Figure 7. Role of the CTD in branched polymer formation.

A. Turbidity time courses for IN^F185H,L242A in the presence of BI-224436. No aggregation was observed in absence of drug, nor the presence of up to three-fold excess of ALLINI. Shown in green is IN^Y15A,F185H. B. SV-AUC analysis of 15 μM IN^F185H,L242A in the presence (purple) and absence (orange) of 100 μM BI-224436. In the absence of drug, species consistent with monomers and dimers are observed (confirmed by SEC-MALS; see SFig1B). In the presence of ALLINI, a ladder of species is observed consistent with the formation of higher-order oligomers. C. Shown is a cartoon schematic depicting the SV-AUC analysis of drug-mediated CCD-CTD interactions using Alexafluor647-labelled HFS-CTD. D. SV-AUC results at 657 nm is shown, with control data in the absence of any drug for both HFS-CTD^L242A alone (red) and a mixture with excess CCD (orange). No evidence of complex formation is observed. F. In the presence of BI-224436, a new ~5S species is formed that is consistent with a higher order HFS-CTD₂•CCD₂•ALLINI₂ complex (~77.8 kD suggested by SE-AUC analysis (See SFig 2 and STable 4). The breadth of the species observed in the presence of drug are consistent with an associating system.