Cardiomyopathy mutations impact the actin-activated power stroke of human cardiac myosin
| Autor: | Wanjian Tang, Jinghua Ge, William C. Unrath, Christopher M. Yengo, Rohini Desetty |
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| Rok vydání: | 2021 |
| Předmět: |
Myosin ATPase
Biophysics Cardiomyopathy macromolecular substances 03 medical and health sciences Adenosine Triphosphate 0302 clinical medicine ATP hydrolysis Myosin medicine Molecular motor Humans Actin 030304 developmental biology 0303 health sciences Chemistry Myosin Subfragments Skeletal muscle Articles medicine.disease Actins medicine.anatomical_structure Förster resonance energy transfer Mutation Cardiomyopathies Cardiac Myosins 030217 neurology & neurosurgery |
| Zdroj: | Biophys J |
| ISSN: | 0006-3495 |
| Popis: | Cardiac muscle contraction is driven by the molecular motor myosin, which uses the energy from ATP hydrolysis to generate a power stroke when interacting with actin filaments, although it is unclear how this mechanism is impaired by mutations in myosin that can lead to heart failure. We have applied a fluorescence resonance energy transfer (FRET) strategy to investigate structural changes in the lever arm domain of human β-cardiac myosin subfragment 1 (M2β-S1). We exchanged the human ventricular regulatory light chain labeled at a single cysteine (V105C) with Alexa 488 onto M2β-S1, which served as a donor for Cy3ATP bound to the active site. We monitored the FRET signal during the actin-activated product release steps using transient kinetic measurements. We propose that the fast phase measured with our FRET probes represents the macroscopic rate constant associated with actin-activated rotation of the lever arm during the power stroke in M2β-S1. Our results demonstrated M2β-S1 has a slower actin-activated power stroke compared with fast skeletal muscle myosin and myosin V. Measurements at different temperatures comparing the rate constants of the actin-activated power stroke and phosphate release are consistent with a model in which the power stroke occurs before phosphate release and the two steps are tightly coupled. We suggest that the actin-activated power stroke is highly reversible but followed by a highly irreversible phosphate release step in the absence of load and free phosphate. We demonstrated that hypertrophic cardiomyopathy (R723G)- and dilated cardiomyopathy (F764L)-associated mutations both reduced actin activation of the power stroke in M2β-S1. We also demonstrate that both mutations alter in vitro actin gliding in the presence and absence of load. Thus, examining the structural kinetics of the power stroke in M2β-S1 has revealed critical mutation-associated defects in the myosin ATPase pathway, suggesting these measurements will be extremely important for establishing structure-based mechanisms of contractile dysfunction. |
| Databáze: | OpenAIRE |
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