Journal: ACS synthetic biology

Article Title: Genetically Encoded Fluorescent Biosensor for Rapid Detection of Protein Expression

doi: 10.1021/acssynbio.0c00407

Figure Lengend Snippet: Sensor for transiently expressed proteins (STEP). (a) Cartoon representation of the STEP. A green fluorescent STEP (gSTEP) is expressed and allowed to mature before expression of a STEPtagged protein of interest (not to scale). Prior to STEPtag binding to the Bim peptide, gSTEP is dimly fluorescent (OFF), while the bound gSTEP emits a strong fluorescence signal (ON). (b) Crystal structure of the circularly permuted GFP from the GCaMP3 genetically encoded calcium indicator (PDB ID: 4IK8).37 The chromophore is shown as sticks, and residues forming the N- and C-terminal amino acid linkers are shown as gray spheres and identified by their one-letter code. (c) Surface of the circularly permuted GFP shows a pore on the barrel surface next to the chromophore phenolate moiety (green sticks). (d) Schematic representation of gSTEP0, gSTEP1, and STEPtag. Linker sequences are shown in gray. Circularly permuted GFP (cpGFP) is shown in green, and residues are numbered according to the sequence of Aequorea victoria GFP. Bc1-Xl is shown in magenta, and residues are numbered according to the UniProt sequence (Q07817). 6×His, mBim, and hBim indicate the histidine tag, mouse Bim, and human Bim peptides, respectively.

Article Snippet: Codon-optimized (E. coli ) and his-tagged (N-terminus) sequences for gSTEP0 and STEPtag ( Supplementary Table S1 ) were purchased from ATUM.

Techniques: Expressing, Binding Assay, Fluorescence, Sequencing

Journal: ACS synthetic biology

Article Title: Genetically Encoded Fluorescent Biosensor for Rapid Detection of Protein Expression

doi: 10.1021/acssynbio.0c00407

Figure Lengend Snippet: gSTEP1 enables rapid detection of protein expression in live bacterial cells. (a) Flow cytometry histograms of a representative biological replicate of gSTEP1 fluorescence in live E. coli cells expressing only STEPtag (negative control), gSTEP1 (OFF state), or both (ON state). The pZA vector constitutively expresses gSTEP1, while STEPtag expression from the pBAD vector is induced using 0.2% arabinose. (b) Time course of protein expression in live E. coli. Cells were grown to the end of the exponential growth phase (OD600 = 1.1), then fluorescence was measured immediately after pBAD vectors containing either STEPtag (for cells constitutively expressing gSTEP1), EGFP, or sfGFP were induced with 0.45% arabinose. Each data set was blanked by the fluorescence signal at 0 min, and smoothed by three passes through a seven-point moving average filter. n = 4, the shaded area represents the mean ± s.d. A negative control experiment is also shown, where cells constitutively expressing gSTEP1 have an empty pBAD vector induced (pBAD-empty, pZA-gSTEP1). These cells were grown to OD600 of 0.6, and the shaded area represents the mean ± s.d. for n = 3. Inset: the same time course is shown, extended to 20 min to show the effects of longer-term induction. The same color scheme is used as in the main figure.

Article Snippet: Codon-optimized (E. coli ) and his-tagged (N-terminus) sequences for gSTEP0 and STEPtag ( Supplementary Table S1 ) were purchased from ATUM.

Techniques: Expressing, Flow Cytometry, Fluorescence, Negative Control, Plasmid Preparation

Journal: ACS synthetic biology

Article Title: Genetically Encoded Fluorescent Biosensor for Rapid Detection of Protein Expression

doi: 10.1021/acssynbio.0c00407

Figure Lengend Snippet: In vitro characterization of gSTEP1. All assays were performed in 20 mM sodium phosphate buffer containing 50 mM NaCl (pH 7.4). (a) Normalized excitation (λem = 550 nm, dotted line) and emission (λex = 485 nm, full line) spectra of gSTEP1 (75 nM) in the presence or absence of saturating STEPtag (10 μM). n = 3, average spectra shown. Inset: the fluorescence intensity at 515 nm (λex = 485 nm) of gSTEP1, in the presence or absence of saturating STEPtag, compared to 75 nM EGFP. n = 6 for gSTEP1, n = 3 for EGFP, mean values are shown as black lines. (b) Binding curves of 75 nM gSTEP1 (green) or cpGFP (gray) with STEPtag. Fluorescence is normalized to the maximum intensity observed for gSTEP1. Dashed lines represent fits of the Hill equation to the data (Hill coefficients of 1.5 or 2.2 for gSTEP1 or cpGFP, respectively). For the gSTEP1 binding curve, n = 18, mean ± SEM shown. For cpGFP, n = 3, mean value shown. Kd and ΔF/F0 values were obtained from the fit and indicated with the 95% confidence interval around the fit values. Inset: emission spectra (λex = 485 nm) of 75 nM gSTEP1 alone and in the presence of 10 μM hen egg white lysozyme or bovine serum albumin (BSA). n = 3, average spectra shown. (c) Rapid-mixing stopped-flow binding kinetics of a representative replicate of gSTEP1 mixed with saturating STEPtag. The black line represents a fit of the integrated rate equation to the data (Methods).

