Fig. 5. Binding of Cdc20^N and Mad1^CTD mutants by fluorescent anisotropy and SEC-MALS.

a A multiple sequence alignment of Cdc20^1–40. Secondary structure prediction by PSIPRED is shown above for the α1 and Box1 predicted helices. The asterisks denote residues which were mutated and tested by fluorescent anisotropy or SEC-MALS in b–d. b Fluorescent anisotropy with preformed AF488-Bub1^CD1:Mad1^CTD complex (20 nM: 2.1 µM) using pMad1^CTD or Mad1^CTD and where various Cdc20^N mutants were titrated. The data were presented as mean values ± SD derived from three independent measurements. c Summary of the calculated K_D of the interactions analysed in (b). d SEC-MALS analysis of Cdc20^N interaction with pMad1^CTD or Mad1^CTD using various mutants. Theoretical masses for Mad1^CTD dimer, Cdc20^N, and Mad1^CTD dimer with a single Cdc20^N bound are 28, 7.8 and 36 kDa respectively. e SDS-PAGE analysis of the SEC-MALS experiments shown in (d), using a 4–12% Bis-Tris Glycine gel with the SeeBlue™ Plus2 Protein Ladder (Thermo Fisher Scientific). Three independent measurements of each sample were completed with similar results.