Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers

Abstract Reliable measurement of the binding kinetics of low molecular weight analytes to their targets is still a challenging task. Often, the introduction of labels is simply impossible in such measurements, and the application of label-free methods is the only reliable choice. By measuring the bi...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Hajnalka Jankovics, Boglarka Kovacs, Andras Saftics, Tamas Gerecsei, Éva Tóth, Inna Szekacs, Ferenc Vonderviszt, Robert Horvath
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/bbc0e199f2464a76b41617e43716222b
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:bbc0e199f2464a76b41617e43716222b
record_format	dspace
spelling	oai:doaj.org-article:bbc0e199f2464a76b41617e43716222b2021-12-02T12:42:27ZGrating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers10.1038/s41598-020-79226-w2045-2322https://doaj.org/article/bbc0e199f2464a76b41617e43716222b2020-12-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-79226-whttps://doaj.org/toc/2045-2322Abstract Reliable measurement of the binding kinetics of low molecular weight analytes to their targets is still a challenging task. Often, the introduction of labels is simply impossible in such measurements, and the application of label-free methods is the only reliable choice. By measuring the binding kinetics of Ni(II) ions to genetically modified flagellin layers, we demonstrate that: (1) Grating-Coupled Interferometry (GCI) is well suited to resolve the binding of ions, even at very low protein immobilization levels; (2) it supplies high quality kinetic data from which the number and strength of available binding sites can be determined, and (3) the rate constants of the binding events can also be obtained with high accuracy. Experiments were performed using a flagellin variant incorporating the C-terminal domain of the nickel-responsive transcription factor NikR. GCI results were compared to affinity data from titration calorimetry. We found that besides the low-affinity binding sites characterized by a micromolar dissociation constant (K d), tetrameric FliC-NikRC molecules possess high-affinity binding sites with K d values in the nanomolar range. GCI enabled us to obtain real-time kinetic data for the specific binding of an analyte with molar mass as low as 59 Da, even at signals lower than 1 pg/mm2.Hajnalka JankovicsBoglarka KovacsAndras SafticsTamas GerecseiÉva TóthInna SzekacsFerenc VondervisztRobert HorvathNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-11 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Hajnalka Jankovics Boglarka Kovacs Andras Saftics Tamas Gerecsei Éva Tóth Inna Szekacs Ferenc Vonderviszt Robert Horvath Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers
description	Abstract Reliable measurement of the binding kinetics of low molecular weight analytes to their targets is still a challenging task. Often, the introduction of labels is simply impossible in such measurements, and the application of label-free methods is the only reliable choice. By measuring the binding kinetics of Ni(II) ions to genetically modified flagellin layers, we demonstrate that: (1) Grating-Coupled Interferometry (GCI) is well suited to resolve the binding of ions, even at very low protein immobilization levels; (2) it supplies high quality kinetic data from which the number and strength of available binding sites can be determined, and (3) the rate constants of the binding events can also be obtained with high accuracy. Experiments were performed using a flagellin variant incorporating the C-terminal domain of the nickel-responsive transcription factor NikR. GCI results were compared to affinity data from titration calorimetry. We found that besides the low-affinity binding sites characterized by a micromolar dissociation constant (K d), tetrameric FliC-NikRC molecules possess high-affinity binding sites with K d values in the nanomolar range. GCI enabled us to obtain real-time kinetic data for the specific binding of an analyte with molar mass as low as 59 Da, even at signals lower than 1 pg/mm2.
format	article
author	Hajnalka Jankovics Boglarka Kovacs Andras Saftics Tamas Gerecsei Éva Tóth Inna Szekacs Ferenc Vonderviszt Robert Horvath
author_facet	Hajnalka Jankovics Boglarka Kovacs Andras Saftics Tamas Gerecsei Éva Tóth Inna Szekacs Ferenc Vonderviszt Robert Horvath
author_sort	Hajnalka Jankovics
title	Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers
title_short	Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers
title_full	Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers
title_fullStr	Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers
title_full_unstemmed	Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers
title_sort	grating-coupled interferometry reveals binding kinetics and affinities of ni ions to genetically engineered protein layers
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/bbc0e199f2464a76b41617e43716222b
work_keys_str_mv	AT hajnalkajankovics gratingcoupledinterferometryrevealsbindingkineticsandaffinitiesofniionstogeneticallyengineeredproteinlayers AT boglarkakovacs gratingcoupledinterferometryrevealsbindingkineticsandaffinitiesofniionstogeneticallyengineeredproteinlayers AT andrassaftics gratingcoupledinterferometryrevealsbindingkineticsandaffinitiesofniionstogeneticallyengineeredproteinlayers AT tamasgerecsei gratingcoupledinterferometryrevealsbindingkineticsandaffinitiesofniionstogeneticallyengineeredproteinlayers AT evatoth gratingcoupledinterferometryrevealsbindingkineticsandaffinitiesofniionstogeneticallyengineeredproteinlayers AT innaszekacs gratingcoupledinterferometryrevealsbindingkineticsandaffinitiesofniionstogeneticallyengineeredproteinlayers AT ferencvonderviszt gratingcoupledinterferometryrevealsbindingkineticsandaffinitiesofniionstogeneticallyengineeredproteinlayers AT roberthorvath gratingcoupledinterferometryrevealsbindingkineticsandaffinitiesofniionstogeneticallyengineeredproteinlayers
_version_	1718393706968514560

Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers

Ejemplares similares