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fluc n hibit  (Addgene inc)


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    Addgene inc fluc n hibit
    a Conceptual framework for an SDR assay using terminal fusion protein of interest. The equilibrium between apo ( i ) and ligand-bound protein ( ii ) can be distinguished by the sensor ( iii ) activity, where a reddish color represents a dynamic apo protein relative to a more static blue ligand-bound protein. Created in BioRender. Inglese, J. (2025) https://BioRender.com/26yvpbw . b Key steps in an SDR assay. In this study the assay was performed in a 7 μL volume (4 μL target protein/ligand plus 3 μL detection reagent) permitting 1536-well plate quantitative high-throughput screening, qHTS (see Methods for full protocol). c SDR assay proof-of-concept using a C -terminal NLuc fusion to firefly luciferase (FLuc- C -NLuc, 1 nM), or d an N - or C -terminal fusion of <t>HiBiT</t> to FLuc (FLuc- N- or -C -HiBiT, 10 nM). The left axes indicate RLU generated by the enzyme activity of FLuc with increasing concentrations of FLuc inhibitor PTC124, for a C -terminal NLuc fusion ( c , black circles), or an N - (black circle) or C - (gray circle) HiBiT fusion ( d ) protein. The right axes indicate ligand-dependent SDR RLU, measured from NLuc ( c ) FLuc- C -NLuc (inverted blue triangle) or reconstituted NLuc ( d ) FLuc N - (blue triangle) or C - (blue square) HiBiT, with increasing PTC124 concentration in the presence (solid symbols) or absence (open symbols) of 10 µM ATP. Error bars are SD, n = 6 experiments. e qHTS waterfall plot illustrating concentration response curves (CRCs) obtained for a library of 1,343 compounds enriched for FLuc inhibitor chemotypes. Black CRCs from FLuc enzyme assay and blue CRCs from the 10 nM FLuc- C -HiBiT SDR assay (+ ATP condition). f Representative CRC from ( e ) for indicated clades, where symbols are activity from: SDR, plus ATP (blue solid square), SDR, no ATP (blue open square), and FLuc enzyme (black circle) assays. For full SAR around clade P see Supplementary Fig. . g Comparative SDR activity of clade representative compounds using the 1 nM FLuc- C -NLuc protein with ATP (blue solid triangle), no ATP (blue open triangle), or 1 nM NLuc without ATP (open red circle), or with ATP (solid red circle). Supplementary Data , support ( e − g ). RLU, relative light units; No., number; Cpd., compound. Source data are provided in a Source Data file.
    Fluc N Hibit, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A general assay platform to study protein pharmacology using ligand-dependent structural dynamics"

    Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

    Journal: Nature Communications

    doi: 10.1038/s41467-025-59658-6

    a Conceptual framework for an SDR assay using terminal fusion protein of interest. The equilibrium between apo ( i ) and ligand-bound protein ( ii ) can be distinguished by the sensor ( iii ) activity, where a reddish color represents a dynamic apo protein relative to a more static blue ligand-bound protein. Created in BioRender. Inglese, J. (2025) https://BioRender.com/26yvpbw . b Key steps in an SDR assay. In this study the assay was performed in a 7 μL volume (4 μL target protein/ligand plus 3 μL detection reagent) permitting 1536-well plate quantitative high-throughput screening, qHTS (see Methods for full protocol). c SDR assay proof-of-concept using a C -terminal NLuc fusion to firefly luciferase (FLuc- C -NLuc, 1 nM), or d an N - or C -terminal fusion of HiBiT to FLuc (FLuc- N- or -C -HiBiT, 10 nM). The left axes indicate RLU generated by the enzyme activity of FLuc with increasing concentrations of FLuc inhibitor PTC124, for a C -terminal NLuc fusion ( c , black circles), or an N - (black circle) or C - (gray circle) HiBiT fusion ( d ) protein. The right axes indicate ligand-dependent SDR RLU, measured from NLuc ( c ) FLuc- C -NLuc (inverted blue triangle) or reconstituted NLuc ( d ) FLuc N - (blue triangle) or C - (blue square) HiBiT, with increasing PTC124 concentration in the presence (solid symbols) or absence (open symbols) of 10 µM ATP. Error bars are SD, n = 6 experiments. e qHTS waterfall plot illustrating concentration response curves (CRCs) obtained for a library of 1,343 compounds enriched for FLuc inhibitor chemotypes. Black CRCs from FLuc enzyme assay and blue CRCs from the 10 nM FLuc- C -HiBiT SDR assay (+ ATP condition). f Representative CRC from ( e ) for indicated clades, where symbols are activity from: SDR, plus ATP (blue solid square), SDR, no ATP (blue open square), and FLuc enzyme (black circle) assays. For full SAR around clade P see Supplementary Fig. . g Comparative SDR activity of clade representative compounds using the 1 nM FLuc- C -NLuc protein with ATP (blue solid triangle), no ATP (blue open triangle), or 1 nM NLuc without ATP (open red circle), or with ATP (solid red circle). Supplementary Data , support ( e − g ). RLU, relative light units; No., number; Cpd., compound. Source data are provided in a Source Data file.
    Figure Legend Snippet: a Conceptual framework for an SDR assay using terminal fusion protein of interest. The equilibrium between apo ( i ) and ligand-bound protein ( ii ) can be distinguished by the sensor ( iii ) activity, where a reddish color represents a dynamic apo protein relative to a more static blue ligand-bound protein. Created in BioRender. Inglese, J. (2025) https://BioRender.com/26yvpbw . b Key steps in an SDR assay. In this study the assay was performed in a 7 μL volume (4 μL target protein/ligand plus 3 μL detection reagent) permitting 1536-well plate quantitative high-throughput screening, qHTS (see Methods for full protocol). c SDR assay proof-of-concept using a C -terminal NLuc fusion to firefly luciferase (FLuc- C -NLuc, 1 nM), or d an N - or C -terminal fusion of HiBiT to FLuc (FLuc- N- or -C -HiBiT, 10 nM). The left axes indicate RLU generated by the enzyme activity of FLuc with increasing concentrations of FLuc inhibitor PTC124, for a C -terminal NLuc fusion ( c , black circles), or an N - (black circle) or C - (gray circle) HiBiT fusion ( d ) protein. The right axes indicate ligand-dependent SDR RLU, measured from NLuc ( c ) FLuc- C -NLuc (inverted blue triangle) or reconstituted NLuc ( d ) FLuc N - (blue triangle) or C - (blue square) HiBiT, with increasing PTC124 concentration in the presence (solid symbols) or absence (open symbols) of 10 µM ATP. Error bars are SD, n = 6 experiments. e qHTS waterfall plot illustrating concentration response curves (CRCs) obtained for a library of 1,343 compounds enriched for FLuc inhibitor chemotypes. Black CRCs from FLuc enzyme assay and blue CRCs from the 10 nM FLuc- C -HiBiT SDR assay (+ ATP condition). f Representative CRC from ( e ) for indicated clades, where symbols are activity from: SDR, plus ATP (blue solid square), SDR, no ATP (blue open square), and FLuc enzyme (black circle) assays. For full SAR around clade P see Supplementary Fig. . g Comparative SDR activity of clade representative compounds using the 1 nM FLuc- C -NLuc protein with ATP (blue solid triangle), no ATP (blue open triangle), or 1 nM NLuc without ATP (open red circle), or with ATP (solid red circle). Supplementary Data , support ( e − g ). RLU, relative light units; No., number; Cpd., compound. Source data are provided in a Source Data file.

    Techniques Used: Activity Assay, High Throughput Screening Assay, Luciferase, Generated, Concentration Assay, Enzymatic Assay

