Supplementary Figure 15 Inhibition of assembly at 2 µM AtzC-SH2, 3 µM pY-AtzA with 0-15 µM inhibitor

Article: Stimulus-responsive Self-Assembly of Protein-Based Fractals by Computational Design

Pre-print: bioRxiv 274183; doi: https://doi.org/10.1101/274183

Figure: S15. Inhibition of assembly at 2 µM AtzC-SH2, 3 µM pY-AtzA with 0-15 µM inhibitor. All DLS traces were performed in triplicate (A) Inhibition graph of SH2-DhaA of 2 µM AtzC-SH2, 3 µM pY-AtzA assembly. Size recorded represents most predominant DLS sizing peak. Data are presented as mean ± 1 standard deviation. IC50 (SH2) = 6.18 µM, IC50 (SH2-DhaA) = 6.13 µM. Adjusted R2 (SH2) = 0.97. Adjusted R2 8 (SH2-DhaA) = 0.99. (B) DLS traces of assembly from 0-15 µM SH2. (C) DLS traces of assembly from 0-15 µM SH2-DhaA

(SI 2.9) Phosphorylation, assembly formation, and disassembly – The phosphorylation protocol was based upon Src kinase activity assay by Sigma (Catalog # S1076). In a final reaction volume of 150μL, 3μM AtzAM1 was mixed into 1X Kinase Activity Buffer (4mM MgCl2, 2.5mM MnCl2, 0.25mM DTT, 5mM MOPS, 2.5mM glycerol-2-phosphate, 1mM EGTA, 400nM EDTA, pH 7.6), 2.5 mM MnCl2,HNG, 2 mM ATP, 800ng Src kinase, and incubated for 7 – 16 hr at 25°C for phosphorylation to occur. After phosphorylating, AtzCM1 was added to a final 2μM concentration. Assembly was allowed to form at 2hr 25°C. Disassembly was performed by adding 4.8μg of YopH phosphatase into the 150μL reaction mixture after assembly formation occurred. Size measurements using DLS were performed to determine assembly formation/disassembly.

(SI 2.10) Dynamic light scattering (DLS) – 50 μL of an assembly sample was used for size determination using a Malvern Zetasizer and a quartz cuvette (ZEN2112, Malvern). Ten spectra measures were recorded for eleven replicates at 25 °C. The standard operating procedure accounted for 5% glycerol in solution.