Features of the structural organization of inactivated actin--an intermediate form of the protein during the folding-unfolding process

The structure of inactivated actin was studied by the methods of intrinsic fluorescence upon stationary and pulse excitation, selective fluorescence quenching with acrylamide, and testing the protein surface with a hydrophobic probe, 8-anilino-1-naphthalenesulfonic acid (ANS). The results are discussed along with earlier data on actin sedimentation, near- and far-UV CD spectra, and fluorescence anisotropy. The thermodynamic stability of inactivated actin, the presence of a secondary structure characteristic of the native protein, and the reversibility of the inactivated actin-completely unfolded actin transition allow inactivated actin to be considered an intermediate form in the process of protein folding into the native globular structure. In vitro actin inactivation is accompanied by specific association of actin macromolecules resulting in the formation of homogeneous stable complexes. The tendency toward aggregation (or specific association, in the case of actin), which is determined by the presence of extended hydrophobic clusters on the molecule surface, appears to be one of the intrinsic properties of any protein in the intermediate state. The mobility of the amino acid side chains in the inactivated actin differs considerably from that in the completely unfolded actin. The relaxation properties of the microenvironment of tryptophan residues determine relatively long-wave fluorescence spectra of the inactivated actin. However, the mobility observed is insufficient to compensate the asymmetry of the microenvironment of aromatic residues, which is confirmed by a characteristic and intense CD spectrum in the near-UV region. The mobility of the indole rings of tryptophans located in the internal regions of the inactivated actin that are solvent-inaccessible although polar is even considerably lower than that in the native actin.