Actin Polymerization Correlates with the Flattening of Actin Molecule

Actin plays the fundamental roles in a variety of cell functions in eukaryotic cells. Actin is a polymerizable protein and in two forms; monomeric G-actin and fibrous F-actin. The polymerization - depolymerization cycle drives essential cell processes, such as cell locomotion and cell division. For understanding of these processes, atomic structures for G-actin and F-actin are essential. Recently, we proposed the atomic model for F-actin structure, and found that actin is in the twisted form in a monomer, and in the untwisted form in a filament. The question when the transition from the twisted to the untwisted form occurs in filament formation remains elusive.In this study, to access the question, we prepared two actin mutants by the use of the insect cell expression system we developed. We focused on the loop A108-P112 crucial for the flattening transition. We introduced site-directed mutations at 108th Ala to Gly; A108G, and 109th Pro to Ala; P109A in the loop. Both mutants were purified to a high homogeneity as well as the wild-type, and were measured the polymerization activity and the actin ATPase activity relevant to polymerization.We demonstrated that A108G had both reduced elongation and ATP hydrolysis rates, and P109A had both accelerated ones on polymerization. These results indicate that A108G and P109A in the loop A108-P112 modulated the polymerization activity of the actin mutants, but did not affect the intrinsic ATPase activity relevant to polymerization. The mutant analyses of A108G and P109A suggest that the polymerization event correlates directly with the flattening of actin molecule.