Abstract
Double mutation D208Q:K450L was introduced in the beta isoform of human cardiac myosin to remove the salt bridge D208:K450 connecting loop 1 and the seven-stranded beta sheet within the myosin head. Beta isoform-specific salt bridge D208:K450, restricting the flexibility of loop 1, was previously discovered in molecular dynamics simulations. Earlier it was proposed that loop 1 modulates nucleotide affinity to actomyosin and we hypothesized that the electrostatic interactions between loop 1 and myosin head backbone regulate ATP binding to and ADP dissociation from actomyosin, and therefore, the time of the strong actomyosin binding. To examine the hypothesis we expressed the wild type and mutant of the myosin head construct (1–843 amino acid residues) in differentiated C2C12 cells, and the kinetics of ATP-induced actomyosin dissociation and ADP release were characterized using stopped-flow spectrofluorometry. Both constructs exhibit a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. We observed a faster rate of ATP-induced actomyosin dissociation with the mutant, compared to the wild type actomyosin. The rate of ADP release from actomyosin remains the same for the mutant and the wild type actomyosin. We conclude that the flexibility of loop 1 is a factor affecting the rate of ATP binding to actomyosin and actomyosin dissociation. The flexibility of loop 1 does not affect the rate of ADP release from human cardiac actomyosin.
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Raw data generated during the current study are available from the corresponding author on reasonable request. All analyzed data are included in this published article and its supplementary information file.
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Origin (OriginLab Corp, Northampton, MA) and Wolfram Mathematica (Champaign, IL) were used in data analysis. Wolfram Mathematica scripts are available in the supplementary information file.
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We thank the anonymous reviewers for critical reviews of an earlier version of the manuscript.
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This work was supported by the National Institutes of Health, Grant No. HL132315.
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YN designed research, AG and YN performed research and wrote the manuscript.
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Actin was produced from rabbit skeletal tissue. All experimental protocols were approved by the Institutional Animal Care and Use Committee of UNC Charlotte and all experiments were performed in accordance with relevant guidelines and regulations.
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Gargey, A., Nesmelov, Y.E. Electrostatic interaction of loop 1 and backbone of human cardiac myosin regulates the rate of ATP induced actomyosin dissociation. J Muscle Res Cell Motil 43, 1–8 (2022). https://doi.org/10.1007/s10974-021-09611-z
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DOI: https://doi.org/10.1007/s10974-021-09611-z