EFFECT OF CA2-MG2+ EXCHANGE ON THE FLEXIBILITY AND()OR CONFORMATION OF THE SMALL DOMAIN IN MONOMERIC ACTIN/

A fluorescence resonance energy transfer (FRET) parameter, f' (defined as the average transfer efficiency, (E), normalized by the actual flu orescence intensity of the donor in the presence of acceptor, F-DA), w as previously shown to be capable of monitoring both changes in local flexibility of the protein matrix and major conformational transitions . The temperature profile of this parameter was used to detect the cha nge of the protein flexibility in the small domain of the actin monome r (G-actin) upon the replacement of Ca2+ by Mg2+. The Cys-374 residue of the actin monomer was labeled with -iodoacetyl-N'-(5-sulfo-1-naphth yl)ethylenediamine (IAEDANS) to introduce a fluorescence donor and the Lys-61 residue with fluorescein-5-isothiocyanate (FITC) to serve as a n acceptor. The f' increases with increasing temperature over the whol e temperature range for Mg-G-actin. This parameter increases similarly in the case of Ca-G-actin up to 26 degrees C, whereas an opposite ten dency appears above this temperature. These data indicate that there i s a conformational change in Ca-G-actin above 26 degrees C that was no t detected in the case of Mg-G-actin. In the temperature range between 6 degrees C and 26 degrees C the slope of the temperature profile of f' is the same for Ca-G-actin and Mg-G-actin, suggesting that the flex ibility of the protein matrix between the two labels is identical in t he two forms of actin.