    a ABL1 kinase domain bound to PD166326 inhibitor (dark blue) and myristic acid (red) (PDB: 1OPK), Nagar B et al. b SDR output of imatinib (blue square) or ATP (blue diamond) binding to 0.5 nM (open symbol) or 10 nM (solid symbol) ABL1- N -HiBiT kinase domain or imatinib inhibition of kinase activity (black circle) using Kinase-Glo Plus reagent (KGP). Error bars represent the SD, n = 3 experiments (Imatinib SDR) or n = 4 experiments (ATP SDR and imatinib kinase activity). c Correlation plot comparing the ABL1 KGP assay pIC 50 vs pSDR 50 (10 nM enzyme without ATP) for all inhibitors identified from the 128-member kinase inhibitor library. Colored symbols reference compounds discussed in Results section. For underlying data see Supplementary Data , , and Supplementary Fig. . d , e SDR output for myristate site ligands asciminib (red solid triangle) and GNF-5 (red open triangle) compared to imatinib (blue square) and nilotinib (open square) for ABL1- C- or - N -HiBiT, respectively. f Effect of allosteric ligands on enzyme catalysis (imatinib, black circle, asciminib, solid red triangle, and GNF-5, open red triangle). Error bars for d , e , and f represent the SEM, n = 2 technical replicates. g Protein kinase A (PKA) bound to H-89 inhibitor (dark blue) (PDB: 1YDT), Engh RA et al. h PKA enzyme activity (black circle) and SDR assay (blue square) CRC for H-89. Error bars represent the SD, n = 3 experiments i Correlation plot comparing the PKA KGP assay pIC 50 to PKA SDR assay pSDR 50 (without ATP). H-89 (blue square) and staurosporine (staur., red circle) indicated. See Supplementary Data and Supplementary Fig. for underlying data and compound information. Supplementary Data – supports panels ( c , i ). Source data are provided in a Source Data file.
    Figure Legend Snippet: a ABL1 kinase domain bound to PD166326 inhibitor (dark blue) and myristic acid (red) (PDB: 1OPK), Nagar B et al. b SDR output of imatinib (blue square) or ATP (blue diamond) binding to 0.5 nM (open symbol) or 10 nM (solid symbol) ABL1- N -HiBiT kinase domain or imatinib inhibition of kinase activity (black circle) using Kinase-Glo Plus reagent (KGP). Error bars represent the SD, n = 3 experiments (Imatinib SDR) or n = 4 experiments (ATP SDR and imatinib kinase activity). c Correlation plot comparing the ABL1 KGP assay pIC 50 vs pSDR 50 (10 nM enzyme without ATP) for all inhibitors identified from the 128-member kinase inhibitor library. Colored symbols reference compounds discussed in Results section. For underlying data see Supplementary Data , , and Supplementary Fig. . d , e SDR output for myristate site ligands asciminib (red solid triangle) and GNF-5 (red open triangle) compared to imatinib (blue square) and nilotinib (open square) for ABL1- C- or - N -HiBiT, respectively. f Effect of allosteric ligands on enzyme catalysis (imatinib, black circle, asciminib, solid red triangle, and GNF-5, open red triangle). Error bars for d , e , and f represent the SEM, n = 2 technical replicates. g Protein kinase A (PKA) bound to H-89 inhibitor (dark blue) (PDB: 1YDT), Engh RA et al. h PKA enzyme activity (black circle) and SDR assay (blue square) CRC for H-89. Error bars represent the SD, n = 3 experiments i Correlation plot comparing the PKA KGP assay pIC 50 to PKA SDR assay pSDR 50 (without ATP). H-89 (blue square) and staurosporine (staur., red circle) indicated. See Supplementary Data and Supplementary Fig. for underlying data and compound information. Supplementary Data – supports panels ( c , i ). Source data are provided in a Source Data file.

