Thermal unfolding of G-actin monitored with the DNase I-inhibition assay -Stabilities of actin isoforms

Actin is one of the proteins that rely on chaperonins for proper folding. T his paper shows that the thermal unfolding of G-actin, as studied by GD and ultraviolet difference spectrometry, coincides with a loss in DNase I-inhi biting activity of the protein. Thus, the DNase I inhibition assay should b e useful for systematic studies of actin unfolding and refolding. Using thi s assay, we have investigated how the thermal stability of actin is affecte d by either Ca2+ or Mg2+ at the high affinity divalent cation binding site, by the concentration of excess nucleotide, and by the nucleotide in differ ent states of phosphorylation (ATP, ADP.P-i, ADP.V-i, ADP.AlF4, ADP.BeFx, a nd ADP). Actin isoforms from different species were also compared, and the effect of profilin on the thermal stability of actin was studied. We conclu de that the thermal unfolding of G-actin is a three-state process, in which an equilibrium exists between native actin with bound nucleotide and an in termediate free of nucleotide. Actins in the Mg-form were less stable than the Ca-forms, and the stability of the different isoforms decreased in the following order: rabbit skeletal muscle alpha-actin = bovine cytoplasmic ga mma-actin > yeast actin > cytoplasmic p-actin. The activation energies for the thermal unfolding reactions were in the range 200-290 kJ.mol(-1), depen ding on the bound ligands. Generally, the stability of the actin depended o n the degree with which the nucleotide contributed to the connectivity betw een the two domains of the protein.