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 fluorescence intensity of the donor in the presence of acceptor, F(DA)), was 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 change of the protein flexibility in the small domain of the actin monomer (G-actin) upon the replacement of Ca2+ by Mg2+. The Cys-374 residue of the actin monomer was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) to introduce a fluorescence donor and the Lys-61 residue with fluorescein-5-isothiocyanate (FITC) to serve as an acceptor. The f' increases with increasing temperature over the whole 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 tendency appears above this temperature. These data indicate that there is a conformational change in Ca-G-actin above 26 degrees C that was not 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 flexibility of the protein matrix between the two labels is identical in the two forms of actin.
Nyitrai
M
M
Research Group of the Hungarian Academy of Sciences at the Department of Biophysics, University Medical School of Pécs, Hungary.
Hild
G
G
Lakos
Z
Z
Somogyi
B
B
D000199:Actins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002138:Calibration; D003545:Cysteine; D004735:Energy Transfer; D005456:Fluorescent Dyes; D008239:Lysine; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D008958:Models, Molecular; D018482:Muscle, Skeletal; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; C006421:1,5-I-AEDANS; D008274:Magnesium; D008239:Lysine; D003545:Cysteine; D002118:Calcium