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Label-free quantification of membrane-ligand interactions using backscattering interferometry

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Abstract

Although membrane proteins are ubiquitous within all living organisms and represent the majority of drug targets, a general method for direct, label-free measurement of ligand binding to native membranes has not been reported. Here we show that backscattering interferometry (BSI) can accurately quantify ligand-receptor binding affinities in a variety of membrane environments. By detecting minute changes in the refractive index of a solution, BSI allows binding interactions of proteins with their ligands to be measured at picomolar concentrations. Equilibrium binding constants in the micromolar to picomolar range were obtained for small- and large-molecule interactions in both synthetic and cell-derived membranes without the use of labels or supporting substrates. The simple and low-cost hardware, high sensitivity and label-free nature of BSI should make it readily applicable to the study of many membrane-associated proteins of biochemical and pharmacological interest.

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Figure 1: Experimental components.
Figure 2: Representative plots of BSI signal versus ligand concentration for the determination of binding constants for the following pairs of molecules (membrane-bound species + ligand).

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Acknowledgements

This work was supported by the US National Institutes of Health (RO1 EB003537-01A2; U01 MH069062; and the Joint Center for Innovative Membrane Protein Technologies, Roadmap Grant GM073197) and The Skaggs Institute for Chemical Biology. We are grateful to J. Garfunkel and D. Boger of The Scripps Research Institute for samples of the FAAH inhibitors, R. Stevens of The Scripps Research Institute for samples of the FAAH protein, M. Hanes and T. Handel of the University of California, San Diego for the samples of the CXCL12 chemokine and K. Kaupmann of Novartis for the GABAB-transfected CHO cell line.

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Authors and Affiliations

  1. Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA

    Michael M Baksh & M G Finn

  2. Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA

    Mauro Mileni

  3. Department of Chemistry, Vanderbilt Institute for Chemical Biology, Vanderbilt University, Nashville, Tennessee, USA

    Amanda K Kussrow & Darryl J Bornhop

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  1. Michael M Baksh
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  2. Amanda K Kussrow
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  3. Mauro Mileni
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  4. M G Finn
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  5. Darryl J Bornhop
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Contributions

M.M.B. developed methods for sample preparation, prepared samples for analysis and processed the raw BSI data; A.K.K. performed BSI measurements and processed the raw data; M.M. prepared the FAAH protein; M.M.B., A.K.K., M.G.F. and D.J.B. designed the project and wrote the manuscript.

Corresponding authors

Correspondence to M G Finn or Darryl J Bornhop.

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Competing interests

D.J.B. has a financial interest in a company that is commercializing BSI. The other authors declare that they have no competing financial interests.

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Supplementary Text and Figures

Supplementary Methods and Supplementary Figs. 1–3 (PDF 854 kb)

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Baksh, M., Kussrow, A., Mileni, M. et al. Label-free quantification of membrane-ligand interactions using backscattering interferometry. Nat Biotechnol 29, 357–360 (2011). https://doi.org/10.1038/nbt.1790

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