Divalent Cation-, Nucleotide-, and Polymerization-Dependent Changes in the Conformation of Subdomain 2 of Actin (original) (raw)

Abstract

Conformational changes in subdomain 2 of actin were investigated using fluorescence probes dansyl cadaverine (DC) or dansyl ethylenediamine (DED) covalently attached to Gln 41 . Examination of changes in the fluorescence emission spectra as a function of time during Ca 2ϩ /Mg 2ϩ and ATP/ADP exchange at the high-affinity site for divalent cation-nucleotide complex in G-actin confirmed a profound influence of the type of nucleotide but failed to detect a significant cationdependent difference in the environment of Gln 41 . No significant difference between Ca-and Mg-actin was also seen in the magnitude of the fluorescence changes resulting from the polymerization of these two actin forms. Evidence is presented that earlier reported cation-dependent differences in the conformation of the loop 38 -52 may be related to time-dependent changes in the conformation of subdomain 2 in DED-or DC-labeled G-actin, accelerated by substitution of Mg 2ϩ for Ca 2ϩ in CaATP-G-actin and, in particular, by conversion of MgATP-into MgADP-G-actin. These spontaneous changes are associated with a denaturation-driven release of the bound nucleotide that is promoted by two effects of DED or DC labeling: lowered affinity of actin for nucleotide and acceleration of ATP hydrolysis on MgATP-G-actin that converts it into a less stable MgADP form. Evidence is presented that the changes in the environment of Gln 41 accompanying actin polymerization result in part from the release of P i after the hydrolysis of ATP on the polymer. A similarity of this change to that accompanying replacement of the bound ATP with ADP in G-actin is discussed.

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