Cargando…

Unraveling the Mechanics of a Repeat-Protein Nanospring: From Folding of Individual Repeats to Fluctuations of the Superhelix

[Image: see text] Tandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive mechanical properties that are proposed to direct their cellular functions. Here, we use single-molecule optical tweezers to study the folding of consensus-de...

Descripción completa

Detalles Bibliográficos
Autores principales: Synakewicz, Marie, Eapen, Rohan S., Perez-Riba, Albert, Rowling, Pamela J. E., Bauer, Daniela, Weißl, Andreas, Fischer, Gerhard, Hyvönen, Marko, Rief, Matthias, Itzhaki, Laura S., Stigler, Johannes
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2022
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8944806/
https://www.ncbi.nlm.nih.gov/pubmed/35258937
http://dx.doi.org/10.1021/acsnano.1c09162
_version_ 1784673808331833344
author Synakewicz, Marie
Eapen, Rohan S.
Perez-Riba, Albert
Rowling, Pamela J. E.
Bauer, Daniela
Weißl, Andreas
Fischer, Gerhard
Hyvönen, Marko
Rief, Matthias
Itzhaki, Laura S.
Stigler, Johannes
author_facet Synakewicz, Marie
Eapen, Rohan S.
Perez-Riba, Albert
Rowling, Pamela J. E.
Bauer, Daniela
Weißl, Andreas
Fischer, Gerhard
Hyvönen, Marko
Rief, Matthias
Itzhaki, Laura S.
Stigler, Johannes
author_sort Synakewicz, Marie
collection PubMed
description [Image: see text] Tandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive mechanical properties that are proposed to direct their cellular functions. Here, we use single-molecule optical tweezers to study the folding of consensus-designed tetratricopeptide repeats (CTPRs), superhelical arrays of short helix-turn-helix motifs. We find that CTPRs display a spring-like mechanical response in which individual repeats undergo rapid equilibrium fluctuations between partially folded and unfolded conformations. We rationalize the force response using Ising models and dissect the folding pathway of CTPRs under mechanical load, revealing how the repeat arrays form from the center toward both termini simultaneously. Most strikingly, we also directly observe the protein’s superhelical tertiary structure in the force signal. Using protein engineering, crystallography, and single-molecule experiments, we show that the superhelical geometry can be altered by carefully placed amino acid substitutions, and we examine how these sequence changes affect intrinsic repeat stability and inter-repeat coupling. Our findings provide the means to dissect and modulate repeat-protein stability and dynamics, which will be essential for researchers to understand the function of natural repeat proteins and to exploit artificial repeats proteins in nanotechnology and biomedical applications.
format Online
Article
Text
id pubmed-8944806
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher American Chemical Society
record_format MEDLINE/PubMed
spelling pubmed-89448062023-03-08 Unraveling the Mechanics of a Repeat-Protein Nanospring: From Folding of Individual Repeats to Fluctuations of the Superhelix Synakewicz, Marie Eapen, Rohan S. Perez-Riba, Albert Rowling, Pamela J. E. Bauer, Daniela Weißl, Andreas Fischer, Gerhard Hyvönen, Marko Rief, Matthias Itzhaki, Laura S. Stigler, Johannes ACS Nano [Image: see text] Tandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive mechanical properties that are proposed to direct their cellular functions. Here, we use single-molecule optical tweezers to study the folding of consensus-designed tetratricopeptide repeats (CTPRs), superhelical arrays of short helix-turn-helix motifs. We find that CTPRs display a spring-like mechanical response in which individual repeats undergo rapid equilibrium fluctuations between partially folded and unfolded conformations. We rationalize the force response using Ising models and dissect the folding pathway of CTPRs under mechanical load, revealing how the repeat arrays form from the center toward both termini simultaneously. Most strikingly, we also directly observe the protein’s superhelical tertiary structure in the force signal. Using protein engineering, crystallography, and single-molecule experiments, we show that the superhelical geometry can be altered by carefully placed amino acid substitutions, and we examine how these sequence changes affect intrinsic repeat stability and inter-repeat coupling. Our findings provide the means to dissect and modulate repeat-protein stability and dynamics, which will be essential for researchers to understand the function of natural repeat proteins and to exploit artificial repeats proteins in nanotechnology and biomedical applications. American Chemical Society 2022-03-08 2022-03-22 /pmc/articles/PMC8944806/ /pubmed/35258937 http://dx.doi.org/10.1021/acsnano.1c09162 Text en © 2022 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Synakewicz, Marie
Eapen, Rohan S.
Perez-Riba, Albert
Rowling, Pamela J. E.
Bauer, Daniela
Weißl, Andreas
Fischer, Gerhard
Hyvönen, Marko
Rief, Matthias
Itzhaki, Laura S.
Stigler, Johannes
Unraveling the Mechanics of a Repeat-Protein Nanospring: From Folding of Individual Repeats to Fluctuations of the Superhelix
title Unraveling the Mechanics of a Repeat-Protein Nanospring: From Folding of Individual Repeats to Fluctuations of the Superhelix
title_full Unraveling the Mechanics of a Repeat-Protein Nanospring: From Folding of Individual Repeats to Fluctuations of the Superhelix
title_fullStr Unraveling the Mechanics of a Repeat-Protein Nanospring: From Folding of Individual Repeats to Fluctuations of the Superhelix
title_full_unstemmed Unraveling the Mechanics of a Repeat-Protein Nanospring: From Folding of Individual Repeats to Fluctuations of the Superhelix
title_short Unraveling the Mechanics of a Repeat-Protein Nanospring: From Folding of Individual Repeats to Fluctuations of the Superhelix
title_sort unraveling the mechanics of a repeat-protein nanospring: from folding of individual repeats to fluctuations of the superhelix
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8944806/
https://www.ncbi.nlm.nih.gov/pubmed/35258937
http://dx.doi.org/10.1021/acsnano.1c09162
work_keys_str_mv AT synakewiczmarie unravelingthemechanicsofarepeatproteinnanospringfromfoldingofindividualrepeatstofluctuationsofthesuperhelix
AT eapenrohans unravelingthemechanicsofarepeatproteinnanospringfromfoldingofindividualrepeatstofluctuationsofthesuperhelix
AT perezribaalbert unravelingthemechanicsofarepeatproteinnanospringfromfoldingofindividualrepeatstofluctuationsofthesuperhelix
AT rowlingpamelaje unravelingthemechanicsofarepeatproteinnanospringfromfoldingofindividualrepeatstofluctuationsofthesuperhelix
AT bauerdaniela unravelingthemechanicsofarepeatproteinnanospringfromfoldingofindividualrepeatstofluctuationsofthesuperhelix
AT weißlandreas unravelingthemechanicsofarepeatproteinnanospringfromfoldingofindividualrepeatstofluctuationsofthesuperhelix
AT fischergerhard unravelingthemechanicsofarepeatproteinnanospringfromfoldingofindividualrepeatstofluctuationsofthesuperhelix
AT hyvonenmarko unravelingthemechanicsofarepeatproteinnanospringfromfoldingofindividualrepeatstofluctuationsofthesuperhelix
AT riefmatthias unravelingthemechanicsofarepeatproteinnanospringfromfoldingofindividualrepeatstofluctuationsofthesuperhelix
AT itzhakilauras unravelingthemechanicsofarepeatproteinnanospringfromfoldingofindividualrepeatstofluctuationsofthesuperhelix
AT stiglerjohannes unravelingthemechanicsofarepeatproteinnanospringfromfoldingofindividualrepeatstofluctuationsofthesuperhelix