The effects of ADF/cofilin and profilin on the conformation of the ATP-binding cleft of monomeric actin

Biophys J. 2009 Mar 18;96(6):2335-43. doi: 10.1016/j.bpj.2008.12.3906.

Abstract

Actin depolymerizing factor (ADF)/cofilin and profilin are small actin-binding proteins, which have central roles in cytoskeletal dynamics in all eukaryotes. When bound to an actin monomer, ADF/cofilins inhibit the nucleotide exchange, whereas most profilins accelerate the nucleotide exchange on actin monomers. In this study the effects of ADF/cofilin and profilin on the accessibility of the actin monomer's ATP-binding pocket was investigated by a fluorescence spectroscopic method. The fluorescence of the actin bound epsilon-ATP was quenched with a neutral quencher (acrylamide) in steady-state and time dependent experiments, and the data were analyzed with a complex form of the Stern-Volmer equation. The experiments revealed that in the presence of ADF/cofilin the accessibility of the bound epsilon-ATP decreased, indicating a closed and more compact ATP-binding pocket induced by the binding of ADF/cofilin. In the presence of profilin the accessibility of the bound epsilon-ATP increased, indicating a more open and approachable protein matrix around the ATP-binding pocket. The results of the fluorescence quenching experiments support a structural mechanism regarding the regulation of the nucleotide exchange on actin monomers by ADF/cofilin and profilin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actin Depolymerizing Factors / chemistry
  • Actin Depolymerizing Factors / metabolism*
  • Actins / chemistry*
  • Actins / metabolism*
  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / metabolism*
  • Algorithms
  • Animals
  • Binding Sites
  • Ethenoadenosine Triphosphate
  • Fungal Proteins / metabolism
  • Profilins / chemistry
  • Profilins / metabolism*
  • Rabbits
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Spectrometry, Fluorescence

Substances

  • Actin Depolymerizing Factors
  • Actins
  • Ethenoadenosine Triphosphate
  • Fungal Proteins
  • Profilins
  • Recombinant Fusion Proteins
  • Adenosine Triphosphate