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

Kanassatega, Rhye-Samuel; Bunch, Thomas A.; Lepak, Victoria C.; Wang, Christopher; Colson, Brett A.

doi:10.1016/j.yjmcc.2022.02.005

Cited by 7 publications

(13 citation statements)

References 51 publications

(75 reference statements)

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“…C0-C2 contains 5 endogenous cysteines, which were strategically removed to allow for introduction of single cysteines (Fig. 1F, 1H) (23). Since Cys249 of the C1 domain is an endogenous, surface-accessible thiol, the remaining 4 Cys were removed to generate this protein (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…On myosin the probe in the tri-helix bundle (Cys330) did show a 24% increase in FRET with myosin when T59A was phosphorylated and a 21% increase in FRET with actin in the unphosphorylated state (Table 1). R282W , in a PKA target sequence, altered phosphorylation and actin interactions (23, 24). Our new FRET assays found no effects on the binding of unphosphorylated R282W to either myosin or actin (FRET was within 10% of wild type for all 3 FRET acceptor probes).…”

Section: Resultsmentioning

confidence: 99%

“…Fluorescence lifetime measurements were acquired using a high-precision fluorescence lifetime plate reader (FLTPR; Fluorescence Innovations, Inc) (23, 24, 37), provided by Photonic Pharma LLC. For TR-FRET experiments, FMAL was excited with a 473-nm microchip laser (Bright Solutions) and emission was filtered with 488-nm long-pass and 517/20-nm band-pass filters (Semrock).…”

Section: Methodsmentioning

confidence: 99%

“…The observed waveforms were convolved with the IRF to determine the lifetime (τ) (Eq. 1) by fitting to a single-exponential decay (23,24,37). The decay of the excited state of the fluorescence FMAL dye attached to myosin at V105C or actin at Cys-374 to the ground state is:…”

Section: Time-resolved Fret (Tr-fret) Data Acquisition and Analysismentioning

confidence: 99%

“…Finally, one of the mutations (T59A in C0) has been reported to influence binding to RLC (16), however alanine is found in this position of C0 in several other species and may simply be a nonpathogenic variant in humans. We previously studied the actin binding properties of three of these mutations (R282W, E334K, and L352P) using an environmentally-sensitive single-probe fluorescent lifetime assay (i.e., time-resolved fluorescence, TR-F) (24) and others have investigated mutation effects on myosin/actin binding or phosphorylation (21)(22)(23).…”

Section: Introductionmentioning

confidence: 99%

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N-terminal cardiac myosin-binding protein C interactions with myosin and actin filaments using time-resolved FRET

Wong

Bunch

Lepak

et al. 2022

Preprint

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Myosin binding protein-C (cMyBP-C) is a sarcomeric protein responsible for normal contraction and relaxation of the heart. We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to resolve the interactions of cardiac myosin and F-actin with cMyBP-C, focusing on the N-terminal region. The results imply roles of these bound protein complexes in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure and hypertrophic cardiomyopathy (HCM). N-terminal cMyBP-C domains C0 through C2 (C0-C2) contain binding regions for interactions with both thick (myosin) and thin (actin) filaments. Phosphorylation by protein kinase A (PKA) in the cMyBP-C motif (M-domain) regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C and either myosin or actin. We engineered intermolecular pairs of labeling sites between donor-labeled myosin regulatory light chain (V105C) or F-actin (C374) and cMyBP-C (S85C in C0, C249 in C1, or P330C in M-domain) to detect interactions. Phosphorylation reduced the interaction of cMyBP-C to both myosin and actin. Further insight was gained from evaluating cMyBP-C HCM mutations T59A, R282W, E334K, and L349R, which revealed increases in myosin-FRET, increases or decreases in actin-FRET, and perturbations of phosphorylation effects. These findings elucidate binding of cMyBP-C to myosin or actin under physiological and pathological conditions, providing new molecular insight into the modulatory role of these protein-protein interactions in cardiac muscle contractility. Further, these findings suggest that the TR-FRET assays are suitable for rapid and accurate determination of quantitative binding for screening physiological conditions and compounds that affect cMyBP-C interactions with myosin or F-actin for therapeutic discovery.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%