Three steps in the thermal unfolding of F-actin: an experimental evidence

The development of differential scanning calorimetry has resulted in an increased interest in studies of the unfolding process in proteins with the aim of identifying domains and interactions with ligands or other proteins. Several of these studies were done with actin and showed that the thermal unfolding of F-actin occurs in at least three steps; this was interpreted as the denaturation of independent domains. In the present work, we have followed the thermal unfolding of F-actin using differential scanning calorimetry (DSC), CD spectroscopy, and probe fluorescence. We found that the three steps revealed through DSC are not the denaturation of independent domains. These three steps are a change in the environment of cys 374 at 49.5 degrees C; a modification at the nucleotide-binding site at 55 degrees C; and the unfolding of the peptide chain at 64 degrees C. Previous interpretations of the thermograms of F-actin were thus erroneous. Since DSC is now widely used to study proteins, our experimental approach and conclusions may also be relevant in denaturation studies of proteins in general.