Visualizing conformational dynamics of proteins in solution and at the cell membrane
Abstract
Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.
Data availability
Data generated or analysed during this study are included in the manuscript and supporting files.
Article and author information
Author details
Funding
National Eye Institute (R01EY017564)
- Sharona E Gordon
National Institute of Mental Health (R01MH102378)
- William N Zagotta
National Institute of General Medical Sciences (R01GM100718)
- William N Zagotta
National Eye Institute (R01EY010329)
- William N Zagotta
National Institute of General Medical Sciences (R01GM125351)
- William N Zagotta
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Leon D Islas, Universidad Nacional Autónoma de México, Mexico
Version history
- Received: April 4, 2018
- Accepted: June 5, 2018
- Accepted Manuscript published: June 20, 2018 (version 1)
- Version of Record published: July 23, 2018 (version 2)
- Version of Record updated: November 30, 2018 (version 3)
Copyright
© 2018, Gordon 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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Further reading
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