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Use of 19F NMR spectroscopy to screen chemical libraries for ligands that bind to proteins
Umeå University, Faculty of Science and Technology, Chemistry.
Umeå University, Faculty of Science and Technology, Chemistry.
Umeå University, Faculty of Science and Technology, Chemistry.
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2004 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, Vol. 2, no 5, 725-731 p.Article in journal (Refereed) Published
Abstract [en]

Identification of compounds from chemical libraries that bind to macromolecules by use of NMR spectroscopy has gained increasing importance during recent years. A simple methodology based on F-19 NMR spectroscopy for the screening of ligands that bind to proteins, which also provides qualitative information about relative binding strengths and the presence of multiple binding sites, is presented here. A library of fluorinated compounds was assembled and investigated for binding to the two bacterial chaperones PapD and FimC, and also to human serum albumin (HSA). It was found that library members which are bound to a target protein could be identified directly from line broadening and/or induced chemical shifts in a single, one-dimensional F-19 NMR spectrum. The results obtained for binding to PapD using F-19 NMR spectroscopy agreed well with independent studies based on surface plasmon resonance, providing support for the versatility and accuracy of the technique. When the library was titrated to a solution of PapD chemical shift and linewidth changes were observed with increasing ligand concentration, which indicated the presence of several binding sites on PapD and enabled the assessment of relative binding strengths for the different ligands. Screening by F-19 NMR spectroscopy should thus be a valuable addition to existing NMR techniques for evaluation of chemical libraries in bioorganic and medicinal chemistry.

Place, publisher, year, edition, pages
2004. Vol. 2, no 5, 725-731 p.
URN: urn:nbn:se:umu:diva-14086DOI: 10.1039/b313166aPubMedID: 14985813OAI: diva2:153757
Available from: 2007-05-22 Created: 2007-05-22 Last updated: 2009-09-24Bibliographically approved
In thesis
1. Studies of protein structure, dynamics and protein-ligand interactions using NMR spectroscopy
Open this publication in new window or tab >>Studies of protein structure, dynamics and protein-ligand interactions using NMR spectroscopy
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the first part of the thesis, protein-ligand interactions were investigated using the chaperone LcrH, from Yersinia as target protein. The structure of a peptide encompassing the amphipathic domain (residue 278-300) of the protein YopD from Yersinia was determined by NMR in 40% TFE. The structure of YopD278-300 is a well defined α-helix with a β-turn at the C-terminus of the helix capping the structure. This turn is crucial for the structure as peptides lacking the residues involved in the turn are unstructured. NMR relaxation indicates that the peptide is not monomeric. This is supported by intermolecular NOEs found from residue Phe280 to Ile288 and Val292 indicative of a multimeric structure with the helical structures oriented in an antiparallel manner with hydrophobic residues forming the oligomer. The interaction with the chaperone LcrH was confirmed by 1H relaxation experiments and induced chemical shift changes in the peptide

Protein-ligand interactions were investigated further in the second paper using a different approach. A wide range of substances were used in screening for affinity against the chaperones PapD and FimC from uropathogenic Escherichia coli using 1H relaxation NMR experiments, surface plasmon resonance and 19F NMR. Fluorine NMR proved to be advantageous as compared to proton NMR as it is straight forward to identify binding ligands due to the well resolved 19F NMR spectra. Several compounds were found to interact with PapD and FimC through induced line-broadening and chemical shift changes for the ligands. Data corroborate well with surface plasmon resonance and proton NMR experiments. However, our results indicate the substances used in this study to have poor specificity for PapD and FimC as the induced chemical shift is minor and hardly no competitive binding is observed.

Paper III and IV is an investigation of the structural features of the allergenic 2S albumin Ber e 1 from Brazil nut. Ber e 1 is a 2S albumin previously identified as the major allergen of Brazil nut. Recent studies have demonstrated that endogenous Brazil nut lipids are required for an immune response to occur in vivo. The structure was obtained from 3D heteronuclear NMR experiments followed by simulated annealing using the software ARIA. Interestingly, the common fold of the 2S albumin family, described as a right-handed super helix with the core composed of a helix bundle, is not found in Ber e 1. Instead the C-terminal region is participating in the formation of the core between helix 3, 4 and 5. The dynamic properties of Ber e 1 were investigated using 15N relaxation experiments and data was analyzed using the model-free approach. The analysis showed that a few residues in the loop between helix 2 and 3 experience decreased mobility, compared to the rest of the loop. This is consistent with NOE data as long range NOEs were found from the loop to the core region of the protein. The anchoring of this loop is a unique feature of Ber e 1, as it is not found in any other structures of 2S albumins. Chemical shift mapping of Ber e 1 upon the addition of lipid extract from Brazil nut identified 4 regions in the protein where chemical shift perturbations were detected. Interestingly, all four structural clusters align along a cleft in the structure formed by helix 1-3 on one side and helix 4-5 on the other. This cleft is big enough to encompass a lipid molecule. It is therefore tempting to speculate whether this cleft is the lipid binding epitope in Ber e 1.

Place, publisher, year, edition, pages
Umeå: Medicinsk kemi och biofysik, 2007. 40 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1147
19F NMR, 2S albumin, PapD, protein-ligand interaction, structure, TFE, Yersinia, YopD, allergy, amphipathic helix, Ber e 1, Brazil nut, dynamics, FimC, LcrH, NMR screening
National Category
urn:nbn:se:umu:diva-1472 (URN)978-91-7264-459-5 (ISBN)
Public defence
2008-01-18, KB3B1, KBC, Campus, Umeå, 10:00 (English)
Available from: 2008-01-07 Created: 2008-01-07 Last updated: 2009-09-24Bibliographically approved

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