The effects of severely decreased hydrophobicity in a subdomain 3/4 loop on the dynamics and stability of yeast G-actin.

The hydrophobicity of the subdomain 3/4 hydrophobic loop (262-274) has been implicated to be essential for actin's function. We previously showed (Kuang, B., and Rubenstein, P. A. (1997) J. Biol. Chem. 272, 1237-1247) that a mutant yeast actin (V266G/L267G) with markedly decreased hydrophobicity in this loop conferred severe cold sensitivity to its polymerization. Here we further tested the mutational effect on the conformation and function of G-actin. This GG mutation caused no significant changes in overall secondary structure or in the microenvironment around actin's tryptophan residues, nor did it alter the dissociation constant of G-actin for ATP. However, it lowers the intrinsic ATPase activity and the melting temperature for Mg-GG actin from 51 to 33 degrees C and transforms the conformation of subdomain 2 and the central cleft of G-actin into an F-monomer-like structure. The results suggest that the hydrophobic plug may not only play a role in actin filament stabilization but also may be important for controlling the stability of G-actin and for promoting the conformational change of the monomer needed for addition to a growing actin filament.