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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1 March 2021
Methods and Approaches|
February 18 2021
A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin
Thomas A. Bunch
,
Thomas A. Bunch
Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ
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Victoria C. Lepak
,
Victoria C. Lepak
Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ
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Kellan M. Bortz
,
Kellan M. Bortz
Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ
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Brett A. Colson
Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ
Correspondence to Brett A. Colson: bcolson@arizona.edu
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Thomas A. Bunch
Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ
Victoria C. Lepak
Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ
Kellan M. Bortz
Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ
Brett A. Colson
Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ
This work is part of a special collection on myofilament function and disease.
Correspondence to Brett A. Colson: bcolson@arizona.edu
Received:
July 17 2020
Accepted:
January 12 2021
Online Issn: 1540-7748
Print Issn: 0022-1295
Funding:
National Institutes of Health
(R00 HL122397)
University of Arizona
(NO AWARD)
© 2021 Bunch et al.
2021
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/).
J Gen Physiol (2021) 153 (3): e202012707.
Article history
Received:
July 17 2020
Accepted:
January 12 2021
Citation
Thomas A. Bunch, Victoria C. Lepak, Kellan M. Bortz, Brett A. Colson; A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin. J Gen Physiol 1 March 2021; 153 (3): e202012707. doi: https://doi.org/10.1085/jgp.202012707
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