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Robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins

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 transitio...

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Autores principales: Kim, Seoyoon, Lee, Daehyo, Wijesinghe, WC Bhashini, Min, Duyoung
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10259496/
https://www.ncbi.nlm.nih.gov/pubmed/37249211
http://dx.doi.org/10.7554/eLife.85882
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author Kim, Seoyoon
Lee, Daehyo
Wijesinghe, WC Bhashini
Min, Duyoung
author_facet Kim, Seoyoon
Lee, Daehyo
Wijesinghe, WC Bhashini
Min, Duyoung
author_sort Kim, Seoyoon
collection PubMed
description 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 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 hr 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 homodimer for 9 hr 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 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.
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spelling pubmed-102594962023-06-13 Robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins Kim, Seoyoon Lee, Daehyo Wijesinghe, WC Bhashini Min, Duyoung eLife Structural Biology and Molecular Biophysics 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 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 hr 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 homodimer for 9 hr 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 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. eLife Sciences Publications, Ltd 2023-05-30 /pmc/articles/PMC10259496/ /pubmed/37249211 http://dx.doi.org/10.7554/eLife.85882 Text en © 2023, Kim, Lee et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Structural Biology and Molecular Biophysics
Kim, Seoyoon
Lee, Daehyo
Wijesinghe, WC Bhashini
Min, Duyoung
Robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins
title Robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins
title_full Robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins
title_fullStr Robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins
title_full_unstemmed Robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins
title_short Robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins
title_sort robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins
topic Structural Biology and Molecular Biophysics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10259496/
https://www.ncbi.nlm.nih.gov/pubmed/37249211
http://dx.doi.org/10.7554/eLife.85882
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