    Techniques Used: Binding Assay, Inhibition, Activity Assay

    a Crystal structures of the apo (PDB 5KGL) and ipglycermide Ce-2d bound (PDB 5KGN) iPGM from Yu H et al. Metal ions are identified as purple spheres. b Saturation binding of Ce-2 to 1 nM (black circle) 0.5 nM (gray circle) or 0.1 nM iPGM (open black circle). c Relative detection sensitivity of the SDR assay (blue circle) compared to a functional couple-enzyme assay (gray/black square) for ipglycermide Ce-2 binding C. elegans iPGM. Error bars for ( b , c ) represent the SEM of 2 experiments. d Correlation plot comparing the binding potencies for ipglycermide analogs from either an FP-based competition binding (10 nM iPGM) or SDR assay (0.5 nM iPGM- C -HiBiT) format. The FP assay uses a fluorescein-labeled Ce-2d analog (Ce-2d-FL). e Correlation plot comparing the binding potencies for ipglycermide analogs between B. malayi and C. elegans iPGM orthologs using the SDR assay. Ipglycermides are identified by symbols to the right. Error bars for ( d , e ) represent the SD, n = 3 experiments. RLU, relative light units; FP, fluorescence polarization; Ce, C. elegans ; Bm, B. malayi . Source data are provided in a Source Data file.
    Figure Legend Snippet: a Crystal structures of the apo (PDB 5KGL) and ipglycermide Ce-2d bound (PDB 5KGN) iPGM from Yu H et al. Metal ions are identified as purple spheres. b Saturation binding of Ce-2 to 1 nM (black circle) 0.5 nM (gray circle) or 0.1 nM iPGM (open black circle). c Relative detection sensitivity of the SDR assay (blue circle) compared to a functional couple-enzyme assay (gray/black square) for ipglycermide Ce-2 binding C. elegans iPGM. Error bars for ( b , c ) represent the SEM of 2 experiments. d Correlation plot comparing the binding potencies for ipglycermide analogs from either an FP-based competition binding (10 nM iPGM) or SDR assay (0.5 nM iPGM- C -HiBiT) format. The FP assay uses a fluorescein-labeled Ce-2d analog (Ce-2d-FL). e Correlation plot comparing the binding potencies for ipglycermide analogs between B. malayi and C. elegans iPGM orthologs using the SDR assay. Ipglycermides are identified by symbols to the right. Error bars for ( d , e ) represent the SD, n = 3 experiments. RLU, relative light units; FP, fluorescence polarization; Ce, C. elegans ; Bm, B. malayi . Source data are provided in a Source Data file.

    Techniques Used: Binding Assay, Functional Assay, Enzymatic Assay, FP Assay, Labeling, Fluorescence

    a DNA ligase crystal structures illustrating the relative positions of the N - and C -termini, DNA and nucleotide binding sites for the E. coli ligase (MW 75 kDa) complexed with NAD + (PDB 5TT5) from Unciuleac MC et al., with NAD + and DNA (PDB 2OWO) from Nandakumar J et al., and bacteriophage T7 ligase (MW 41.1 kDa) complexed with ATP (1A0I) from Subramanya HS et al. The structure of NAD + is shown. b Agarose gel electrophoresis of Hin dIII-digested λDNA repair by ligases from E. coli and bacteriophage T7 containing N - or C -terminal HiBiT α-peptide. Ligase concentrations used: 1, NEB E. coli ligase, 0.5 U/μL; 2, E. coli N -HiBiT, 1 µM; 3, E. coli C -HiBiT, 100 nM; 4, NEB T7 ligase, 150 U/ μL; 5, T7 N -HiBiT, 100 nM; 6, T7 C -HiBiT, 1 μM. Control ligases were from NEB and used as directed. Gel is representative of 2 replicates. Concentration-response curves obtained for the SDR assay for 0.5 nM E. coli Lig- N -HiBiT (solid circle), 1 nM E. coli Lig- C -HiBiT (open circle) and 1 nM T7 Lig- N / C -HiBiT (solid/open squares, respectively). c , d a 22-mer dsDNA oligo; e , f NAD + ; g , h ATP. Error bars are the SD, n = 3 experiments. Supplementary Table supports panels ( c − h ). kb, kilobase; Lig, ligase. The uncropped gel and source data are provided in a Source Data file.
    Figure Legend Snippet: a DNA ligase crystal structures illustrating the relative positions of the N - and C -termini, DNA and nucleotide binding sites for the E. coli ligase (MW 75 kDa) complexed with NAD + (PDB 5TT5) from Unciuleac MC et al., with NAD + and DNA (PDB 2OWO) from Nandakumar J et al., and bacteriophage T7 ligase (MW 41.1 kDa) complexed with ATP (1A0I) from Subramanya HS et al. The structure of NAD + is shown. b Agarose gel electrophoresis of Hin dIII-digested λDNA repair by ligases from E. coli and bacteriophage T7 containing N - or C -terminal HiBiT α-peptide. Ligase concentrations used: 1, NEB E. coli ligase, 0.5 U/μL; 2, E. coli N -HiBiT, 1 µM; 3, E. coli C -HiBiT, 100 nM; 4, NEB T7 ligase, 150 U/ μL; 5, T7 N -HiBiT, 100 nM; 6, T7 C -HiBiT, 1 μM. Control ligases were from NEB and used as directed. Gel is representative of 2 replicates. Concentration-response curves obtained for the SDR assay for 0.5 nM E. coli Lig- N -HiBiT (solid circle), 1 nM E. coli Lig- C -HiBiT (open circle) and 1 nM T7 Lig- N / C -HiBiT (solid/open squares, respectively). c , d a 22-mer dsDNA oligo; e , f NAD + ; g , h ATP. Error bars are the SD, n = 3 experiments. Supplementary Table supports panels ( c − h ). kb, kilobase; Lig, ligase. The uncropped gel and source data are provided in a Source Data file.

