title: Polymerization and structure of nucleotide-free actin filaments.
abstract: Two factors have limited studies of the properties of nucleotide-free actin (NFA). First, actin lacking bound nucleotide denatures rapidly without stabilizing agents such as sucrose; and second, without denaturants such as urea, it is difficult to remove all of the bound nucleotide. We used apyrase, EDTA and Dowex-1 to prepare actin that is stable in sucrose and approximately 99 % free of bound nucleotide. In high concentrations of sucrose where NFA is stable, it polymerizes more favorably with a lag phase shorter than ATP-actin and a critical concentration close to zero. NFA filaments are stable, but depolymerize at low sucrose concentrations due to denaturation of subunits when they dissociate from filament ends. By electron microscopy of negatively stained specimens, NFA forms long filaments with a persistence length 1.5 times greater than ADP-actin filaments. Three-dimensional helical reconstructions of NFA and ADP-actin filaments at 2.5 nm 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 peak of NFA filaments is near, but does not coincide with, the vacated nucleotide binding site. The empty nucleotide binding site in these NFA filaments is not accessible to free nucleotide in the solution. The affinity of NFA filaments for rhodamine phalloidin is lower than that of native actin filaments, due to a lower association rate. This work confirms that bound nucleotide 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 hydrolysis and phosphate release, and to provide an internal timer for the age of the filament.
authors.affiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD, 21205, USA.
authors.forename: E M
authors.initials: EM
authors.lastname: De La Cruz
authors.forename: A
authors.initials: A
authors.lastname: Mandinova
authors.forename: M O
authors.initials: MO
authors.lastname: Steinmetz
authors.forename: D
authors.initials: D
authors.lastname: Stoffler
authors.forename: U
authors.initials: U
authors.lastname: Aebi
authors.forename: T D
authors.initials: TD
authors.lastname: Pollard
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D000244:Adenosine Diphosphate / Q000737:chemistry*; D001704:Biopolymers; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D008024:Ligands; D008854:Microscopy, Electron; D011487:Protein Conformation
chemical_list: D000199:Actins; D001704:Biopolymers; D008024:Ligands; D000244:Adenosine Diphosphate