Article Snippet: Codon-optimized (E. coli ) and his-tagged (N-terminus) sequences for gSTEP0 and STEPtag ( Supplementary Table S1 ) were purchased from ATUM.

Techniques: In Vitro, Fluorescence, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene

doi: 10.3390/ijms22094345

Figure Lengend Snippet: Three-dimensional model of VvROMT. ( A ) The homodimeric closed conformation of the VvROMT model is shown in ribbon representation with semitransparent surface. ( B ) Surface representation of the active site with binding site residues shown as gray sticks. H261 and E323 are the catalytic residues. F24 and W20 (marked with an asterisk) are contributed by the sister monomer and green labeled residues were engineered by site-directed mutagenesis. SAM is shown in orange sticks and resveratrol in green sticks. Predicted hydrogen bonds are shown as cyan dashed lines, and atoms are colored according to their atom type (carbon, grey; nitrogen, blue; oxygen, red; and sulfur, yellow). Image generated with PyMOL version 2.3.5.

Article Snippet: An E. coli codon-optimized sequence of resveratrol O-methyltransferase, accession no. FM178870 (GenBank), from Vitis vinifera was purchased from GenScript (Piscataway, NJ, USA) .

Techniques: Binding Assay, Labeling, Mutagenesis, Generated

Journal: International Journal of Molecular Sciences

Article Title: Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene

doi: 10.3390/ijms22094345

Figure Lengend Snippet: In silico identification of critical residues in the VvROMT substrate-binding site. The binding site was defined to include all residues at 6 Å distance from resveratrol. Buried surface area (BSA, green), ConSurf conservation score (gray), and VvROMT-resveratrol binding affinity (orange) were calculated. BSA of each residue in contact with resveratrol was calculated with dr_sasa software. The conservation score was estimated by ConSurf. The KDEEP server was used to evaluate the complex’s interaction affinity (ΔΔG) between the wildtype enzyme and the respective alanine variant of each residue. Positive ΔΔG values indicate a loss of binding affinity for the respective variant.

Article Snippet: An E. coli codon-optimized sequence of resveratrol O-methyltransferase, accession no. FM178870 (GenBank), from Vitis vinifera was purchased from GenScript (Piscataway, NJ, USA) .

Techniques: In Silico, Binding Assay, Residue, Software, Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene

doi: 10.3390/ijms22094345

Figure Lengend Snippet: Enzymatic activity of VvROMT alanine variants 1 .

Article Snippet: An E. coli codon-optimized sequence of resveratrol O-methyltransferase, accession no. FM178870 (GenBank), from Vitis vinifera was purchased from GenScript (Piscataway, NJ, USA) .

Techniques: Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene

doi: 10.3390/ijms22094345

Figure Lengend Snippet: Schematic design of the VvROMT variants. For the construction of VvROMT variants (grey) we selected two regions that interact with resveratrol according to our VvROMT substrate-binding site model. The residue substitution is color-coded and is related to the type-I OMTs (blue) included in the analysis; their regioselectivity is indicated in parentheses. As an example, the substitution F311L (cyan) is found in the enzymes PsPMT2 and RhOOMT4, while the substitution F311W (green) is found in the enzyme ROMT13 and the previously reported variant F311W .

Article Snippet: An E. coli codon-optimized sequence of resveratrol O-methyltransferase, accession no. FM178870 (GenBank), from Vitis vinifera was purchased from GenScript (Piscataway, NJ, USA) .

Techniques: Binding Assay, Residue, Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene

doi: 10.3390/ijms22094345

Figure Lengend Snippet: Enzymatic conversion of VvROMT variants. ( A ) Enzymatic reactions started with 350 µM of resveratrol or ( B ) pinostilbene. Reaction conditions: 5 µM of purified enzyme and 700 µM of SAM, incubated in darkness at 30 °C for 24 h and 600 rpm. Reaction products were quantified by HPLC-UV at 306 nm, with a calibration curve using the respective standards. Values represent averages of two replicates. * An undefined product was also observed with this variant.

