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Modeled active site of AtzA.
No full text<p>A homology model of the active site of AtzA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039822#pone.0039822-Scott1" target="_blank">[20]</a> was used to illustrate the positions of five of the nine amino acid differences between AtzA and TriA. Shown here are the AtzA substrate (atrazine; green), amino acids identical in both AtzA and TriA (Q71, W87, L88, Q96, N126, M155, A216, A220, E246 and D250; white), and amino acids that differ between AtzA and TriA (positions 84, 217, 219, 328 and 331; purple).</p
Evolution from AtzA to TriA (adapted from reference [34]).
No full text(A) AtzA catalyzes the dechlorination of atrazine (AtzA activity). TriA catalyzes the deamination of melamine (TriA activity). TriA catalyzed the dechlorination reaction promiscuously. Deamination by AtzA could not be detected. (B) A possible uphill evolutionary trajectory from AtzA to TriA determined by Noor et al. In each round of evolution, a single point mutation was added in the order shown in (C)—(F) (see also S8 Table). (C)—(F) Effect of all single point mutations separating AtzA and TriA (S9 and S10 Tables). (C) Effect of mutations in the AtzA background on AtzA activity and (D) TriA activity. (E) Effect of mutations in the TriA background on TriA activity and (F) AtzA activity. Activities are expressed as kcat/KM values. Relative activities could not be calculated because several variants do not have detectable activity. Amino acids found in AtzA are shown in lower-case italics.</p
Supplementary Figure 7 Phosphorylation of SH2 peptide AtzA fusion (pY-AtzA) by Src kinase
No full textArticle: Stimulus-responsive Self-Assembly of Protein-Based Fractals by Computational Design
Pre-print: bioRxiv 274183; doi: https://doi.org/10.1101/274183
Figure: S7. Phosphorylation of SH2 peptide AtzA fusion (pY-AtzA) by Src kinase. In order to verify phosphorylation of AtzA by Src kinase into phosphorylated SH2 peptide AtzA fusion (pY-AtzA), ELISA with (1:4000 dilution) antiphosphotyrosine-horseradish peroxidase conjugate was performed on pY-AtzA samples either with Src kinase (+) or without Src kinase (-), in phosphorylation reaction buffer at 1.25 µg/mL pY-AtzA or 20 µg/mL pY-AtzA. Data is presented as mean ± 1 standard deviation.
(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 2.7) 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.****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 for additional details.</p
Supplementary Figure 10 Biolayer interferometry (BLI) binding profiles of AtzC wildtype SH2 fusion (AtzC9 wtSH2) and AtzC superbinder SH2 fusion (AtzC-SH2) to phosphorylated SH2 binding peptide AtzA fusion (pY-AtzA)
No full textArticle: Stimulus-responsive Self-Assembly of Protein-Based Fractals by Computational Design
Pre-print: bioRxiv 274183; doi: https://doi.org/10.1101/274183
Figure: S10. Biolayer interferometry (BLI) binding profiles of AtzC wildtype SH2 fusion (AtzC9 wtSH2) and AtzC superbinder SH2 fusion (AtzC-SH2) to phosphorylated SH2 binding peptide AtzA fusion (pY-AtzA). (A) Binding profile of AtzC-wtSH2 to pY-AtzA. PY-AtzA was loaded onto the biosensor via a streptavidin-biotin interaction. AtzC-wtSH2 was flowed into the sample. KD = 41.79 ± 0.32 nM. (B) Binding profile of AtzCM1 (superbinder) to pY-AtzA. PY AtzA was loaded onto the biosensor via a streptavidin-biotin interaction. AtzC-SH2 was flowed into the sample. KD = 7.67 ± 0.52 nM.
Comment. KD was calculated using ForteBio BLItz software. KD statistical analysis provided in datafile.
(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).****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.</p
Supplementary Figure 9 Experimental selection of AtzA, AtzC subunits for characterization
No full textArticle: 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.****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.</p
Ongoing functional evolution of the bacterial atrazine chlorohydrolase AtzA
No full textTriazine herbicides such as atrazine and simazine which were heavily used in the latter half of the twentieth century constituted a rich new source of nitrogen for soil microbes. An atzA dechlorinase active against both atrazine and simazine was isolate
Partial step-wise laboratory evolution of TriA to AtzA.
No full text<p>Circles indicate the variants for which the <i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub> values (s<sup>−1</sup>.M<sup>−1</sup>; values in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039822#pone-0039822-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039822#pone.0039822.s003" target="_blank">Table S2</a>) for atrazine dechlorination and melamine deamination were determined, and are color coded as follows: TriA, red; generation 1, orange; generation 2, green; generation 3, blue; AtzA, violet. Lines are used to link variants differing by one substitution (thick lines link optimal variants; thin lines link the optimal variants to suboptimal variants – suboptimal variants were not used to generate subsequent variants). Amino acid substitutions discussed in the text have been labelled for clarity, as have the wild-type AtzA and TriA enzymes.</p
Seasonal detection of atrazine and atzA in man-made waterways receiving agricultural runoff in a subtropical, semi-arid environment (Hidalgo County, Texas, USA)
No full textAtrazine is a widely-used herbicide that can impact non-target organisms in the environment but can be biologically degraded by several types of microorganisms. In this study, the gene atzA, which encodes for the initial step in bacterially-mediated atrazine degradation, was used as an indicator of atrazine pollution in agricultural canals located in Hidalgo County, Texas, USA. The concentration of atrazine and atzA were monitored once per month for 12 months during 2010–2011. Atrazine was measured using an enzyme-linked immunosorbent assay; atzA abundance was monitored using Quantitative Polymerase Chain Reaction (Q-PCR) analyses. Abundance of atrazine and atzA were compared with rainy versus dry months and during planting versus non-planting months. Results showed that atrazine levels varied from below detection to 0.43 ppb and were not influenced by precipitation or planting season. Concentrations of the gene atzA were significantly different in rainy versus dry months; during planting versus non-planting times of the year; and in the interaction of precipitation and planting season. The highest concentration of atzA, approx. 4.57 × 108 gene copies ml−1, was detected in July 2010—a rainy, planting month in Hidalgo County, South Texas. However, atrazine was below detection during that month. We conclude that Q-PCR using atzA as an indicator gene is a potential method for monitoring low levels of atrazine pollution in environmental samples
Tecnica combinata (fibrobroncoscopio introdotto attraverso broncospio rigido) per la rimozione dei corpi estranei endobronchiali in età pediatrica
No full textReaction schemes for melamine deaminase (TriA) and atrazine dechlorinase (AtzA).
No full text<p>The hydrolytic deamination of melamine to ammeline by TriA and the dechlorination of atrazine to 2-hydroxyatrazine by AtzA are shown. TriA also possesses a low level of atrazine dechlorinase activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039822#pone.0039822-Seffernick2" target="_blank">[17]</a>.</p
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