title: Cysteine engineering of actin self-assembly interfaces.
abstract: The Holmes model of filamentous actin (F-actin) and recent structural studies suggest specific atomic interactions between F-actin subunits. We tested these interactions through a cysteine-engineering approach with the goal of inhibiting filament formation by introducing chemical groups at sites important for polymerization. We substituted surface amino acids on the actin molecule with cysteine residues and tested the effect of producing these actin mutant proteins in a yeast expression system. The intrinsic folding and polymerization characteristics of the cysteine-engineered actin proteins were measured. The effect of chemical modification of the introduced cysteine residues on the polymerization of the actin mutant proteins was also examined. Modification of cysteine residues with large hydrophobic reagents resulted in polymerization inhibition. We examined the finding that the D288C actin protein does not polymerize under oxidizing conditions and forms protein aggregates when magnesium and EGTA are present. Chemical crosslinking experiments revealed the presence of a lower dimer when only D288C actin was present. When both D288C and A204C actin were present, crosslinking experiments support the proximity of Asp288 on the barbed end of one subunit to Ala204 on the pointed end of a neighboring subunit in the Holmes model of F-actin.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada.
authors.forename: Kate
authors.initials: K
authors.lastname: Pengelly
authors.forename: Ana
authors.initials: A
authors.lastname: Loncar
authors.forename: Alex A
authors.initials: AA
authors.lastname: Perieteanu
authors.forename: John F
authors.initials: JF
authors.lastname: Dawson
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000235:genetics; D001704:Biopolymers / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D016296:Mutagenesis; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000737:chemistry
chemical_list: D000199:Actins; D001704:Biopolymers; D003545:Cysteine