Article Snippet: An E. coli codon-optimized sequence of resveratrol O-methyltransferase, accession no. FM178870 (GenBank), from Vitis vinifera was purchased from GenScript (Piscataway, NJ, USA) .

Techniques: Purification, Incubation, Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene

doi: 10.3390/ijms22094345

Figure Lengend Snippet: Specific activity of VvROMT variants 1 .

Article Snippet: An E. coli codon-optimized sequence of resveratrol O-methyltransferase, accession no. FM178870 (GenBank), from Vitis vinifera was purchased from GenScript (Piscataway, NJ, USA) .

Techniques: Activity Assay

Journal: Journal of Chemical Theory and Computation

Article Title: Molecular Dynamics-Assisted Optimization of Protein NMR Relaxation Analysis

doi: 10.1021/acs.jctc.1c01165

Figure Lengend Snippet: Internal autocorrelation function for the amide H–N bond vector of Arg 74 in the C-terminal tail of ubiquitin. 15 N R 1 , R 2 , and NOE values were calculated, utilizing the internal autocorrelation function derived from 2 μs CHARMM36 simulations (green) and an isotropic global tumbling time of 4.10 ns. Those derived relaxation values are precisely fitted at 600 MHz (blue) and 900 MHz (red), utilizing the ( S f 2 ,τ s , S 2 ) representation with parameter sets (0.767, 1.252 ns, 0.398) and (0.790, 0.813 ns, 0.423), respectively.

Article Snippet: An E. coli -optimized codon sequence for ubiquitin (Genscript) was cloned into the expression vector pET11a and transformed into BL21-STAR (DE3) (Invitrogen).

Techniques: Plasmid Preparation, Derivative Assay

Journal: Journal of Chemical Theory and Computation

Article Title: Molecular Dynamics-Assisted Optimization of Protein NMR Relaxation Analysis

doi: 10.1021/acs.jctc.1c01165

Figure Lengend Snippet: Internal bond vector autocorrelation functions for the C-terminal tail (Leu 73 to Gly 76) and mobile internal loop (Leu 8 to Thr 12) of ubiquitin as calculated from 2 μs of simulation with CHARMM36 (panels A and C) and AMBER 14SB (panels B and D).

Article Snippet: An E. coli -optimized codon sequence for ubiquitin (Genscript) was cloned into the expression vector pET11a and transformed into BL21-STAR (DE3) (Invitrogen).

Techniques: Plasmid Preparation

Journal: Journal of Chemical Theory and Computation

Article Title: Molecular Dynamics-Assisted Optimization of Protein NMR Relaxation Analysis

doi: 10.1021/acs.jctc.1c01165

Figure Lengend Snippet: Standard deviations for the back-prediction of the six force field-derived internal autocorrelation functions at 600 MHz 1 H for the nine mobile residues of ubiquitin utilizing the ( S f 2 , S H 2 , S N 2 ) and ( S f 2 , S 2 ,τ s ) representations (panels A and B, respectively), as averaged over the time interval between 30 ps and the assumed global tumbling time τ M .

Article Snippet: An E. coli -optimized codon sequence for ubiquitin (Genscript) was cloned into the expression vector pET11a and transformed into BL21-STAR (DE3) (Invitrogen).

Techniques: Derivative Assay

Journal: Journal of Chemical Theory and Computation

Article Title: Molecular Dynamics-Assisted Optimization of Protein NMR Relaxation Analysis

doi: 10.1021/acs.jctc.1c01165

Figure Lengend Snippet: Standard deviations for the back-prediction of the six force field-derived internal autocorrelation functions at 600 MHz (panel A) and 900 MHz (panel B) for the nine mobile ubiquitin residues, utilizing the optimized time constant-constrained triexponential (TCCT) representation ( S f 2 , S h 2 , S x 2 ) as averaged over the time interval between 30 ps and the assumed global tumbling time τ M .

Article Snippet: An E. coli -optimized codon sequence for ubiquitin (Genscript) was cloned into the expression vector pET11a and transformed into BL21-STAR (DE3) (Invitrogen).

Techniques: Derivative Assay

Journal: Journal of Chemical Theory and Computation

Article Title: Molecular Dynamics-Assisted Optimization of Protein NMR Relaxation Analysis

doi: 10.1021/acs.jctc.1c01165

Figure Lengend Snippet: Experimentally determined values of the internal bond vector autocorrelation functions of the ubiquitin backbone at 4.10 ns are displayed as rainbow coloring, with dark blue set to 1.0, green set to 0.69, and red set to 0.0. Residues lacking experimental measurements are set to dark blue.

