Supplementary Figure 9 Experimental selection of AtzA, AtzC subunits for characterization

Article: Stimulus-responsive Self-Assembly of Protein-Based Fractals by Computational Design

Pre-print: bioRxiv 274183; doi: https://doi.org/10.1101/274183

Figure: Fig. S9. Experimental selection of AtzA, AtzC subunits for characterization. (A) ELISA screening of AtzA designs to determine phosphorylation levels. (B) DLS size distribution of AtzA designs with AtzCM0. (C) DLS size distribution of AtzC-SH2 designs with pY-AtzA. Samples prepared at 3 µM pY-AtzA, 2 µM AtzC-SH2 design. Only AtzCM1 and AtzCM3 showed assembly formation with pY-AtzA. Volume distribution reported. (D) BLI binding traces of AtzC2 SH2 designs with pY-AtzA. AtzC-SH2 designs were screened for binding with BLI, using pY3 AtzA as the load. Out of all AtzC-SH2 designs prepared, AtzCM1 had the highest binding affinity to pY-AtzA. Based on the assembly formation and binding data, AtzCM1 was chosen for further investigation.

Figure supports Fig S8 Experimental selection process for pY-AtzA and AtzC-SH2.
Fig. S8. Experimental selection process for pY-AtzA and AtzC-SH2. Five N-terminal SH2 binding peptide AtzA fusions (AtzAM1-AtzAM5) and five C-terminal SH2 binding domain AtzC fusions (AtzCM1-AtzCM5) were selected, cloned, expressed, and purified. AtzAM1-M5 were screened for having the ability to be phosphorylated via ELISA with anti-phosphotyrosine. Only two AtzA designs, AtzAM1 and AtzAM3, showed strong phosphorylation. The ability for assembly formation to occur with a direct C-terminal SH2 binding domain AtzC fusion (no mutations; AtzCM0) was used to select the best AtzA design. AtzAM1 was chosen for superior assembly formation ability, becoming pY-AtzA. The five AtzC designs AtzCM1-AtzCM5 were screened for the ability to effectively bind and assemble with pY-AtzA. The combination of pY- 35 AtzA and AtzCM1 (which we call AtzC-SH2) showed the strongest binding and the most robust assembly formation. This pair was then chosen for further characterization.

Comment:

A) ELISA protocol provided in SI 2.7. AtzA M1-M5 was phosphorylated and ELISA performed in triplicate. Values represent average reads for absorbance at 450 nm. Negative control (no kinase added, no phosphorylation) is provided in data table.

B) DLS between AtzAM1, M3 and AtzCM0. DLS was performed in triplicate and averages of the % volume distribution is reported. Negative control data provided. Graphs for both negative control and assembly are provided in data file as well.

C) DLS between AtzAM1 and AtzCM. DLS was performed in triplicate and averages of the % volume distribution is reported.

D) BLI between AtzAM1 and AtzCM1-M5. Buffer trace provided as control. See BLI protocol for details (SI 2.8).

(SI 2.8) Bio-layer interferometry (BLI) – AtzAM1 was phosphorylated using the conditions described below. pY-AtzAM1 was then biotinylated at 10mM Sulfo-NHS-Biotin (APExBIO) for 30min at 25°C. Excess biotin was buffer exchanged with a PD-10 desalting column (GE Healthcare) equilibrated with HNG. Biotinylated pY-AtzAM1 was loaded onto streptavidin (SA) coated biosensors (ForteBio) and used for BLI. AtzCM1 was flowed in from 4nM to 4μM. BLI experiments were performed using the BLItz System (ForteBio).

(SI 2.7) Enzyme-linked immunosorbent assay (ELISA) – Phosphorylated AtzAM1 (pY27 AtzAM1) was loaded onto clear flat-bottom immuno 96-well plates (Thermo Scientific item #442404) at 20μg/mL and 1.25μg/mL in 50μL 1X PBS (Gibco pH 7.4, #10010023) overnight at 4◦C. Plates were rinsed twice in 200μL 1X TBS (Biorad #1706435). 1% BSA in TBS 0.05% Tween 20 was used to block wells at 200μL block solution for 1.5hr at 25°C under gentle agitation. Anti-phosphotyrosine 4G10 Platinum HRP conjugate (EMD #16-316) was diluted 1:5000 in 1% BSA TBS 0.05% Tween 20 and loaded onto the well at 25°C for 1.5hr under gentle agitation. Excess anti-phosphotyrosine was washed off with 200μL of TBS 0.05% Tween 20 in triplicate. To detect bound antibody, 100μL of TMB substrate reagent (Biolegend #421101) was added to each well and incubated for 5 minutes at 25°C. 100μL of TMB stop solution (Biolegend #423001) was added to the wells. Absorbance was read at 450nm using the Tecan Infinite M200 Pro plate reader.

(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.
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Note: This work was part of the Rutgers Biomod 2016 Project (M. Liu, A. Permaul, O. Dineen, M. Khalid, M. Shea, G.L. Bilker). See reference below.

