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>

论文：基于计算设计的蛋白质基分形的刺激响应自组装 预印本：bioRxiv 274183；doi：https://doi.org/10.1101/274183
补充图S17：组装形成速率依赖于三磷酸腺苷（ATP）浓度。(A) 0–1500秒内样品的体积均值。每个数据点代表三次重复实验的平均值。(B) 0–1500秒内样品的数量均值。每个数据点代表三次重复实验的平均值。曲线拟合采用带平滑参数（p）的倾斜样条函数完成，调整后决定系数（adjusted R²）详见表S1（两张图中时间0处从顶部到底部依次对应ATP浓度从高到低）。补充图S17的曲线拟合数据详见表S2。请注意，本图为图2E提供数据支持（补充信息2.9）。

磷酸化、组装形成及解组装——本磷酸化实验方案基于Sigma Src激酶活性检测试剂盒（货号S1076）。在总体积150μL的反应体系中，将3μM AtzAM1与1×激酶活性缓冲液（含4mM氯化镁、2.5mM氯化锰、0.25mM二硫苏糖醇、5mM吗啉乙磺酸、2.5mM甘油-2-磷酸、1mM乙二醇双(2-氨基乙基醚)四乙酸、400nM乙二胺四乙酸，pH 7.6）、2.5mM氯化锰、HNG、2mM ATP、800ng Src激酶混合，于25℃孵育7–16小时以完成磷酸化。磷酸化完成后，加入AtzCM1至终浓度2μM。随后于25℃静置2小时以允许组装形成。组装完成后，向150μL反应体系中加入4.8μg YopH磷酸酶以执行解组装实验。采用动态光散射（Dynamic Light Scattering, DLS）进行粒径测量以表征组装形成与解组装过程。（补充信息2.10）

动态光散射（DLS）——取50μL组装样品，使用马尔文（Malvern）Zetasizer仪器及石英比色皿（ZEN2112，马尔文（Malvern））进行粒径测定。在25℃下对11次重复实验各记录10条光谱。标准操作流程考虑了溶液中5%甘油的影响。收集并报告体积百分比数据。（补充信息2.13）

ATP浓度梯度DLS动力学实验——按照前述方法配制3μM non35 pyAtzA与2μM AtzCM1的组装混合液，并经0.22μm滤膜针式过滤。向每个50μL反应体系中加入1.2μg Src激酶。使用马尔文（Malvern）Zetasizer Nano ZS仪器及低体积石英粒径比色皿（ZEN2112，马尔文（Malvern）），于25℃下连续监测粒径变化30分钟，每样品体积50μL。实验设置三次重复。每个时间点的单次测量均采集25秒内的5次读数并取平均值。使用MATLAB（R2016b；MathWorks）中的倾斜样条函数进行曲线拟合，平滑参数可调节。采用调整后决定系数评估模型有效性。