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Proteolytic cleavage of actin within the DNase-I-binding loop changes the conformation of F-actin and its sensitivity

Y S Borovikov1, J Moraczewska, M I Khoroshev

  • 1Laboratory of Mechanisms of Cell Motility, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, St. Petersburg, Russia. boroviko@link.cytspb.rssi.ru

Biochimica Et Biophysica Acta
|March 17, 2000
PubMed

Insights

Subtilisin cleavage of actin alters F-actin structure and myosin binding dynamics. This modification impacts filament flexibility and myosin head movement, crucial for muscle contraction.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Muscle Physiology

Background:

  • Actin filaments form the structural basis of muscle contraction, interacting with myosin.
  • Myosin subfragment-1 (S1) binding to actin triggers conformational changes essential for force generation.
  • Subtilisin cleavage site between residues 47-48 in actin offers a tool to probe structural and dynamic roles.

Purpose of the Study:

  • To investigate the impact of subtilisin cleavage of actin on F-actin conformation.
  • To analyze how actin cleavage affects conformational changes upon myosin subfragment-1 (S1) binding.
  • To elucidate the role of actin loop 38-52 in F-actin structure and myosin interaction.

Main Methods:

  • Utilized polarized fluorescence spectroscopy on rhodamine-phalloidin- or 1,5-IAEDANS-labeled actin filaments.
  • Employed myosin-free muscle fibers (ghost fibers) for experiments.
  • Measured fluorescence from labeled actin and labeled S1 bound to actin.

Main Results:

  • Subtilisin cleavage induced differences in probe orientation, C-terminus mobility, and filament flexibility.
  • Cleavage diminished changes in filament flexibility and fluorophore orientation upon S1 binding (without ATP).
  • Myosin head orientation and mobility on cleaved actin filaments differed from intact actin.

Conclusions:

  • Actin loop 38-52 is vital for maintaining F-actin structure.
  • This loop is critical for actin's conformational transitions during strong myosin binding.
  • These transitions are likely essential for force and movement generation in actin-myosin interactions.

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