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.<br> 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.****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.