    Techniques Used: Binding Assay, Agarose Gel Electrophoresis, Control, Concentration Assay

    a DHFR bound to folate (red molecular surface) and NADP + (blue molecular surface) (PDB 4M6K) from Bhabha G et al. with N - and C -termini indicated. b Co-factor dependent SDR saturation binding curves for methotrexate (MTX) binding to 5 nM (solid symbol) and 0.5 nM (open symbol) DHFR- C -HiBiT in the presence (square) or absence (circle) of saturating NADPH. c SDR assay concentration response curves for MTX binding to 0.5 nM DHFR- C -HiBiT for various [NADPH]. Data normalized to 32.5 μM MTX response. d Correlation analysis of antifolate chemotherapeutic affinities as determined by a functional DHFR- C -HiBiT assay (100 nM, 75 μM NADPH) versus SDR assay using DHFR- C -HiBiT (0.5 nM, 5 μM NADPH), respectively. e SDR assay concentration response curves for MTX binding in 1:100 cellular lysate from DHFR C -terminus HiBiT gene edited HEK293 cells for indicated NADPH concentrations. Data normalized to vehicle control. f Correlation analysis of antifolate affinities determined by the SDR assay for a 1:100 lysate (~ 0.2 nM enzyme) without added NADPH or with 1 μM NADPH, respectively. The antifolates tested were methotrexate (M), γ-fluoromethotrexate (F), pralatrexate (Pr), aminopterin (A), trimetrexate (T), and pemetrexed (Pm). Error bars represent the SEM, n = 2 technical replicates, representative of 2 ( b ) or 3 ( c ) experiments, or SD, n = 3 experiments ( d , e , and f ). g Aligned minimal energy solution NMR conformer structures of apo (cyan, PDB: 2L28) and holo MTX-bound (raspberry, PDB: 1AO8) L. casei DHFR illustrating the conformational shift induced by the association of MTX (green). h Aligned solution NMR conformer ensemble structures of apo (cyan, 25 conformers, PDB: 2L28) and holo MTX-bound (raspberry, 21 conformers, PDB: 1AO8) L. casei DHFR illustrating the structural dynamics shift in DHFR upon MTX (green) binding as shown by the coalescence of conformers in the ensemble. RLU, relative light units. Source data are provided in a Source Data file.
    Figure Legend Snippet: a DHFR bound to folate (red molecular surface) and NADP + (blue molecular surface) (PDB 4M6K) from Bhabha G et al. with N - and C -termini indicated. b Co-factor dependent SDR saturation binding curves for methotrexate (MTX) binding to 5 nM (solid symbol) and 0.5 nM (open symbol) DHFR- C -HiBiT in the presence (square) or absence (circle) of saturating NADPH. c SDR assay concentration response curves for MTX binding to 0.5 nM DHFR- C -HiBiT for various [NADPH]. Data normalized to 32.5 μM MTX response. d Correlation analysis of antifolate chemotherapeutic affinities as determined by a functional DHFR- C -HiBiT assay (100 nM, 75 μM NADPH) versus SDR assay using DHFR- C -HiBiT (0.5 nM, 5 μM NADPH), respectively. e SDR assay concentration response curves for MTX binding in 1:100 cellular lysate from DHFR C -terminus HiBiT gene edited HEK293 cells for indicated NADPH concentrations. Data normalized to vehicle control. f Correlation analysis of antifolate affinities determined by the SDR assay for a 1:100 lysate (~ 0.2 nM enzyme) without added NADPH or with 1 μM NADPH, respectively. The antifolates tested were methotrexate (M), γ-fluoromethotrexate (F), pralatrexate (Pr), aminopterin (A), trimetrexate (T), and pemetrexed (Pm). Error bars represent the SEM, n = 2 technical replicates, representative of 2 ( b ) or 3 ( c ) experiments, or SD, n = 3 experiments ( d , e , and f ). g Aligned minimal energy solution NMR conformer structures of apo (cyan, PDB: 2L28) and holo MTX-bound (raspberry, PDB: 1AO8) L. casei DHFR illustrating the conformational shift induced by the association of MTX (green). h Aligned solution NMR conformer ensemble structures of apo (cyan, 25 conformers, PDB: 2L28) and holo MTX-bound (raspberry, 21 conformers, PDB: 1AO8) L. casei DHFR illustrating the structural dynamics shift in DHFR upon MTX (green) binding as shown by the coalescence of conformers in the ensemble. RLU, relative light units. Source data are provided in a Source Data file.

