Figure 2: Assembly Formation, Dissolution and Inhibition in vitro

Article: Stimulus-responsive Self-Assembly of Protein-Based Fractals by Computational Design 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 647TM-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 Rutgers Biomod 2016 project (M. Liu, A. Permaul, O. Dineen, M. Shea, M. Khalid, G.L. Bilker). See reference for additional details.

论文：基于计算设计的蛋白质分形刺激响应自组装 预印本：bioRxiv 274183；DOI：https://doi.org/10.1101/274183

图2。(A) 利用Src激酶可将AtzAM1磷酸化（得到pY-AtzAM1），将其与AtzCM1-SH2共孵育即可形成组装体；而磷酸酶YOP可实现组装体的解聚。
(B) 与(C) 预期分别形成（B）与溶解（C）的组装体4，该结果经动态光散射（Dynamic Light Scattering, DLS）实验验证。
(D) 将组装组分与不同浓度的游离SH2结构域以及一种不同的（单价）SH2融合蛋白共孵育，可实现显著的组装抑制。
(E) ATP浓度可调控组装体的形成速率（0时刻从顶部到底部依次对应ATP浓度从高到低，共8组样本）
(F) 与(G) 组装体的形成对组分化学计量比具有高度敏感性。相较于“超强结合型”SH2结构域（G），改变化学计量比（F、G）以及使用结合力更弱的SH2-肽相互作用（F），会扰乱组装形成区域11。
(H) 光学显微镜下观察到的分形类结构。
(I) 经Alexa Fluor 647™标记的AtzCM1-SH2与pY-AtzAM1形成的组装体的荧光显微镜图像。

(SI 2.9) 磷酸化、组装形成与解聚——磷酸化实验方案基于Sigma公司的Src激酶活性检测方法（货号：S1076）。在总体积150μL的反应体系中，将3μM AtzAM1加入至1×激酶活性缓冲液（Kinase Activity Buffer, KAB，含4mM MgCl₂、2.5mM MnCl₂、0.25mM DTT、5mM MOPS、2.5mM甘油-2-磷酸、1mM EGTA、400nM EDTA，pH 7.6），随后加入2.5mM MnCl₂、HNG、2mM ATP、800ng Src激酶，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，于25℃孵育2小时以形成组装体。组装完成后，向150μL反应体系中加入4.8μg YopH磷酸酶以实现解聚。通过动态光散射（DLS）检测粒径以判定组装体形成/解聚情况。

(SI 2.10) 动态光散射（DLS）——取50μL组装体样品，使用马尔文Zetasizer仪器与石英比色皿（ZEN2112，马尔文）进行粒径测定。于25℃下对11个重复样本各采集10条光谱数据。标准操作流程需考虑溶液中5%的甘油含量。

(SI 2.11) DLS抑制实验——将6μM pY-AtzAM1置于75μL反应体系中进行磷酸化（含1×KAB、2mM ATP、1mM DTT、HNG、1μg Src激酶），于25℃孵育过夜。向每个样品中加入终浓度分别为0μM、3μM、6μM、9μM、12μM、15μM、18μM的SH2或SH2-DhaA，于25℃孵育1小时以“封闭”pY10 AtzAM1上的结合位点。随后向每个样品中加入AtzCM1至终浓度2μM。因此所有组分的终浓度为：3μM pyAtzA、1μM AtzCM1、0~18μM SH2或SH2-DhaA。将样品于25℃孵育2小时后，通过DLS分析组装体粒径。DLS检测条件为：25℃、每个样品体积50μL，使用低体积石英粒径比色皿（马尔文；ZEN2112）与Zetasizer Nano ZS仪器（马尔文）。每个样品重复测定3次，每次读取15条数据并取平均值。该实验方案以终浓度1μM pyAtzA、0.66μM AtzCM1、0~6μM SH2-DhaA重复进行。使用MATLAB（R2016b；MathWorks）进行曲线拟合，拟合模型为：f(x) = A/(1+e^(-k(x-x0))) + B，其中A、B、k、x0为常数。采用校正R²判定模型有效性。半抑制浓度（IC50）基于使组装体粒径达到100nm的抑制剂浓度计算得到。

(SI 2.12) DLS滴定实验——将6μM、3μM、1.5μM、0.5μM、0.1μM的pyAtzA按前述方法进行磷酸化，于25℃孵育过夜。向每个样品中分别加入终浓度为2μM、1μM、0.5μM、0.25μM、0.50μM的野生型AtzCM1（WT）或超强结合型AtzCM1（SB），于25℃孵育2小时。因此所有组分的终浓度范围为：3μM~0.05μM pyAtzA、2μM~0.05μM AtzCM1-WT或AtzCM1-SB。DLS检测条件为：25℃、每个样品体积50μL，使用低体积石英粒径比色皿（马尔文；ZEN2112）与Zetasizer Nano ZS仪器（马尔文）。每个样品重复测定2次，每次读取15条数据并取平均值。

本研究部分内容隶属于罗格斯大学（Rutgers）Biomod 2016项目（作者：M. Liu、A. Permaul、O. Dineen、M. Shea、M. Khalid、G.L. Bilker），更多细节可参见相关参考文献。