Key Points
- To characterize the effects of antibodies targeting actin sequence 18-29 on actin polymerization and conformational dynamics across different structural subdomains.
- Assessed actin polymerization kinetics in the presence of magnesium chloride, calcium chloride, and sodium chloride after binding of peptide antibodies targeting the 18-29 sequence.
- Conducted sedimentation assays to measure IgG binding affinities across diverse actin forms (Mg-F, Mg-G, Ca-F, and Ca-G) under varying ionic strengths.
- Performed spectroscopic assays using fluorescent probes (epsilon-ATP in the nucleotide cleft, dansyl at Gln-41, and pyrene at Cys-374) to monitor localized conformational shifts.
- Binding of the 18-29 antibody strongly inhibited MgCl2-induced polymerization of G-actin, weakly impacted CaCl2-induced polymerization, and had minimal effect on NaCl-induced polymerization.
- Antibody binding affinity was higher for Mg-associated F- and G-actins compared to their Ca-associated counterparts, with F-actin binding decreasing sharply at higher ionic strength.
- Antibody interaction induced conformational changes that slowed nucleotide exchange, increased epsilon-ATP fluorescence, and altered probe fluorescence at Gln-41 (DNase I loop) and Cys-374 (C-terminus).
Structured PICO
PPopulationActin (G-actin and F-actin)
IIntervention18-29 peptide antibodies
OOutcomePolymerization and conformation of actinsurrogate
Antibody binding to the 18-29 site of actin inhibits polymerization and induces conformational changes, highlighting actin plasticity in actomyosin interactions.