    Techniques Used: Binding Assay, Concentration Assay, Functional Assay, Control



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    a Conceptual framework for an SDR assay using terminal fusion protein of interest. The equilibrium between apo ( i ) and ligand-bound protein ( ii ) can be distinguished by the sensor ( iii ) activity, where a reddish color represents a dynamic apo protein relative to a more static blue ligand-bound protein. Created in BioRender. Inglese, J. (2025) https://BioRender.com/26yvpbw . b Key steps in an SDR assay. In this study the assay was performed in a 7 μL volume (4 μL target protein/ligand plus 3 μL detection reagent) permitting 1536-well plate quantitative high-throughput screening, qHTS (see Methods for full protocol). c SDR assay proof-of-concept using a C -terminal NLuc fusion to firefly luciferase (FLuc- C -NLuc, 1 nM), or d an N - or C -terminal fusion of <t>HiBiT</t> to FLuc (FLuc- N- or -C -HiBiT, 10 nM). The left axes indicate RLU generated by the enzyme activity of FLuc with increasing concentrations of FLuc inhibitor PTC124, for a C -terminal NLuc fusion ( c , black circles), or an N - (black circle) or C - (gray circle) HiBiT fusion ( d ) protein. The right axes indicate ligand-dependent SDR RLU, measured from NLuc ( c ) FLuc- C -NLuc (inverted blue triangle) or reconstituted NLuc ( d ) FLuc N - (blue triangle) or C - (blue square) HiBiT, with increasing PTC124 concentration in the presence (solid symbols) or absence (open symbols) of 10 µM ATP. Error bars are SD, n = 6 experiments. e qHTS waterfall plot illustrating concentration response curves (CRCs) obtained for a library of 1,343 compounds enriched for FLuc inhibitor chemotypes. Black CRCs from FLuc enzyme assay and blue CRCs from the 10 nM FLuc- C -HiBiT SDR assay (+ ATP condition). f Representative CRC from ( e ) for indicated clades, where symbols are activity from: SDR, plus ATP (blue solid square), SDR, no ATP (blue open square), and FLuc enzyme (black circle) assays. For full SAR around clade P see Supplementary Fig. . g Comparative SDR activity of clade representative compounds using the 1 nM FLuc- C -NLuc protein with ATP (blue solid triangle), no ATP (blue open triangle), or 1 nM NLuc without ATP (open red circle), or with ATP (solid red circle). Supplementary Data , support ( e − g ). RLU, relative light units; No., number; Cpd., compound. Source data are provided in a Source Data file.
    Fluc N Hibit, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    plasmid fluc- n -hibit  (Addgene inc)
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    a Conceptual framework for an SDR assay using terminal fusion protein of interest. The equilibrium between apo ( i ) and ligand-bound protein ( ii ) can be distinguished by the sensor ( iii ) activity, where a reddish color represents a dynamic apo protein relative to a more static blue ligand-bound protein. Created in BioRender. Inglese, J. (2025) https://BioRender.com/26yvpbw . b Key steps in an SDR assay. In this study the assay was performed in a 7 μL volume (4 μL target protein/ligand plus 3 μL detection reagent) permitting 1536-well plate quantitative high-throughput screening, qHTS (see Methods for full protocol). c SDR assay proof-of-concept using a C -terminal NLuc fusion to firefly luciferase (FLuc- C -NLuc, 1 nM), or d an N - or C -terminal fusion of <t>HiBiT</t> to FLuc (FLuc- N- or -C -HiBiT, 10 nM). The left axes indicate RLU generated by the enzyme activity