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.

研究论文：基于计算设计的刺激响应型蛋白质基分形自组装
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，调整决定系数(adjusted R²)值详见补充表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补充表S2。注：本图为图2E提供实验数据。（补充信息2.9）

磷酸化、组装形成与解组装——磷酸化实验流程基于Sigma公司的Src激酶活性检测试剂盒（货号：S1076）建立。在终体积为150μL的反应体系中，将3μM的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(DTT)、5mM 3-(N-吗啉代)丙磺酸(MOPS)、2.5mM甘油-2-磷酸、1mM乙二醇双(2-氨基乙醚)四乙酸(EGTA)、400nM乙二胺四乙酸(EDTA)，pH 7.6）、2.5mM氯化锰、HNG、2mM三磷酸腺苷(ATP)、800ng Src激酶混合，于25℃孵育7~16小时以完成磷酸化反应。磷酸化完成后，加入AtzCM1至终浓度为2μM，于25℃下静置2小时以启动组装过程。组装完成后，向150μL的反应体系中加入4.8μg YopH磷酸酶以启动解组装反应。采用动态光散射(Dynamic Light Scattering, DLS)进行粒径检测，以表征组装与解组装过程。

（补充信息2.10）动态光散射(DLS)检测——取50μL组装样品，使用马尔文Zetasizer粒径分析仪及石英比色皿（ZEN2112，马尔文公司）进行粒径测定。于25℃下对11次重复实验分别采集10次光谱测量数据。标准操作流程已考虑体系中5%甘油的影响，最终报告体积占比数据。

（补充信息2.13）动态光散射动力学（不同ATP浓度）实验——按照前述方法配制含3μM non35 pyAtzA与2μM AtzCM1的组装混合体系，经0.22μm针头滤膜过滤除菌。向每个50μL的反应体系中加入1.2μg Src激酶。使用马尔文Zetasizer Nano ZS粒径分析仪，在低体积石英粒径比色皿（马尔文；ZEN2112）中，以每样品50μL的体系于25℃下连续监测30分钟的粒径变化。实验设置三次生物学重复，每个样品在单个时间点的25秒内进行5次读数并取平均值。使用MATLAB（R2016b版；MathWorks公司）的倾斜样条函数进行曲线拟合，平滑参数可调整。采用调整决定系数(adjusted R²)评估模型拟合有效性。