The Effect of Actin-Binding Proteins on the Dynamics of Monomeric Actin

In eukaryotic cells, actin, which is the main component of the microfilament network, can be found in monomer (globular) and polymer (filament) form as well. The balance between these two forms is maintained by the assistance of the intracellular actin-binding proteins. Cofilin and profilin are small actin-binding proteins that can be found in nearly all eukaryotic cells. Cofilin can induce the disaggregation of the microfilament system by splitting the actin filaments and increasing their depolymerisation. Profilin has an opposite effect by increasing the rate of polymerisation at the plus end of the actin filaments. Cofilin and profilin are able to modify the rate of nucleotide exchange on G-actin via closing and opening the nucleotide binding cleft.Fluorescence resonance energy transfer (FRET) measurements were completed to answer the question of what kind of dynamic and conformational changes can be identified behind the functions of these actin-binding proteins. Fluorescent donor and acceptor molecules were attached to the actin monomers at Lys61 and Cys374. The possible conformational actions and the flexibility of the protein matrix between the probes were investigated by temperature dependent FRET experiments. The distance didn't change significantly between the labeled residues, while the protein matrix became more rigid after the binding of cofilin and profilin as well.With the help of the applied measurements it is possible to demonstrate that although the cofilin and profilin have an opposite effect on the conformation of the nucleotide binding cleft, both proteins modify the flexibility between the subdomain I and II in the same direction. These results can demonstrate that the small domain on actin behaves as a rigid unit during the opening and closing of the nucleotide binding pocket in the presence of profilin and cofilin as well.