Download the full-sized PDF
Permanent link (DOI): https://doi.org/10.7939/R3VT1H30T
This file is in the following communities:
|Chemistry, Department of|
This file is in the following collections:
|Journal Articles (Chemistry)|
Protein-glycolipid interactions studied in vitro using ESI-MS and nanodiscs: Insights into the mechanisms and energetics of binding Open Access
- Author or creator
Han, Ling Han
Kitova, Elena N.
Boraston, Alisdair B.
Klassen, John S.
- Additional contributors
- Type of item
- Journal Article (Published)
Electrospray ionization-mass spectrometry (ESI-MS) analysis combined with the use of nanodiscs (NDs) to solubilize glycolipids (GLs) has recently emerged as a promising analytical method for detecting protein–GL interactions in vitro and, when applied to libraries of GLs, ranking their affinities. However, there is uncertainty regarding the mechanism(s) of complex formation in solution and the extent to which the relative abundances of protein–glycolipid complexes observed by ESI-MS reflect the relative concentrations in solution. Here, we describe the results of a systematic ESI-MS study aimed at elucidating the processes that influence binding of water-soluble proteins to GLs incorporated into NDs and to exploit these insights to quantify the binding energetics. The interactions between the cholera toxin B subunit homopentamer (CTB5) and its native ganglioside receptor, β-D-Gal-(1 → 3)-β-D-GalNAc-(1 → 4)-[α-D-Neu5Ac-(2 → 3)]-β-D-Gal-(1 → 4)-β-D-Glc-ceramide (GM1), and between a recombinant fragment of family 51 carbohydrate-binding module (CBM), originating from S. pneumoniae, with a synthetic B type 2 neoglycolipid, α-D-Gal-(1 → 3)-[α-L-Fuc-(1 → 2)]-β-D-Gal-(1 → 4)-β-D-GlcNAc-1,2-di-O-dodecyl-sn-glycero (B2NGL) served as model protein–GL complexes for this study. The results of the ESI-MS measurements reveal that proteins bind reversibly to ND-bound GLs and that proteins possessing multiple ligand binding sites are able to interact with GLs originating from different NDs. Experimental evidence suggests that the diffusion of GLs between NDs is rapid and influences the nature of the protein–GL complexes that are detected. Using a newly developed ESI-MS assay, the proxy ligand method, the association constants for the CBM-B2NGL and CTB5–GM1 interactions were quantified and found to be slightly smaller than those for the corresponding oligosaccharides in solution.
- Date created
- License information
- © 2015 Han, L., Kitova, E. N., Li, J., Nikjah, S., Lin, H., Pluvinage, B., Boraston, A. B., & Klassen, J. S. This version of this article is open access and can be downloaded and shared. The original author(s) and source must be cited.
- Citation for previous publication
Han, L., Kitova, E. N., Li, J., Nikjah, S., Lin, H., Pluvinage, B., Boraston, A. B., & Klassen, J. S. (2015). Protein-glycolipid interactions studied in vitro using ESI-MS and nanodiscs: Insights into the mechanisms and energetics of binding. Analytical Chemistry, 87(9), 4888-4896. http://doi.org/10.1021/acs.analchem.5b00678
- Link to related item
- Date Uploaded
- Date Modified
- Audit Status
- Audits have not yet been run on this file.
File format: pdf (Portable Document Format)
Mime type: application/pdf
File size: 4649541
Last modified: 2017:06:24 01:06:32-06:00
Original checksum: 80add51254dd4aee641883c0c0f4e0ef
Well formed: true
File title: Table 2
File author: Igor Sinelnikov
Page count: 72
File language: en-US