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A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin

Binding properties of actin-binding proteins are typically evaluated by cosedimentation assays. However, this method is time-consuming, involves multiple steps, and has a limited throughput. These shortcomings preclude its use in screening for drugs that modulate actin-binding proteins relevant to h...

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Autores principales: Bunch, Thomas A., Lepak, Victoria C., Bortz, Kellan M., Colson, Brett A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Rockefeller University Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898471/
https://www.ncbi.nlm.nih.gov/pubmed/33600558
http://dx.doi.org/10.1085/jgp.202012707
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author Bunch, Thomas A.
Lepak, Victoria C.
Bortz, Kellan M.
Colson, Brett A.
author_facet Bunch, Thomas A.
Lepak, Victoria C.
Bortz, Kellan M.
Colson, Brett A.
author_sort Bunch, Thomas A.
collection PubMed
description Binding properties of actin-binding proteins are typically evaluated by cosedimentation assays. However, this method is time-consuming, involves multiple steps, and has a limited throughput. These shortcomings preclude its use in screening for drugs that modulate actin-binding proteins relevant to human disease. To develop a simple, quantitative, and scalable F-actin–binding assay, we attached fluorescent probes to actin's Cys-374 and assessed changes in fluorescence lifetime upon binding to the N-terminal region (domains C0–C2) of human cardiac myosin-binding protein C (cMyBP-C). The lifetime of all five probes tested decreased upon incubation with cMyBP-C C0–C2, as measured by time-resolved fluorescence (TR-F), with IAEDANS being the most sensitive probe that yielded the smallest errors. The TR-F assay was compared with cosedimentation to evaluate in vitro changes in binding to actin and actin–tropomyosin arising from cMyBP-C mutations associated with hypertrophic cardiomyopathy (HCM) and tropomyosin binding. Lifetime changes of labeled actin with added C0–C2 were consistent with cosedimentation results. The HCM mutation L352P was confirmed to enhance actin binding, whereas PKA phosphorylation reduced binding. The HCM mutation R282W, predicted to disrupt a PKA recognition sequence, led to deficits in C0–C2 phosphorylation and altered binding. Lastly, C0–C2 binding was found to be enhanced by tropomyosin and binding capacity to be altered by mutations in a tropomyosin-binding region. These findings suggest that the TR-F assay is suitable for rapidly and accurately determining quantitative binding and for screening physiological conditions and compounds that affect cMyBP-C binding to F-actin for therapeutic discovery.
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spelling pubmed-78984712021-09-01 A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin Bunch, Thomas A. Lepak, Victoria C. Bortz, Kellan M. Colson, Brett A. J Gen Physiol Methods and Approaches Binding properties of actin-binding proteins are typically evaluated by cosedimentation assays. However, this method is time-consuming, involves multiple steps, and has a limited throughput. These shortcomings preclude its use in screening for drugs that modulate actin-binding proteins relevant to human disease. To develop a simple, quantitative, and scalable F-actin–binding assay, we attached fluorescent probes to actin's Cys-374 and assessed changes in fluorescence lifetime upon binding to the N-terminal region (domains C0–C2) of human cardiac myosin-binding protein C (cMyBP-C). The lifetime of all five probes tested decreased upon incubation with cMyBP-C C0–C2, as measured by time-resolved fluorescence (TR-F), with IAEDANS being the most sensitive probe that yielded the smallest errors. The TR-F assay was compared with cosedimentation to evaluate in vitro changes in binding to actin and actin–tropomyosin arising from cMyBP-C mutations associated with hypertrophic cardiomyopathy (HCM) and tropomyosin binding. Lifetime changes of labeled actin with added C0–C2 were consistent with cosedimentation results. The HCM mutation L352P was confirmed to enhance actin binding, whereas PKA phosphorylation reduced binding. The HCM mutation R282W, predicted to disrupt a PKA recognition sequence, led to deficits in C0–C2 phosphorylation and altered binding. Lastly, C0–C2 binding was found to be enhanced by tropomyosin and binding capacity to be altered by mutations in a tropomyosin-binding region. These findings suggest that the TR-F assay is suitable for rapidly and accurately determining quantitative binding and for screening physiological conditions and compounds that affect cMyBP-C binding to F-actin for therapeutic discovery. Rockefeller University Press 2021-02-18 /pmc/articles/PMC7898471/ /pubmed/33600558 http://dx.doi.org/10.1085/jgp.202012707 Text en © 2021 Bunch et al. http://www.rupress.org/terms/https://creativecommons.org/licenses/by-nc-sa/4.0/This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Methods and Approaches
Bunch, Thomas A.
Lepak, Victoria C.
Bortz, Kellan M.
Colson, Brett A.
A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin
title A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin
title_full A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin
title_fullStr A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin
title_full_unstemmed A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin
title_short A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin
title_sort high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein c to f-actin
topic Methods and Approaches
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898471/
https://www.ncbi.nlm.nih.gov/pubmed/33600558
http://dx.doi.org/10.1085/jgp.202012707
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