. Methods in Molecular Biology, vol 2039. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9678-0_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9678-0_13
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9677-3
Online ISBN: 978-1-4939-9678-0
eBook Packages: Springer Protocols