Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy

Autores
Marcano Garcia, Luis Fernando; Zaza, Cecilia; Dalby, Olivia P. L.; Joseph, Megan D.; Cappellari, María Victoria; Simoncelli, Sabrina; Aramendia, Pedro Francisco
Año de publicación
2024
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Current methods for determining equilibrium constants often operate in three-dimensional environments, which may not accurately reflect interactions with membrane-bound proteins. With our technique, based on single-molecule localization microscopy (SMLM), we directly determine protein–protein association (Ka) and dissociation (Kd) constants in cellular environments by quantifying associated and isolated molecules and their interaction area. We introduce Kernel Surface Density (ks-density,) a novel method for determining the accessible area for interacting molecules, eliminating the need for user-defined parameters. Simulation studies validate our method’s accuracy across various density and affinity conditions. Applying this technique to T cell signaling proteins, we determine the 2D association constant of T cell receptors (TCRs) in resting cells and the pseudo-3D dissociation constant of pZAP70 molecules from phosphorylated intracellular tyrosine-based activation motifs on the TCR-CD3 complex. We address challenges of multiple detection and molecular labeling efficiency. This method enhances our understanding of protein interactions in cellular environments, advancing our knowledge of complex biological processes.
Fil: Marcano Garcia, Luis Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina
Fil: Zaza, Cecilia. University College London; Estados Unidos
Fil: Dalby, Olivia P. L.. University College London; Estados Unidos
Fil: Joseph, Megan D.. University College London; Estados Unidos
Fil: Cappellari, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina
Fil: Simoncelli, Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina
Fil: Aramendia, Pedro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina
Materia
equilibrium constant
single-molecule localization microscopy
protein−protein interactions
DNA-PAINT
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/265011
Acceder
id CONICETDig_07e3c7dee969891ac2d588951812da3b
oai_identifier_str oai:ri.conicet.gov.ar:11336/265011
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization MicroscopyMarcano Garcia, Luis FernandoZaza, CeciliaDalby, Olivia P. L.Joseph, Megan D.Cappellari, María VictoriaSimoncelli, SabrinaAramendia, Pedro Franciscoequilibrium constantsingle-molecule localization microscopyprotein−protein interactionsDNA-PAINThttps://purl.org/becyt/ford/1.4https://purl.org/becyt/ford/1Current methods for determining equilibrium constants often operate in three-dimensional environments, which may not accurately reflect interactions with membrane-bound proteins. With our technique, based on single-molecule localization microscopy (SMLM), we directly determine protein–protein association (Ka) and dissociation (Kd) constants in cellular environments by quantifying associated and isolated molecules and their interaction area. We introduce Kernel Surface Density (ks-density,) a novel method for determining the accessible area for interacting molecules, eliminating the need for user-defined parameters. Simulation studies validate our method’s accuracy across various density and affinity conditions. Applying this technique to T cell signaling proteins, we determine the 2D association constant of T cell receptors (TCRs) in resting cells and the pseudo-3D dissociation constant of pZAP70 molecules from phosphorylated intracellular tyrosine-based activation motifs on the TCR-CD3 complex. We address challenges of multiple detection and molecular labeling efficiency. This method enhances our understanding of protein interactions in cellular environments, advancing our knowledge of complex biological processes.Fil: Marcano Garcia, Luis Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Zaza, Cecilia. University College London; Estados UnidosFil: Dalby, Olivia P. L.. University College London; Estados UnidosFil: Joseph, Megan D.. University College London; Estados UnidosFil: Cappellari, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Simoncelli, Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Aramendia, Pedro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaAmerican Chemical Society2024-10info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/265011Marcano Garcia, Luis Fernando; Zaza, Cecilia; Dalby, Olivia P. L.; Joseph, Megan D.; Cappellari, María Victoria; et al.; Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy; American Chemical Society; Nano Letters; 24; 43; 10-2024; 13834-138421530-6984CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://pubs.acs.org/doi/10.1021/acs.nanolett.4c04394info:eu-repo/semantics/altIdentifier/doi/10.1021/acs.nanolett.4c04394info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:09:05Zoai:ri.conicet.gov.ar:11336/265011instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 11:09:05.284CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy
title Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy
spellingShingle Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy
Marcano Garcia, Luis Fernando
equilibrium constant
single-molecule localization microscopy
protein−protein interactions
DNA-PAINT
title_short Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy
title_full Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy
title_fullStr Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy
title_full_unstemmed Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy
title_sort Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy
dc.creator.none.fl_str_mv Marcano Garcia, Luis Fernando
Zaza, Cecilia
Dalby, Olivia P. L.
