Figure 6.

DBLβ domains of BC12a and J1a bind ICAM-1^D1D5 with nanomolar affinity. Size exclusion chromatographs and purity by SDS-PAGE are shown for BC12^DBLβ (A) and J1a^DBLβ (B). The majority of BC12^DBLβ is present in aggregated form with a small proportion of monomeric protein (A). J1a^DBLβ is predominantly monomeric with some aggregated protein evidenced by presence of a large shoulder (B). mAu, miliabsorbance units. The purified monomeric samples used in the SPR studies are shown in the gels to the right of the chromatographs in (A) and (B), with full gels shown in Fig. S1 (BC12) and Fig. S2 (J1a). (C) CD analysis of secondary structure. CD spectra were recorded between 195 and 260 nm at 20 °C. For each protein, four measurements were averaged and corrected for buffer absorption. D-F, ICAM-1^D1D5 was coupled to a sensor chip surface (1100 RU) and DBLβ domains were injected at 30 µl/min with an association time of 240 seconds and a dissociation time of 600 seconds. Shown are sensorgrams for the binding to ICAM-1^D1D5 of BC12a^DBLβ (D), J1a^DBLβ (E) and IT4var13^DBLβ (F). Data (black lines) are modelled to a 1:1 global interaction model (red lines). Shown are sensorgrams for the sequential binding of IT4var13^DBLβ and BC12a^DBLβ to ICAM-1^D1D5 (G) and IT4var41^DBLβ and J1a^DBLβ to ICAM-1^D1D5 (H), blue lines. The response to the second domain alone is shown in red. Data for Fig. 6 is shown in S2–S4 Data.