Purification and characterization of a nonpolymerizing long-pitch actin dimer.
Atomic resolution structures of filamentous actin have not been obtained owing to the self-association of actin under crystallization conditions. Obtaining short filamentous actin complexes of defined lengths is therefore a highly desirable goal. Here we report the production and isolation of a long-pitch actin dimer employing chemical crosslinking between wild-type actin and Q41C/C374A mutant actin. The Q41C/C374A mutant actin possessed altered polymerization properties, with a 2-fold reduction in the rate of elongation and an increased critical concentration relative to wild-type actin. The Q41C/C374A mutant actin also displayed an increase in the IC50 for DNase I, a pointed-end actin-binding protein. The long-pitch dimer was bound by DNase I to prevent polymerization and purified. It was found that each actin dimer is bound by 2 DNase I molecules, 1 likely bound to each of the actin protomers. The long-pitch dimer bound by DNase I did not form short F actin structures, as assessed by the binding of rhodamine-phalloidin.
Sweeting
Braden
B
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada.
Dawson
John F
JF
D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D003850:Deoxyribonuclease I / Q000378:metabolism; D019281:Dimerization; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification; D017354:Point Mutation; D011108:Polymers; D015202:Protein Engineering; D015003:Yeasts
D000199:Actins; D003432:Cross-Linking Reagents; D050505:Mutant Proteins; D011108:Polymers; D003850:Deoxyribonuclease I