e., 89 interface sequences from 39 species) to identify pairs of interface segments with the following properties: (1) they share Screening Library datasheet the same symmetry center (position 111), (2) each contains amino acids of opposite charge

at interface residues flanking the symmetry center (i.e., positions 109 and 112), and (3) the charges at positions 109 and 112 in one interface are the opposite of those found at the other interface (Figures 1B and 1C). By swapping parts of interfaces with these properties, we reasoned that we could create chimeric interface segments that would disrupt self-pairing, while simultaneously directing pairing to a complementary yet different interface chimera. One example of such an interface chimera is shown in Figure 1B. A Drosophila Ig2 and silkworm Ig2 interface share an asparagine at position 111, the Drosophila sequence has an aspartic acid at position 109 and a lysine at 112, and the silkworm sequence has an arginine at position 109 and an aspartic acid at 112. Two unique half-interface C646 research buy segments were then created by flanking the shared symmetry center with amino acids 108–110 and 112–114 from the Drosophila and silkworm sequences, respectively. We predicted

that the resulting chimeras would not support self-binding due to charge incompatibility ( Figure 1B) but that the two chimeras would

bind to each other, because the contacts on each half-interface were seen in a wild-type interface. Two pairs of complementary chimeric interface segments (indicated Ig2.3C/Ig2.4C and Ig2.10C/Ig2.11C) were introduced through mutagenesis of Drosophila Ig2 domains with the most similar interfaces ( Figures 1B and 1C; also see sequence alignment in Figure 1D). To test the binding specificity of each altered variable domain, we inserted complementary Thiamine-diphosphate kinase pairs of Ig2 interfaces into ectodomains comprising the same Ig3 and Ig7 domains to generate pairs of closely related chimeric isoforms. We first assessed interactions by using the ELISA-based binding assay in which Dscam1 protein ectodomains were clustered in cis in a limited fashion (presumably tetramers) ( Wojtowicz et al., 2007). The binding of two ectodomains each comprising the N-terminal ten domains was tested as previously described ( Wojtowicz et al., 2007). Wild-type isoforms exhibited strong homophilic interaction, but homophilic binding of each chimera was reduced to background levels ( Figure 1D). Importantly, heterophilic binding of each chimera pair was observed at a similar level to that observed with homophilic binding of the control wild-type isoforms. To gain a more quantitative measure of binding specificity, we performed analytical ultracentrifugation (AUC).