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Probing actin-activated ATP turnover kinetics of human cardiac myosin II by single molecule fluorescence
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.ORCID iD: 0000-0003-0225-8227
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.ORCID iD: 0000-0001-6662-8886
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2024 (English)In: Cytoskeleton, ISSN 1949-3584, Vol. 81, no 12, p. 883-901Article in journal (Refereed) Published
Abstract [en]

Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit from functional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero-length cross-linking of human cardiac myosin II motor fragments (sub-fragment 1 long [S1L]) to surface-immobilized actin filaments in a configuration with maintained actin-activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin-activated rate constant (k(cat)). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin-activated ATPase, but also a third exponential process consistent with non-specific ATP-binding outside the active site.

Place, publisher, year, edition, pages
John Wiley & Sons, 2024. Vol. 81, no 12, p. 883-901
Keywords [en]
actin, actin-activated ATPase, Alexa 647 ATP, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, single molecule fluorescence, beta-Cardiac myosin II
National Category
Biophysics Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
URN: urn:nbn:se:lnu:diva-129124DOI: 10.1002/cm.21858ISI: 001203081500001PubMedID: 38623952Scopus ID: 2-s2.0-85191016814OAI: oai:DiVA.org:lnu-129124DiVA, id: diva2:1855597
Available from: 2024-05-02 Created: 2024-05-02 Last updated: 2025-02-04Bibliographically approved
In thesis
1. Single-molecule biochemical characterization of human β-cardiac myosin purified from non-virally transfected C2C12 cells
Open this publication in new window or tab >>Single-molecule biochemical characterization of human β-cardiac myosin purified from non-virally transfected C2C12 cells
2025 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Myosin II is a molecular motor that primarily functions in muscle contraction where it generates force and motion through cyclic interaction with actin filaments, driven by ATP turnover. Gaining mechanistic insights into actomyosin energy transduction is essential, particularly in the context of mutations associated with hypertrophic cardiomyopathy (HCM), a genetic condition linked to altered cardiac function. The current approach for studying mutations in the myosin motor domain relies on protein expression in C2C12 cells using an adenovirus-based transfection system. However, this method is constrained by slow turnaround time and labor-intensive protocols. This thesis presents a virus-free transfection method to express human β-cardiac myosin subfragment-1 (denoted as S1L) in C2C12 cells using commercially available chemical reagents – JetPrime and GenJet.  The purified S1L proteins exhibited actin-activated ATPase and sliding velocities (using in vitro motility assay) were comparable to those obtained using the virus-based system. Our new alternative method provides a faster and less complex approach for screening a wide range of HCM mutations. Furthermore, a highly miniaturized single-molecule ATPase assay was developed to leverage the advantage of the virus-free expression system. Using total internal reflection fluorescence microscopy (TIRF), fluorescent ATP turnover rate constants for myosin were determined for both basal and actin-activated conditions. The latter was obtained by crosslinking S1L proteins to surface-immobilized actin filaments via EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) crosslinker. The observed turnover rates were consistent with those obtained from solution-based kinetic assays. By integrating the virus-free expression system with the single-molecule ATPase assay, several S1L point mutations, including R243E, R243E+E466R, R243C, and R243H were biochemically characterized alongside the classical HCM mutations R403Q and R453C. The R243 mutants are of particular interest, as this residue plays an important role in the secondary Pi binding site and may influence the multistep process of inorganic phosphate release. Initial findings suggest that the R243C mutation could serve as a promising model for investigating orthophosphate release, potentially offering deeper insights into the molecular mechanisms underlying HCM pathogenesis.

Place, publisher, year, edition, pages
Linnaeus University Press, 2025. p. 89
Series
Linnaeus University Dissertations ; 556/2025
Keywords
Human β-cardiac myosin, mutations, hypertrophic cardiomyopathy, C2C12, virus-free transfection, expression, single-molecule ATPase, actin-activated ATPase, crosslinking, basal ATPase, total internal reflection fluorescence microscopy, phosphate release.
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-134689 (URN)9789180822558 (ISBN)9789180822565 (ISBN)
Public defence
2025-02-07, Vi 2166 (Azur), Linnaeus University, Building Vita, Kalmar, 16:28 (English)
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Available from: 2025-01-21 Created: 2025-01-20 Last updated: 2025-01-21Bibliographically approved

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Berg, AlbinVelayuthan, Lok PriyaTågerud, SvenUšaj, MarkoMånsson, Alf

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