Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors

Changes in microscopic viscosity and macromolecular crowding accompany the transition of proteins from their monomeric forms into highly organised fibrillar states. Previously, we have demonstrated that viscosity sensitive fluorophores termed ‘molecular rotors’, when freely mixed with monomers of in...

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Detalles Bibliográficos
Autores: Kubánková, Markéta, López Duarte, Ismael, Bull, James A., Vadukul, Devkee M., Serpell, Louise C., Victor, Marie de Saint, Stride, Eleanor, Kuimova, Marina K.
Tipo de recurso: artículo
Fecha de publicación:2017
País:España
Institución:Universidad Complutense de Madrid (UCM)
Repositorio:Docta Complutense
Idioma:inglés
OAI Identifier:oai:docta.ucm.es:20.500.14352/114924
Acceso en línea:https://hdl.handle.net/20.500.14352/114924
Access Level:acceso abierto
Palabra clave:615.31
615:54
Amyloid aggregation
Live cells
Microviscosity
Fluorescence lifetime imaging microscopy (FLIM)
Sensors for Ab(1-42) aggregates
Química farmaceútica
23 Química
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oai_identifier_str oai:docta.ucm.es:20.500.14352/114924
network_acronym_str ES
network_name_str España
repository_id_str
spelling Probing supramolecular protein assembly using covalently attached fluorescent molecular rotorsKubánková, MarkétaLópez Duarte, IsmaelBull, James A.Vadukul, Devkee M.Serpell, Louise C.Victor, Marie de SaintStride, EleanorKuimova, Marina K.615.31615:54Amyloid aggregationLive cellsMicroviscosityFluorescence lifetime imaging microscopy (FLIM)Sensors for Ab(1-42) aggregatesQuímica farmaceútica23 QuímicaChanges in microscopic viscosity and macromolecular crowding accompany the transition of proteins from their monomeric forms into highly organised fibrillar states. Previously, we have demonstrated that viscosity sensitive fluorophores termed ‘molecular rotors’, when freely mixed with monomers of interest, are able to report on changes in microrheology accompanying amyloid formation, and measured an increase in rigidity of approximately three orders of magnitude during aggregation of lysozyme and insulin. Here we extend this strategy by covalently attaching molecular rotors to several proteins capable of assembly into fibrils, namely lysozyme, fibrinogen and amyloid-b peptide (Ab(1e42)). We demonstrate that upon covalent attachment the molecular rotors can successfully probe supramolecular assembly in vitro. Importantly, our new strategy has wider applications in cellulo and in vivo, since covalently attached molecular rotors can be successfully delivered in situ and will colocalise with the aggregating protein, for example inside live cells. This important advantage allowed us to follow the microscopic viscosity changes accompanying blood clotting and during Ab(1e42) aggregation in live SHSY5Y cells. Our results demonstrate that covalently attached molecular rotors are a widely applicable tool to study supramolecular protein assembly and can reveal microrheological features of aggregating protein systems both in vitro and in cellulo not observable through classical fluorescent probes operating in light switch mode.ElsevierUniversidad Complutense de Madrid20172017-09-0120172017-09-01journal articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/20.500.14352/114924reponame:Docta Complutenseinstname:Universidad Complutense de Madrid (UCM)Inglésengopen accesshttp://purl.org/coar/access_right/c_abf2Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:docta.ucm.es:20.500.14352/1149242026-06-02T12:44:21Z
dc.title.none.fl_str_mv Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors
title Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors
spellingShingle Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors
Kubánková, Markéta
615.31
615:54
Amyloid aggregation
Live cells
Microviscosity
Fluorescence lifetime imaging microscopy (FLIM)
Sensors for Ab(1-42) aggregates
Química farmaceútica
23 Química
title_short Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors
title_full Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors
title_fullStr Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors
title_full_unstemmed Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors
title_sort Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors
dc.creator.none.fl_str_mv Kubánková, Markéta
López Duarte, Ismael
Bull, James A.
Vadukul, Devkee M.
Serpell, Louise C.
Victor, Marie de Saint
Stride, Eleanor
Kuimova, Marina K.
author Kubánková, Markéta
author_facet Kubánková, Markéta
López Duarte, Ismael
Bull, James A.
Vadukul, Devkee M.
Serpell, Louise C.
Victor, Marie de Saint
Stride, Eleanor
Kuimova, Marina K.
author_role author
author2 López Duarte, Ismael
Bull, James A.
Vadukul, Devkee M.
Serpell, Louise C.
Victor, Marie de Saint
Stride, Eleanor
Kuimova, Marina K.
author2_role author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Universidad Complutense de Madrid
dc.subject.none.fl_str_mv 615.31
615:54
Amyloid aggregation
Live cells
Microviscosity
Fluorescence lifetime imaging microscopy (FLIM)
Sensors for Ab(1-42) aggregates
Química farmaceútica
23 Química
topic 615.31
615:54
Amyloid aggregation
Live cells
Microviscosity
Fluorescence lifetime imaging microscopy (FLIM)
Sensors for Ab(1-42) aggregates
Química farmaceútica
23 Química
description Changes in microscopic viscosity and macromolecular crowding accompany the transition of proteins from their monomeric forms into highly organised fibrillar states. Previously, we have demonstrated that viscosity sensitive fluorophores termed ‘molecular rotors’, when freely mixed with monomers of interest, are able to report on changes in microrheology accompanying amyloid formation, and measured an increase in rigidity of approximately three orders of magnitude during aggregation of lysozyme and insulin. Here we extend this strategy by covalently attaching molecular rotors to several proteins capable of assembly into fibrils, namely lysozyme, fibrinogen and amyloid-b peptide (Ab(1e42)). We demonstrate that upon covalent attachment the molecular rotors can successfully probe supramolecular assembly in vitro. Importantly, our new strategy has wider applications in cellulo and in vivo, since covalently attached molecular rotors can be successfully delivered in situ and will colocalise with the aggregating protein, for example inside live cells. This important advantage allowed us to follow the microscopic viscosity changes accompanying blood clotting and during Ab(1e42) aggregation in live SHSY5Y cells. Our results demonstrate that covalently attached molecular rotors are a widely applicable tool to study supramolecular protein assembly and can reveal microrheological features of aggregating protein systems both in vitro and in cellulo not observable through classical fluorescent probes operating in light switch mode.
publishDate 2017
dc.date.none.fl_str_mv 2017
2017-09-01
2017
2017-09-01
dc.type.none.fl_str_mv journal article
http://purl.org/coar/resource_type/c_6501
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://hdl.handle.net/20.500.14352/114924
url https://hdl.handle.net/20.500.14352/114924
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:Docta Complutense
instname:Universidad Complutense de Madrid (UCM)
instname_str Universidad Complutense de Madrid (UCM)
reponame_str Docta Complutense
collection Docta Complutense
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,811543

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