Polymerization and structure of nucleotide-free actin filaments

Two factors have limited studies of the properties of nucleotide-free actin (NFA). First, actin lacking bound nucleotide denatures rapidly without sta bilizing agents such as sucrose; and second, without denaturants such as ur ea, it is difficult to remove all of the bound nucleotide. We used apyrase, EDTA, and Dowex-l to prepare actin that is stable in sucrose and similar t o 99 % free of bound nucleotide. In high concentrations of sucrose where NF A is stable, it polymerizes more favorably with a lag phase shorter than AT P-actin and a critical concentration close to zero. NFA filaments are stabl e, but depolymerize at low sucrose concentrations due to denaturation of su bunits when they dissociate from filament ends. By electron microscopy of n egatively stained specimens, NFA forms long filaments with a persistence le ngth 1.5 times greater than ADP-actin filaments. Three-dimensional helical reconstructions of NFA and ADP-actin filaments at 2.5 nn resolution reveal similar intersubunit contacts along the two long-pitch helical strands but statistically significant less mass density between the two strands of NFA filaments. When compared with ADP-actin filaments, the major difference pea k of NFA filaments is near, but does not coincide with, the vacated nucleot ide binding site. The empty nucleotide binding site in these NFA filaments is not accessible to free nucleotide in the solution. The affinity of NFA f ilaments for rhodamine phalloidin is lower than that of native actin filame nts, due to a lower association rate. This work confirms that bound nucleot ide is not essential for actin polymerization, so the main functions of the nucleotide are to stabilize monomers, modulate the mechanical and dynamic properties of filaments through ATP I hydrolysis and phosphate release, and to provide an internal timer for the age of the filament. (C) 2000 Academi c Press.