1. Structural Biology and Molecular Biophysics
Download icon

A label-free approach to detect ligand binding to cell surface proteins in real time

  1. Verena Burtscher
  2. Matej Hotka
  3. Yang Li
  4. Michael Freissmuth
  5. Walter Sandtner  Is a corresponding author
  1. Medical University of Vienna, Austria
Tools and Resources
  • Cited 9
  • Views 2,996
  • Annotations
Cite this article as: eLife 2018;7:e34944 doi: 10.7554/eLife.34944

Abstract

Electrophysiological recordings allow for monitoring the operation of proteins with high temporal resolution down to the single molecule level. This technique has been exploited to track either ion flow arising from channel opening or the synchronized movement of charged residues and/or ions within the membrane electric field. Here, we describe a novel type of current by using the serotonin transporter (SERT) as a model. We examined transient currents elicited on rapid application of specific SERT inhibitors. Our analysis shows that these currents originate from ligand binding and not from a long-range conformational change. The Gouy-Chapman model predicts that adsorption of charged ligands to surface proteins must produce displacement currents and related apparent changes in membrane capacitance. Here we verified these predictions with SERT. Our observations demonstrate that ligand binding to a protein can be monitored in real time and in a label-free manner by recording the membrane capacitance.

Article and author information

Author details

  1. Verena Burtscher

    Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  2. Matej Hotka

    Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  3. Yang Li

    Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  4. Michael Freissmuth

    Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  5. Walter Sandtner

    Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria
    For correspondence
    walter.sandtner@meduniwien.ac.at
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3637-260X

Funding

Austrian Science Fund (P28090)

  • Walter Sandtner

Austrian Science Fund (F3510)

  • Michael Freissmuth

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Baron Chanda, University of Wisconsin-Madison, United States

Publication history

  1. Received: January 9, 2018
  2. Accepted: April 25, 2018
  3. Accepted Manuscript published: April 26, 2018 (version 1)
  4. Version of Record published: June 7, 2018 (version 2)
  5. Version of Record updated: March 6, 2019 (version 3)

Copyright

© 2018, Burtscher et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,996
    Page views
  • 453
    Downloads
  • 9
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

  1. Further reading

Further reading

    1. Structural Biology and Molecular Biophysics
    Rahul Chadda et al.
    Research Article

    Over two-thirds of integral membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl-/H+ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid a thinned-membrane defect formed by hydrophobic mismatch at their exposed dimerization interfaces. In this defect, lipids are strongly tilted and less densely packed than in the bulk, with a larger degree of entanglement between opposing leaflets and greater water penetration into the bilayer interior. Dimerization restores the membrane to a near-native state and therefore, appears to be driven by the larger free-energy cost of lipid solvation of the dissociated protomers. Supporting this theory, we demonstrate that addition of short-chain lipids strongly shifts the dimerization equilibrium towards the monomeric state, and show that the cause of this effect is that these lipids preferentially solvate the defect. Importantly, we show that this shift requires only minimal quantities of short-chain lipids, with no measurable impact on either the macroscopic physical state of the membrane or the protein's biological function. Based on these observations, we posit that free-energy differentials for local lipid solvation define membrane-protein association equilibria. With this, we argue that preferential lipid solvation is a plausible cellular mechanism for lipid regulation of oligomerization processes, as it can occur at low concentrations and does not require global changes in membrane properties.

    1. Structural Biology and Molecular Biophysics
    Achinta Sannigrahi et al.
    Research Article

    Aggregation of Cu-Zn superoxide dismutase (SOD1) is implicated in the motor neuron disease, ALS. Although more than 140 disease mutations of SOD1 are available, their stability or aggregation behaviors in membrane environment are not correlated with disease pathophysiology. Here, we use multiple mutational variants of SOD1 to show that the absence of Zn, and not Cu, significantly impacts membrane attachment of SOD1 through two loop regions facilitating aggregation driven by lipid induced conformational changes. These loop regions influence both the primary (through Cu intake) and the gain of function (through aggregation) of SOD1 presumably through a shared conformational landscape. Combining experimental and theoretical frameworks using representative ALS disease mutants, we develop a 'co-factor derived membrane association model' wherein mutational stress closer to the Zn (but not to the Cu) pocket is responsible for membrane association mediated toxic aggregation and survival time scale after ALS diagnosis.