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

Abstract

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.

Human cardiac myosin-binding protein C phosphorylation- and mutation-dependent structural dynamics monitored by time-resolved FRET

“…We evaluated C0-C2 Cys225.Cys330 and wild type C0-C2 actin binding by using a filamentous actin (F-actin) cosedimentation assay. We and others have found that wild type C0-C2 binding increases in a concentration-dependent manner and is significantly reduced by PKA phosphorylation [25,27]. The most useful comparisons are found at submaximal binding levels (MyBP-C:actin; 1:7) [25], consistent with the ratios found in cardiac muscle.…”

“…C0-C2 was treated with 7.5 ng PKA/ μg C0-C2 for 30 min at 30 • C. We previously determined that maximal phosphorylation is achieved at 2.5 ng PKA/μg C0C2 [42]. For quantifying sub-maximal phosphorylation levels, C0-C2 was treated with 0.125, 0.25, and 0.5 ng PKA/μg C0-C2 for 30 min at 30 • C [25].…”

“…6B), indicating that phosphorylation is normal in the biosensor. Testing phosphorylation over a range of PKA concentrations previously shown to cause submaximal phosphorylation [25] showed that C0-C2 and C0-C2 Cys225. Cys330 were equally phosphorylated at each concentration (Fig.…”

“…Upon β-adrenergic stimulation, protein kinase A (PKA) phosphorylates cMyBP-C [24] in the M-domain (connecting C1 to C2, Fig. 1B-D), which results in a marked reduction in the binding of N-terminal cMyBP-C to actin and myosin filaments [13,[25][26][27]. This phosphorylation of cMyBP-C, as well as cardiac troponin-I (cTnI) [21] and titin [28], modulates cardiac performance via β-adrenergic signaling on a beat-to-beat basis in healthy myocardium.…”

Toward an understanding of myofibrillar function in health and disease

“…To address these issues, efforts to develop therapeutic interventions that mimic or inhibit the effects of cMyBP-C phosphorylation or its binding to specific partners in the thick or thin filaments have increased in recent years. The paper by Bunch et al (2021) in this issue reports the development of an innovative method to assess protein binding to actin, with emphasis on the binding of the C0–C2 domain of cMyBP-C. Their method involves the use of a novel time-resolved fluorescence assay to assess binding, with results that compare favorably to the more laborious cosedimentation assays that are typically used. The new method has potential value in studies to identify cMyBP-C binding partners and to also screen candidate therapeutics designed to enhance or repress specific binding events.…”