Supplementary Figure 17 Rate of assembly formation is dependent on ATP concentration

Article: Stimulus-responsive Self-Assembly of Protein-Based Fractals by Computational Design Pre-print: bioRxiv 274183; doi: https://doi.org/10.1101/274183 Fig. S17. Rate of assembly formation is dependent on ATP concentration. (A) Volume mean of sample from 0 – 1500 sec. Each point represents average of triplicates. (B) Number mean of sample from 0 – 1500 sec. Each point represents average of triplicates. Curve fitting performed using sloping spline with smoothness parameter (p) and adjusted R2 5 value given in Table S1 (highest concentration of ATP to lowest, starting from top to bottom at time 0 for both graphs). See Table S2 for curve fitting data for figure S17. Note that this figure provides data for Fig 2E. (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 2.13) DLS Kinetics (varying ATP) Experiment – An assembly mixture of 3 µM non35 pyAtzA and 2 µM AtzCM1 was prepared (as described previously) and syringe-filtered at 0.22 µm. To each 50 µL reaction volume, 1.2 µg of src kinase was added. Size was monitored continuously for 30 min at 25°C in a low-volume quartz sizing cuvette (Malvern; ZEN2112) using a Zetasizer Nano ZS (Malvern) at 50 µL/sample. Measurements were performed in triplicates. Each sample was read and averaged five times over the course of 25 seconds for a single time point. Curve fitting was performed in MATLAB (R2016b; Mathworks) using sloping spline function, with varying smoothing parameters. Adjusted R2 was used to determine model validity.<br>