Divalent cation-, nucleotide-, and polymerization-dependent changes in theconformation of subdomain 2 of actin

Conformational changes in subdomain 2 of actin were investigated using fluo rescence probes dansyl cadaverine (DC) or dansyl ethylenediamine (DED) cova lently attached to Gln(41). Examination of changes in the fluorescence emis sion spectra as a function of time during Ca2+/Mg2+ and ATP/ADP exchange at the high-affinity site for divalent cation-nucleotide complex in G-actin c onfirmed a profound influence of the type of nucleotide but failed to detec t a significant cation-dependent difference in the environment of Gin(41). No significant difference between Ca- and Mg-actin was also seen in the mag nitude of the fluorescence changes resulting from the polymerization of the se two actin forms. Evidence is presented that earlier reported cation-depe ndent differences in the conformation of the loop 38-52 may be related to t ime-dependent changes in the conformation of subdomain 2 in DED- or DC-labe led G-actin, accelerated by substitution of Mg2+ for Ca2+ in CaATP-G-actin and, in particular, by conversion of MgATP- into MgADP-G-actin. These spont aneous changes are associated with a denaturation-driven release of the bou nd nucleotide that is promoted by two effects of DED or DC labeling: lowere d affinity of actin for nucleotide and acceleration of ATP hydrolysis on Mg ATP-G-actin that converts it into a less stable MgADP form. Evidence is pre sented that the changes in the environment of Gln(41) accompanying actin po lymerization 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 repla cement of the bound ATP with ADP in G-actin is discussed.