Joseph, Megan D.
Cappellari, María Victoria
Simoncelli, Sabrina
Aramendia, Pedro Francisco
author Marcano Garcia, Luis Fernando
author_facet Marcano Garcia, Luis Fernando
Zaza, Cecilia
Dalby, Olivia P. L.
Joseph, Megan D.
Cappellari, María Victoria
Simoncelli, Sabrina
Aramendia, Pedro Francisco
author_role author
author2 Zaza, Cecilia
Dalby, Olivia P. L.
Joseph, Megan D.
Cappellari, María Victoria
Simoncelli, Sabrina
Aramendia, Pedro Francisco
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv equilibrium constant
single-molecule localization microscopy
protein−protein interactions
DNA-PAINT
topic equilibrium constant
single-molecule localization microscopy
protein−protein interactions
DNA-PAINT
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.4
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Current methods for determining equilibrium constants often operate in three-dimensional environments, which may not accurately reflect interactions with membrane-bound proteins. With our technique, based on single-molecule localization microscopy (SMLM), we directly determine protein–protein association (Ka) and dissociation (Kd) constants in cellular environments by quantifying associated and isolated molecules and their interaction area. We introduce Kernel Surface Density (ks-density,) a novel method for determining the accessible area for interacting molecules, eliminating the need for user-defined parameters. Simulation studies validate our method’s accuracy across various density and affinity conditions. Applying this technique to T cell signaling proteins, we determine the 2D association constant of T cell receptors (TCRs) in resting cells and the pseudo-3D dissociation constant of pZAP70 molecules from phosphorylated intracellular tyrosine-based activation motifs on the TCR-CD3 complex. We address challenges of multiple detection and molecular labeling efficiency. This method enhances our understanding of protein interactions in cellular environments, advancing our knowledge of complex biological processes.
Fil: Marcano Garcia, Luis Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina
Fil: Zaza, Cecilia. University College London; Estados Unidos
Fil: Dalby, Olivia P. L.. University College London; Estados Unidos
Fil: Joseph, Megan D.. University College London; Estados Unidos
Fil: Cappellari, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina
Fil: Simoncelli, Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina
Fil: Aramendia, Pedro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina
description Current methods for determining equilibrium constants often operate in three-dimensional environments, which may not accurately reflect interactions with membrane-bound proteins. With our technique, based on single-molecule localization microscopy (SMLM), we directly determine protein–protein association (Ka) and dissociation (Kd) constants in cellular environments by quantifying associated and isolated molecules and their interaction area. We introduce Kernel Surface Density (ks-density,) a novel method for determining the accessible area for interacting molecules, eliminating the need for user-defined parameters. Simulation studies validate our method’s accuracy across various density and affinity conditions. Applying this technique to T cell signaling proteins, we determine the 2D association constant of T cell receptors (TCRs) in resting cells and the pseudo-3D dissociation constant of pZAP70 molecules from phosphorylated intracellular tyrosine-based activation motifs on the TCR-CD3 complex. We address challenges of multiple detection and molecular labeling efficiency. This method enhances our understanding of protein interactions in cellular environments, advancing our knowledge of complex biological processes.
publishDate 2024
dc.date.none.fl_str_mv 2024-10
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/265011
Marcano Garcia, Luis Fernando; Zaza, Cecilia; Dalby, Olivia P. L.; Joseph, Megan D.; Cappellari, María Victoria; et al.; Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy; American Chemical Society; Nano Letters; 24; 43; 10-2024; 13834-13842
1530-6984
CONICET Digital
CONICET
url http://hdl.handle.net/11336/265011
identifier_str_mv Marcano Garcia, Luis Fernando; Zaza, Cecilia; Dalby, Olivia P. L.; Joseph, Megan D.; Cappellari, María Victoria; et al.; Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy; American Chemical Society; Nano Letters; 24; 43; 10-2024; 13834-13842
1530-6984
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://pubs.acs.org/doi/10.1021/acs.nanolett.4c04394
info:eu-repo/semantics/altIdentifier/doi/10.1021/acs.nanolett.4c04394
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv American Chemical Society
publisher.none.fl_str_mv American Chemical Society
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1846781430276816896
score 12.982451

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