Article Snippet: An E. coli -optimized codon sequence for ubiquitin (Genscript) was cloned into the expression vector pET11a and transformed into BL21-STAR (DE3) (Invitrogen).

Techniques: Plasmid Preparation

Journal: Journal of Chemical Theory and Computation

Article Title: Molecular Dynamics-Assisted Optimization of Protein NMR Relaxation Analysis

doi: 10.1021/acs.jctc.1c01165

Figure Lengend Snippet: Experimentally determined internal bond vector autocorrelation functions for the C-terminal tail of a ubiquitin sample having an average τ M of 4.10 ns and an ellipsoidal asymmetry of 1.22 utilizing the optimized TCCT representation, with data from 600 MHz indicated in blue and the 900 MHz data indicated in red. The Monte Carlo-derived 95% confidence limits (dashes) were determined, assuming uncertainties of 1%, 1%, 0.01, and 1% for the R 1 , R 2 , NOE, and bond-specific τ M values, respectively.

Article Snippet: An E. coli -optimized codon sequence for ubiquitin (Genscript) was cloned into the expression vector pET11a and transformed into BL21-STAR (DE3) (Invitrogen).

Techniques: Plasmid Preparation, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Characterization of an Atypical Trypanosoma brucei Hsp70 Demonstrates Its Cytosolic-Nuclear Localization and Modulation by Quercetin and Methylene Blue

doi: 10.3390/ijms22136776

Figure Lengend Snippet: TbHsp70 and TbHsp70.4 directly interact with TbSTi1. Interaction between the TbHsp70s, TbHsp70 and TbHsp70.4 and TbSTi1 was investigated using far western analysis. TbHsp70 (50 µg); control protein, BSA (50 µg) and TbSTi1 at various concentrations (25 µg, 50 µg, 75 µg, 100 µg) were resolved by SDS-PAGE and transferred onto a blot and the blot was probed using rabbit polyclonal anti-TbHsp70.4 ( A ; top panel). A similar blot was then overlaid with TbHsp70 and it was probed using anti-TbHsp70 ( A ; bottom panel). ( B ) TbHsp70.4 protein (50 µg); control protein, BSA (50 µg) and TbSTi1 protein at various concentrations (25 µg, 50 µg, 75 µg, 100 µg) were resolved by SDS-PAGE and transferred to a blot and the blot was probed using rabbit polyclonal anti-TbHsp70.4 ( B ; top panel). A similar blot was then overlaid with TbHsp70.4 (75 µg) and it was probed using anti-TbHsp70.4 ( B ; bottom panel). The blots are representative of independent replicates.

Article Snippet: The full-length E. coli codon-optimized sequence of TBSTI1 (TriTrypDB accession number: Tb927.5.2190) was synthesized and supplied by the GenScript Corporation (Piscataway, NJ, USA) and inserted into the pQE60 (Qiagen, Germantown, MD, USA) between the Bam HI and Hind III restriction sites to generate the pQE60-TbSTi expression plasmid with an N-terminal HA epitope.

Techniques: Western Blot, Control, SDS Page

Journal: International Journal of Molecular Sciences

Article Title: Characterization of an Atypical Trypanosoma brucei Hsp70 Demonstrates Its Cytosolic-Nuclear Localization and Modulation by Quercetin and Methylene Blue

doi: 10.3390/ijms22136776

Figure Lengend Snippet: TbSTi1 interacts with TbHsp70 but not with TbHsp70.c. Far western analysis was used to investigate the interactions between TbHsp70.c ( A ) and TbHsp70 ( B ) with TbSTi1. BSA (20 µg/mL) as negative control protein, TbSTi1 (20 µg/mL) as positive control protein, and TbHsp70 or TbHsp70.c proteins at various concentrations (2.5 µg/mL, 5 µg/mL, 10 µg/mL, and 20 µg/mL) were resolved by SDS-PAGE and transferred to the respective blots that were then probed using anti-HA antibody (top panel), an epitope of TbSTi1. Similarly transferred blots were overlaid with TbSTi1 then also probed with anti-HA antibody (bottom panel). The blots are representative of independent replicates.

Article Snippet: The full-length E. coli codon-optimized sequence of TBSTI1 (TriTrypDB accession number: Tb927.5.2190) was synthesized and supplied by the GenScript Corporation (Piscataway, NJ, USA) and inserted into the pQE60 (Qiagen, Germantown, MD, USA) between the Bam HI and Hind III restriction sites to generate the pQE60-TbSTi expression plasmid with an N-terminal HA epitope.

Techniques: Western Blot, Negative Control, Positive Control, SDS Page