Robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins
- Ulsan National Institute of Science and Technology, Republic of Korea
Abstract
Single-molecule tweezers, such as magnetic tweezers, are powerful tools for probing nm-scale structural changes in single membrane proteins under force. However, the weak molecular tethers used for the membrane protein studies have limited the observation of long-time, repetitive molecular transitions due to force-induced bond breakage. The prolonged observation of numerous transitions is critical in reliable characterizations of structural states, kinetics, and energy barrier properties. Here, we present a robust single-molecule tweezer method that uses dibenzocyclooctyne (DBCO) cycloaddition and traptavidin binding, enabling the estimation of the folding 'speed limit' of helical membrane proteins. This method is >100 times more stable than a conventional linkage system regarding the lifetime, allowing for the survival for ~12 h at 50 pN and ~1000 pulling cycle experiments. By using this method, we were able to observe numerous structural transitions of a designer single-chained transmembrane (TM) homodimer for 9 h at 12 pN, and reveal its folding pathway including the hidden dynamics of helix-coil transitions. We characterized the energy barrier heights and folding times for the transitions using a model-independent deconvolution method and the hidden Markov modeling (HMM) analysis, respectively. The Kramers rate framework yields a considerably low speed limit of 21 ms for a helical hairpin formation in lipid bilayers, compared to μs scale for soluble protein folding. This large discrepancy is likely due to the highly viscous nature of lipid membranes, retarding the helix-helix interactions. Our results offer a more valid guideline for relating the kinetics and free energies of membrane protein folding.
Data availability
All data and analysis codes that support the findings of this study are available in the manuscript, figure supplements, source data, and source code files.
Article and author information
Author details
Funding
National Research Foundation of Korea (2020R1C1C1003937)
- Duyoung Min
Ulsan National Institute of Science and Technology (1.190147.01)
- Duyoung Min
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2023, Kim 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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Robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins
eLife 12:e85882.
https://doi.org/10.7554/eLife.85882
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