Supplementary Figure 14 Inhibition of assembly at 0.66 µM AtzC-SH2, 1 µM pY-AtzA, 0-6 µM SH2-DhaA

Article: Stimulus-responsive Self-Assembly of Protein-Based Fractals by Computational Design Pre-print: bioRxiv 274183; doi: https://doi.org/10.1101/274183 Figure: S14. Inhibition of assembly at 0.66 µM AtzC-SH2, 1 µM pY-AtzA, 0-6 µM SH2-DhaA. (A) Inhibition graph of SH2-DhaA on 0.66 µM AtzC-SH2, 1 µM pY-AtzA assembly. Size recorded represents most predominant DLS sizing peak. Data are presented as mean ± 1 standard deviation. IC50 = 3.05 µM. Adjusted R2 5 = 0.98. (B) DLS traces of assembly from 0 - 6 µM SH2-DhaA. DLS traces are of triplicate samples, each read 3 times. (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.研究论文：基于计算设计的刺激响应型蛋白质分形自组装 预印本：bioRxiv 274183；DOI：https://doi.org/10.1101/274183 图S14：在0.66 μM AtzC-SH2、1 μM pY-AtzA、0~6 μM SH2-DhaA条件下的组装抑制实验。 (A) SH2-DhaA对0.66 μM AtzC-SH2与1 μM pY-AtzA组装过程的抑制曲线。所记录的粒径为动态光散射（Dynamic Light Scattering, DLS）最显著的峰值。数据以平均值±1倍标准差形式呈现，半最大抑制浓度（Half-maximal inhibitory concentration, IC50）=3.05 μM，校正决定系数（Adjusted R²）=0.98。 (B) 0~6 μM SH2-DhaA对应的组装动态光散射轨迹。该轨迹来自三次重复样本，每个样本重复读取3次。 （补充材料2.9）磷酸化、组装形成与解组装——本研究的磷酸化实验方案基于Sigma公司Src激酶活性检测试剂盒（货号S1076）。在终体积150 μL的反应体系中，将3 μM AtzAM1与1×激酶活性缓冲液（含4 mM氯化镁、2.5 mM氯化锰、0.25 mM二硫苏糖醇、5 mM MOPS、2.5 mM甘油-2-磷酸、1 mM EGTA、400 nM EDTA，pH 7.6）、2.5 mM氯化锰、HNG、2 mM ATP、800 ng Src激酶混合，于25℃孵育7~16小时以完成磷酸化。磷酸化反应完成后，加入AtzCM1至终浓度为2 μM。随后于25℃孵育2小时以形成组装体。组装体形成后，向150 μL的反应体系中加入4.8 μg YopH磷酸酶以触发解组装。通过动态光散射（DLS）测定粒径以表征组装体的形成与解组装过程。 （补充材料2.10）动态光散射（DLS）——取50 μL组装样本，使用马尔文Zetasizer仪器及石英比色皿（ZEN2112，马尔文）进行粒径测定。于25℃下对11次重复样本各采集10条光谱数据。标准操作流程已考虑溶液中5%甘油的影响。