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

• Published in: The Journal of general physiology Vol. 153; no. 3; p. 1
• Main Authors: Bunch, Thomas A, Lepak, Victoria C, Bortz, Kellan M, Colson, Brett A
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 01.03.2021
• Subjects: Actin; Actin Cytoskeleton - metabolism; Actins - metabolism; Biochemistry; Biophysics; Cardiomyopathy; Carrier Proteins - genetics; Carrier Proteins - metabolism; Contraction and cell motility; Drug screening; Fluorescence; Fluorescent indicators; Humans; Methods and Approaches; Mutation; Myofilament Special Issue, 2020; Myosin; Myosin-binding protein C; Phosphorylation; Protein Binding; Protein C; Protein structure and dynamics; Tropomyosin
• ISSN: 0022-1295; 1540-7748
• DOI: 10.1085/JGP.202012707

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.