Single-molecule force spectroscopy of protein-membrane interactions
- Yale University School of Medicine, United States
- Columbia University, United States
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.
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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.
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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Single-molecule force spectroscopy of protein-membrane interactions
eLife 6:e30493.
https://doi.org/10.7554/eLife.30493
