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Actin's view of actomyosin interface

C J Miller1, P Cheung, P White

  • 1Department of Chemistry and Biochemistry, University of California, Los Angeles 90024, USA.

Biophysical Journal
|April 1, 1995
PubMed
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Charged amino acid pairs D24/D25 and E99/E100 in actin are crucial for myosin binding and filament stability. Mutations in these pairs, unlike D80/D81 and E83/K84, significantly alter actomyosin interactions and nucleotide exchange.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Mechanics

Background:

  • Actomyosin interactions are fundamental to cellular processes like muscle contraction and cell motility.
  • Understanding the specific roles of actin residues in myosin binding is key to elucidating these mechanisms.
  • Previous studies have implicated charged residues in protein-protein interactions, but specific roles in actomyosin dynamics remain to be fully defined.

Purpose of the Study:

  • To investigate the functional significance of specific charged amino acid pairs in yeast actin mutants.
  • To determine the role of residues D24/D25, E99/E100, D80/D81, and E83/K84 in actomyosin interactions and actin filament stability.
  • To analyze the impact of these mutations on myosin subfragment-1 (S1) binding, polymerization kinetics, and nucleotide exchange.

Main Methods:

Related Experiment Videos

  • Construction and characterization of yeast actin mutants with alanine substitutions for charged amino acid pairs.
  • In vitro motility assays to measure filament velocity and stability in the presence and absence of methylcellulose.
  • Biochemical assays to quantify myosin subfragment-1 (S1) binding affinity and polymerization rates.
  • Subtilisin cleavage assays to probe the local structure of actin subdomain-2.
  • Nucleotide exchange rate measurements.

Main Results:

  • Actin filaments with D24A/D25A or E99A/E100A mutations exhibited wild-type velocities in motility assays with methylcellulose.
  • Without methylcellulose, D24A/D25A and E99A/E100A filaments dissociated upon ATP addition, unlike D80/D81 and E83/K84 mutants.
  • Myosin S1 binding affinity was reduced threefold for D24A/D25A and twofold for E99A/E100A actins, while monomeric actin binding remained unchanged.
  • Polymerization rates and extents by S1 were reduced for D24A/D25A and E99A/E100A actins.
  • Subdomain-2 structure remained unaltered, but D24A/D25A actin showed a twofold decrease in nucleotide exchange.

Conclusions:

  • Residues D24/D25 and E99/E100 are critical for the weak binding interaction between myosin and actin.
  • These specific residues influence actin filament stability in the presence of ATP.
  • Residues D80/D81 and E83/K84 do not appear to modulate actomyosin interactions under the tested conditions.
  • The D24/D25 mutation affects nucleotide exchange, suggesting a link between nucleotide dynamics and actomyosin binding regulation.