Supplementary Figure 20 Average size of particle formed by pY-AtzA and super-binder AtzC-SH2

Article: Stimulus-responsive Self-Assembly of Protein-Based Fractals by Computational Design Pre-print: bioRxiv 274183; doi: https://doi.org/10.1101/274183 Figure: Fig. S20. Average size of particle formed by pY-AtzA and super-binder AtzC-SH2. (A) Heat map showing volume-weighted mean size of particles found from 50-3000 nM pY-AtzA and 50-2000 nM AtzC-SH2. Value shown is average of two physical samples. Histogram illustrates distribution of sizes found on heatmap. (B) Volume distributions of heat map. Distributions shown are representative of other traces in the sample. (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. % volume was collected and reported. (SI 2.12) DLS Titration Experiment – 6 µM, 3 µM, 1.5 µM, 0.5 µM, 0.1 µM pyAtzA was phosphorylated (as described previously) with an incubation time of overnight at 25°C. Either AtzCM1 wildtype (WT) or AtzCM1 superbinder (SB) was added to each sample at 2 µM, 1 µM, 0.5 µM, 0.25 µM, 0.50 µM final concentration. The sample was allowed to incubate for 2 hr at 25°C. Therefore, the final concentrations of all components was from 3 µM – 0.05 µM pyAtzA, 2 µM – 0.05 µM AtzCM1-WT or AtzCM1-SB. DLS was performed at 25°C, 50 µL/sample volume, in a low-volume quartz sizing cuvette (Malvern; ZEN2112) using a Zetasizer Nano ZS (Malvern). Measurements were performed in duplicate with each sample read and averaged 15 times.<br>