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Quantitative time-resolved measurement of membrane protein–ligand interactions using microcantilever array sensors

Nature Nanotechnology volume 4, pages 179–185 (2009)Cite this article

Abstract

Membrane proteins are central to many biological processes, and the interactions between transmembrane protein receptors and their ligands are of fundamental importance in medical research. However, measuring and characterizing these interactions is challenging. Here we report that sensors based on arrays of resonating microcantilevers can measure such interactions under physiological conditions. A protein receptor—the FhuA receptor of Escherichia coli—is crystallized in liposomes, and the proteoliposomes then immobilized on the chemically activated gold-coated surface of the sensor by ink-jet spotting in a humid environment, thus keeping the receptors functional. Quantitative mass-binding measurements of the bacterial virus T5 at subpicomolar concentrations are performed. These experiments demonstrate the potential of resonating microcantilevers for the specific, label-free and time-resolved detection of membrane protein–ligand interactions in a micro-array format.

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Figure 1: Preparation of FhuA receptors for cantilever functionalization.
Figure 2: Humidity chamber for functionalization of cantilevers with membrane proteins.
Figure 3: Functionalization of the upper cantilever surface with FhuA–proteoliposomes.
Figure 4: Schematic of the dynamic-mode measurement setup.
Figure 5: Docking of T5 phages to FhuA-functionalized cantilevers.

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Acknowledgements

We thank A. Engel for the use of facilities for protein reconstitution at the Biozentrum, University of Basel, and M. Chami for fruitful discussions. We thank D. Mathys, M. Düggelin and M. Dürrenberger for their help with the SEM analysis. Financial and general support is acknowledged from Swiss National Science Foundation (NCCR Nanoscale Science), the Commission for Technology and Innovation (CTI) (TOPNANO21), the European Learning and Teaching Mobility Regio Network, the G.H. Endress Foundation, the Novartis Foundation and the Science Foundation Ireland CSET programme. M.K.G. thanks the Swiss National Science Foundation and the Novartis Foundation for a research fellowship.

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Author notes

  1. Thomas Braun and Murali Krishna Ghatkesar: These authors contributed equally to this work

Authors and Affiliations

  1. School of Physics and Centre for Research on Adaptive Nanostructures and Nanodevices (CRANN), Naughton Institute, Trinity College Dublin, Dublin 2, Ireland

    Thomas Braun, Wilfried Grange & Martin Hegner

  2. California Institute of Technology, Pasadena, 91107, California, USA

    Murali Krishna Ghatkesar

  3. National Centre of Competence for Research in Nanoscience, Institute of Physics, University of Basel, Klingelbergstrasse 82, Basel, 4056, Switzerland

    Natalija Backmann, Hans-Peter Lang, Alex Bietsch & Christoph Gerber

  4. Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, UMR CNRS 8619, Université, Paris-Sud XI, 91405, Orsay, France

    Pascale Boulanger & Lucienne Letellier

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Contributions

T.B. and M.H. conceived and designed the experiments; T.B. and M.K.G. performed the experiments; T.B., M.K.G., W.G. and M.H. analysed the data; P.B. and L.L. contributed expertise in membrane proteins and phages; A.B. and T.B. were responsible for the ink-jet spotting of the membrane proteins; T.B., N.B., W.G., H.P.L. and M.H. co-wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Martin Hegner.

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Braun, T., Ghatkesar, M., Backmann, N. et al. Quantitative time-resolved measurement of membrane protein–ligand interactions using microcantilever array sensors. Nature Nanotech 4, 179–185 (2009). https://doi.org/10.1038/nnano.2008.398

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