Figure 2: Assembly Formation, Dissolution and Inhibition in vitro

Article: Stimulus-responsive Self-Assembly of Protein-Based Fractals by Computational Design<br> Pre-print: bioRxiv 274183; doi: https://doi.org/10.1101/274183 Figure 2. (A) Using Src kinase, the AtzAM1 can be phosphorylated (pY-AtzAM1) and incubated with AtzCM1-SH2 to form an assembly. The phosphatase (YOP) enzyme can be used for disassembly. (B and C) Assemblies 4 were expected to form (B) and dissolve (C), respectively, as confirmed by DLS measurements. (D) Incubation of assembling components with various concentrations of free SH2 domain and a different (monovalent) SH2 fusion protein led to robust inhibition. (E) ATP concentration was shown to control the rate of assembly formation (highest concentration of ATP to lowest, starting 8 from top to bottom at time 0) (F and G) Assembly formation is highly sensitive to stoichiometry of the components. Varying the stoichiometry (F and G) and the use of a weaker binding SH2-peptide interaction (F) leads to a perturbation of the assembly formation zone 11 compared to the “superbinder” SH2 (G). (H) The fractal-like structure observed by light microscopy. (I) Fluorescence microscopy image of assembly formed by Alexa Fluor 647^TM-labeled AtzCM1-SH2 and pY-AtzAM1. (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 (SI 2.11) DLS Inhibition Experiment - 6 µM pY-AtzAM1 was phosphorylated (1X KAB, 2 mM ATP, 1 mM DTT, HNG, 1 µg Src kinase) in a reaction volume of 75 µL. Incubation time was overnight at 25°C. SH2 or SH2-DhaA was added to each sample at 0 µM, 3 µM, 6 µM, 9 µM, 12 µM, 15 µM, 18 µM final concentration and allowed to “block” binding sites on the pY10 AtzAM1 for 1 hr at 25°C. AtzCM1 was added to each sample at 2 µM final concentration. Therefore, the final concentrations of all components was 3 µM pyAtzA, 1 µM AtzCM1, 0 µM - 18 µM SH2 or SH2-DhaA. The sample was incubated for 2 hr at 25°C. DLS was performed to analyze assembly sizes. 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 triplicates while each sample was read and averaged 15 times. This protocol was repeated at a final concentration of 1 µM pyAtzA, 0.66 µM AtzCM1, 0 µM -6 µM SH2-DhaA. Curve fitting was performed in MATLAB (R2016b; Mathworks) using the general model: f(x) = A/(1+e^(-k(x-x0))) + B; where A, B, k, x0 are constants. Adjusted R2 was used to determine model validity. Inhibition concentration 50 (IC50) was determined based upon concentration of inhibitor that resulted in assembly size of 100nm measured. (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.*****Some of this work was part of the <b>Rutgers Biomod 2016</b> project (M. Liu, A. Permaul, O. Dineen, M. Shea, M. Khalid, G.L. Bilker). See reference for additional details. <br>