文章：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：补充图S9 用于表征的AtzA、AtzC亚基实验筛选。(A) 酶联免疫吸附测定（ELISA）筛选AtzA设计变体以确定磷酸化水平。(B) 搭载AtzCM0的AtzA设计变体的动态光散射（DLS）粒径分布。(C) 搭载pY-AtzA的AtzC-SH2设计变体的动态光散射粒径分布。实验样品以3 µM pY-AtzA、2 µM AtzC-SH2设计变体配制。仅AtzCM1与AtzCM3可与pY-AtzA发生自组装，结果以体积分布形式呈现。(D) AtzC2 SH2设计变体与pY-AtzA的生物层干涉术（BLI）结合曲线。以pY3 AtzA为负载物，通过生物层干涉术筛选AtzC-SH2设计变体的结合能力。在所制备的全部AtzC-SH2设计变体中，AtzCM1与pY-AtzA的结合亲和力最高。基于自组装形成情况与结合数据，最终选择AtzCM1开展后续研究。

本图支撑补充图S8中pY-AtzA与AtzC-SH2的实验筛选流程。补充图S8：pY-AtzA与AtzC-SH2的实验筛选流程。本研究选取5种N端SH2结合肽AtzA融合蛋白（AtzAM1~AtzAM5）与5种C端SH2结合结构域AtzC融合蛋白（AtzCM1~AtzCM5），分别进行克隆、表达与纯化。通过抗磷酸酪氨酸ELISA筛选AtzAM1~M5的磷酸化能力，仅AtzAM1与AtzAM3两种AtzA设计变体呈现出较强的磷酸化水平。以直接融合C端SH2结合结构域的AtzC（无突变；AtzCM0）的自组装形成能力作为筛选标准，选取最优AtzA设计变体。因AtzAM1的自组装能力更优异，将其改造为pY-AtzA。随后针对5种AtzC设计变体AtzCM1~AtzCM5，筛选其与pY-AtzA有效结合并发生自组装的能力。pY-AtzA与AtzCM1（本研究中将其命名为AtzC-SH2）的组合呈现出最强的结合能力与最稳定的自组装效果，因此选取该组合开展后续表征研究。

评论：
(A) ELISA实验流程详见补充材料2.7。对AtzAM1~M5进行磷酸化处理，并进行三次重复ELISA实验，结果以450 nm处吸光度的平均读数表示。阴性对照（未添加激酶、无磷酸化过程）的相关数据已在数据表中给出。
(B) AtzAM1、AtzAM3与AtzCM0的动态光散射检测。实验进行三次重复，结果以体积百分比分布的平均值呈现，同时提供了阴性对照数据。阴性对照与自组装组的相关图谱均已包含在数据文件中。
(C) AtzAM1与AtzCM的动态光散射检测。实验进行三次重复，结果以体积百分比分布的平均值呈现。
(D) AtzAM1与AtzCM1~AtzCM5的生物层干涉术检测，以缓冲液扫描曲线作为对照。详细实验流程参见BLI操作方案（补充材料2.8）。

（补充材料2.8）生物层干涉术（BLI）——按照下述条件对AtzAM1进行磷酸化处理。随后将pY-AtzAM1与10 mM Sulfo-NHS-生物素（APExBIO）在25℃下孵育30分钟以完成生物素标记。使用经HNG平衡的PD-10脱盐柱（GE Healthcare）去除过量的生物素。将生物素标记后的pY-AtzAM1加载至包被有链霉亲和素（SA）的生物传感器（ForteBio）上，用于BLI实验。以4 nM至4 μM的浓度梯度注入AtzCM1。BLI实验使用BLItz系统（ForteBio）完成。

（补充材料2.7）酶联免疫吸附测定（ELISA）——将磷酸化的AtzAM1（pY27 AtzAM1）以20 μg/mL与1.25 μg/mL的浓度，在50 μL 1×磷酸盐缓冲液（PBS，Gibco，pH 7.4，货号#10010023）中包被至透明平底免疫96孔板（Thermo Scientific，货号#442404），4℃孵育过夜。用200 μL 1×Tris缓冲盐溶液（TBS，Bio-Rad，货号#1706435）洗涤板孔两次。使用含1%牛血清白蛋白（BSA）与0.05%吐温20的TBS溶液作为封闭液，每孔加入200 μL，在25℃下轻柔振荡孵育1.5小时以封闭非特异性结合位点。将抗磷酸酪氨酸4G10 Platinum辣根过氧化物酶（HRP）偶联物（EMD，货号#16-316）以1:5000的比例稀释于含1% BSA与0.05%吐温20的TBS中，每孔加入该稀释液后在25℃下轻柔振荡孵育1.5小时。用200 μL含0.05%吐温20的TBS洗涤孔三次以去除未结合的抗磷酸酪氨酸抗体。为检测结合的抗体，每孔加入100 μL四甲基联苯胺（TMB）底物试剂（Biolegend，货号#421101），25℃下孵育5分钟。随后每孔加入100 μL TMB终止液（Biolegend，货号#423001），使用Tecan Infinite M200 Pro酶标仪读取450 nm处的吸光度值。

（补充材料2.9）磷酸化、自组装与解组装——本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）。在总反应体积150 μL体系中，将3 μM AtzAM1加入至1×激酶活性缓冲液（含4 mM MgCl2、2.5 mM MnCl2、0.25 mM DTT、5 mM MOPS、2.5 mM 甘油-2-磷酸、1 mM EGTA、400 nM EDTA，pH 7.6）、2.5 mM MnCl2、HNG、2 mM ATP与800 ng Src激酶中，25℃下孵育7~16小时以完成磷酸化。磷酸化完成后，加入AtzCM1至终浓度2 μM，25℃下孵育2小时以诱导自组装形成。自组装完成后，向150 μL反应体系中加入4.8 μg YopH磷酸酶以实现解组装。通过动态光散射（DLS）检测粒径变化，以确定自组装/解组装效果。

（补充材料2.10）动态光散射（DLS）——取50 μL自组装样品，使用Malvern Zetasizer粒径分析仪与石英比色皿（ZEN2112，Malvern）进行粒径检测。在25℃下进行11次重复实验，每次实验记录10条光谱数据。实验标准操作流程已考虑溶液中5%甘油的影响，结果以体积百分比形式收集并呈现。

注：本研究为罗格斯大学（Rutgers）2016年Biomod项目的一部分（参与人员：M. Liu、A. Permaul、O. Dineen、M. Khalid、M. Shea、G.L. Bilker）。相关参考文献见下文。