Structural transition at actin's N-terminus in the actomyosin cross-bridge cycle

Biochemistry. 2000 Feb 22;39(7):1792-9. doi: 10.1021/bi991873c.

Abstract

Force and motion generation by actomyosin involves the cyclic formation and transition between weakly and strongly bound complexes of these proteins. Actin's N-terminus is believed to play a greater role in the formation of the weakly bound actomyosin states than in the formation of the strongly bound actomyosin states. It has been the goal of this project to determine whether the interaction of actin's N-terminus with myosin changes upon transition between these two states. To this end, a yeast actin mutant, Cys-1, was constructed by the insertion of a cysteine residue at actin's N-terminus and replacement of the C-terminal cysteine with alanine. The N-terminal cysteine was labeled stoichiometrically with pyrene maleimide, and the properties of the modified mutant actin were examined prior to spectroscopic measurements. Among these properties, actin polymerization, strong S1 binding, and the activation of S1 ATPase by pyrenyl-Cys-1 actin were not significantly different from those of wild-type yeast actin, while small changes were observed in the weak S1 binding and the in vitro motility of actin filaments. Fluorescence changes upon binding of S1 to pyrenyl-Cys-1 actin were measured for the strongly (with or without ADP) and weakly (with ATP and ATPgammaS) bound acto-S1 states. The fluorescence increased in each case, but the increase was greater (by about 75%) in the presence of MgATP and MgATPgammaS than in the rigor state. This demonstrates a transition at the S1 contact with actin's N-terminus between the weakly and strongly bound states, and implies either a closer proximity of the pyrene probe on Cys-1 to structural elements on S1 (most likely the loop of residues 626-647) or greater S1-induced changes at the N-terminus of actin in the weakly bound acto-S1 states.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actin Cytoskeleton / chemistry
  • Actin Cytoskeleton / genetics
  • Actin Cytoskeleton / metabolism
  • Actins / chemistry*
  • Actins / genetics
  • Actins / metabolism
  • Actomyosin / chemistry*
  • Actomyosin / metabolism
  • Biopolymers / chemistry
  • Biopolymers / genetics
  • Biopolymers / metabolism
  • Cysteine / genetics
  • Fluorescent Dyes / metabolism
  • Molecular Motor Proteins / chemistry
  • Molecular Motor Proteins / genetics
  • Molecular Motor Proteins / metabolism
  • Mutagenesis, Site-Directed
  • Myosin Subfragments / chemistry
  • Myosin Subfragments / metabolism
  • Peptide Fragments / chemistry*
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Plasmids / chemical synthesis
  • Plasmids / metabolism
  • Protein Binding / genetics
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / genetics
  • Spectrometry, Fluorescence

Substances

  • Actins
  • Biopolymers
  • Fluorescent Dyes
  • Molecular Motor Proteins
  • Myosin Subfragments
  • Peptide Fragments
  • Actomyosin
  • Cysteine