The effects of ADF/cofilin and profilin on the conformation of the ATP-binding cleft of monomeric actin.
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.
Kardos
Roland
R
University of Pécs, Faculty of Medicine, Department of Biophysics, Pécs, Hungary.
Pozsonyi
Kinga
K
Nevalainen
Elisa
E
Lappalainen
Pekka
P
Nyitrai
Miklós
M
Hild
Gábor
G
D051339:Actin Depolymerizing Factors / Q000737:chemistry / Q000378:metabolism*; D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000465:Algorithms; D000818:Animals; D001665:Binding Sites; D004985:Ethenoadenosine Triphosphate; D005656:Fungal Proteins / Q000378:metabolism; D051304:Profilins / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D051339:Actin Depolymerizing Factors; D000199:Actins; D004985:Ethenoadenosine Triphosphate; D005656:Fungal Proteins; D051304:Profilins; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate