Fluorescence energy transfer between points in G-actin: the nucleotide-binding site, the metal-binding site and Cys-373 residue

Abstract

Fluorescence energy transfers were studied in order to investigate the spatial relationships between the nucleotide-binding site, the metal-binding site and the Cys-373 residue in the G-actin molecule. When 1- N 6-ethenoadenosine-5′-triphosphate (ε-ATP) in the nucleotide-binding site and Co 2+ or Ni 2+ in the metal-binding site were used as fluorescence donor and acceptor, respectively, the fluorescence intensity of ε-ATP was perfectly quenched by Ni 2+ or Co 2+. This indicated that the nucleotide-binding site is very close to the metal-binding site; the distance should be less than 10 Å. When N-iodoacetyl- N′-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) bound to Cys-373 residue and Co 2+ in the metal-binding site were used as a fluorescence donor and an acceptor, respectively, the transfer efficiency was equal to 5± 1%. The corresponding distance was calculated to be 23–32 Å, assuming a random orientation factor K 2 = 2 3 .