1. Structural Biology and Molecular Biophysics
  2. Cell Biology
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Single-molecule force spectroscopy of protein-membrane interactions

  1. Lu Ma
  2. Yiying Cai
  3. Yanghui Li
  4. Junyi Jiao
  5. Zhenyong Wu
  6. Ben O'Shaughnessy
  7. Pietro De Camilli  Is a corresponding author
  8. Erdem Karatekin  Is a corresponding author
  9. Yongli Zhang  Is a corresponding author
  1. Yale University School of Medicine, United States
  2. Columbia University, United States
Research Article
  • Cited 30
  • Views 4,490
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Cite this article as: eLife 2017;6:e30493 doi: 10.7554/eLife.30493

Abstract

Many biological processes rely on protein-membrane interactions in the presence of mechanical forces, yet high resolution methods to quantify such interactions are lacking. Here, we describe a single-molecule force spectroscopy approach to quantify membrane binding of C2 domains in Synaptotagmin-1 (Syt1) and Extended Synaptotagmin-2 (E-Syt2). Syts and E-Syts bind the plasma membrane via multiple C2 domains, bridging the plasma membrane with synaptic vesicles or endoplasmic reticulum to regulate membrane fusion or lipid exchange, respectively. In our approach, single proteins attached to membranes supported on silica beads are pulled by optical tweezers, allowing membrane binding and unbinding transitions to be measured with unprecedented spatiotemporal resolution. C2 domains from either protein resisted unbinding forces of 2-7 pN and had binding energies of 4-14 kBT per C2 domain. Regulation by bilayer composition or Ca2+ recapitulated known properties of both proteins. The method can be widely applied to study protein-membrane interactions.

Article and author information

Author details

  1. Lu Ma

    Department of Cell Biology, Yale University School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Yiying Cai

    Department of Cell Biology, Yale University School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Yanghui Li

    Department of Cell Biology, Yale University School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Junyi Jiao

    Department of Cell Biology, Yale University School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Zhenyong Wu

    Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Ben O'Shaughnessy

    Department of Chemical Engineering, Columbia University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Pietro De Camilli

    Department of Cell Biology, Yale University School of Medicine, New Haven, United States
    For correspondence
    pietro.decamilli@yale.edu
    Competing interests
    The authors declare that no competing interests exist.
  8. Erdem Karatekin

    Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, United States
    For correspondence
    erdem.karatekin@yale.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5934-8728
  9. Yongli Zhang

    Department of Cell Biology, Yale University School of Medicine, New Haven, United States
    For correspondence
    yongli.zhang@yale.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7079-7973

Funding

National Institutes of Health (R01GM093341)

  • Yongli Zhang

Brain Research Foundation

  • Yongli Zhang

Kavli Foundation

  • Pietro De Camilli
  • Erdem Karatekin

Raymond and Beverly Sackler Institute for Biological, Physical and Engineering Sciences, Yale University (Seed Grant)

  • Erdem Karatekin
  • Yongli Zhang

National Institutes of Health (R01NS36251)

  • Pietro De Camilli

National Institutes of Health (DA018343)

  • Pietro De Camilli

National Institutes of Health (R01GM108954)

  • Erdem Karatekin

National Institutes of Health (R01GM114513)

  • Erdem Karatekin

National Institutes of Health (R01GM120193)

  • Yongli Zhang

Kavli Foundation (Kavli Neuroscience Scholar Award)

  • Erdem Karatekin

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

Reviewing Editor

  1. Taekjip Ha, Johns Hopkins University School of Medicine, United States

Publication history

  1. Received: July 17, 2017
  2. Accepted: October 29, 2017
  3. Accepted Manuscript published: October 30, 2017 (version 1)
  4. Version of Record published: November 16, 2017 (version 2)

Copyright

© 2017, Ma 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.

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