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Muscle myosin performance measured with a synthetic nanomachine reveals a class‐specific Ca 2+ ‐sensitivity of the frog myosin II isoform
Author(s) -
Pertici Irene,
Bianchi Giulio,
Bongini Lorenzo,
Cojoc Dan,
Taft Manuel H.,
Manstein Dietmar J.,
Lombardi Vincenzo,
Bianco Pasquale
Publication year - 2021
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jp280976
Subject(s) - myosin , heavy meromyosin , biophysics , meromyosin , actin , gelsolin , protein filament , sarcomere , molecular motor , gene isoform , skeletal muscle , chemistry , myosin head , anatomy , biology , biochemistry , myosin light chain kinase , microbiology and biotechnology , myocyte , gene
Key points A nanomachine made of an ensemble of seven heavy‐meromyosin (HMM) fragments of muscle myosin interacting with an actin filament is able to mimic the half‐sarcomere generating steady force and constant‐velocity shortening. To preserve Ca 2+ as a free parameter, the Ca 2+ ‐insensitive gelsolin fragment TL40 is used to attach the correctly oriented actin filament to the laser‐trapped bead acting as a force transducer. The new method reveals that the performance of the nanomachine powered by myosin from frog hind‐limb muscles depends on [Ca 2+ ], an effect mediated by a Ca 2+ ‐binding site in the regulatory light chain of HMM. The Ca 2+ ‐sensitivity is class‐specific because the performance of the nanomachine powered by mammalian skeletal muscle myosin is Ca 2+ independent. A model simulation is able to interface the nanomachine performance with that of the muscle of origin and provides a molecular explanation of the functional diversity of muscles with different orthologue isoforms of myosin.Abstract An ensemble of seven heavy‐meromyosin (HMM) fragments of myosin‐II purified from the hindlimb muscles of the frog ( Rana esculenta ) is used to drive a synthetic nanomachine that pulls an actin filament in the absence of confounding effects of other sarcomeric proteins. In the present version of the nanomachine the +end of the actin filament is attached to the laser trapped bead via the Ca 2+ ‐insensitive gelsolin fragment TL40, making [Ca 2+ ] a free parameter. Frog myosin performance in 2 m m ATP is affected by Ca 2+ : in 0.1 mm Ca 2+ , the isometric steady force ( F 0 , 15.25 pN) is increased by 50% ( P = 0.004) with respect to that in Ca 2+ ‐free solution, the maximum shortening velocity ( V 0 , 4.6 μm s –1 ) is reduced by 27% ( P = 0.46) and the maximum power ( P max , 7.6 aW) is increased by 21% ( P = 0.17). V 0 reduction is not significant for the paucity of data at low force, although it is solidified by a similar decrease (33%, P < 0.0001) in the velocity of actin sliding as indicated by an in vitro motility assay ( V f ). The rate of ATP‐hydrolysis in solution (φ) exhibits a similar calcium dependence. Ca 2+ titration curves for V f and φ give K d values of ∼30 μ m . All the above mechanical and kinetic parameters are independent of Ca 2+ when HMM from rabbit psoas myosin is used, indicating that the Ca 2+ ‐sensitivity is a class‐specific property of muscle myosin. A unique multiscale model allows interfacing of the nanomachine performance to that of the muscle of origin and identifies the kinetic steps responsible for the Ca 2+ ‐sensitivity of frog myosin.