of FLuc with increasing concentrations of FLuc inhibitor PTC124, for a C -terminal NLuc fusion ( c , black circles), or an N - (black circle) or C - (gray circle) HiBiT fusion ( d ) protein. The right axes indicate ligand-dependent SDR RLU, measured from NLuc ( c ) FLuc- C -NLuc (inverted blue triangle) or reconstituted NLuc ( d ) FLuc N - (blue triangle) or C - (blue square) HiBiT, with increasing PTC124 concentration in the presence (solid symbols) or absence (open symbols) of 10 µM ATP. Error bars are SD, n = 6 experiments. e qHTS waterfall plot illustrating concentration response curves (CRCs) obtained for a library of 1,343 compounds enriched for FLuc inhibitor chemotypes. Black CRCs from FLuc enzyme assay and blue CRCs from the 10 nM FLuc- C -HiBiT SDR assay (+ ATP condition). f Representative CRC from ( e ) for indicated clades, where symbols are activity from: SDR, plus ATP (blue solid square), SDR, no ATP (blue open square), and FLuc enzyme (black circle) assays. For full SAR around clade P see Supplementary Fig. . g Comparative SDR activity of clade representative compounds using the 1 nM FLuc- C -NLuc protein with ATP (blue solid triangle), no ATP (blue open triangle), or 1 nM NLuc without ATP (open red circle), or with ATP (solid red circle). Supplementary Data , support ( e − g ). RLU, relative light units; No., number; Cpd., compound. Source data are provided in a Source Data file.
    Plasmid Fluc N Hibit, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a Conceptual framework for an SDR assay using terminal fusion protein of interest. The equilibrium between apo ( i ) and ligand-bound protein ( ii ) can be distinguished by the sensor ( iii ) activity, where a reddish color represents a dynamic apo protein relative to a more static blue ligand-bound protein. Created in BioRender. Inglese, J. (2025) https://BioRender.com/26yvpbw . b Key steps in an SDR assay. In this study the assay was performed in a 7 μL volume (4 μL target protein/ligand plus 3 μL detection reagent) permitting 1536-well plate quantitative high-throughput screening, qHTS (see Methods for full protocol). c SDR assay proof-of-concept using a C -terminal NLuc fusion to firefly luciferase (FLuc- C -NLuc, 1 nM), or d an N - or C -terminal fusion of HiBiT to FLuc (FLuc- N- or -C -HiBiT, 10 nM). The left axes indicate RLU generated by the enzyme activity of FLuc with increasing concentrations of FLuc inhibitor PTC124, for a C -terminal NLuc fusion ( c , black circles), or an N - (black circle) or C - (gray circle) HiBiT fusion ( d ) protein. The right axes indicate ligand-dependent SDR RLU, measured from NLuc ( c ) FLuc- C -NLuc (inverted blue triangle) or reconstituted NLuc ( d ) FLuc N - (blue triangle) or C - (blue square) HiBiT, with increasing PTC124 concentration in the presence (solid symbols) or absence (open symbols) of 10 µM ATP. Error bars are SD, n = 6 experiments. e qHTS waterfall plot illustrating concentration response curves (CRCs) obtained for a library of 1,343 compounds enriched for FLuc inhibitor chemotypes. Black CRCs from FLuc enzyme assay and blue CRCs from the 10 nM FLuc- C -HiBiT SDR assay (+ ATP condition). f Representative CRC from ( e ) for indicated clades, where symbols are activity from: SDR, plus ATP (blue solid square), SDR, no ATP (blue open square), and FLuc enzyme (black circle) assays. For full SAR around clade P see Supplementary Fig. . g Comparative SDR activity of clade representative compounds using the 1 nM FLuc- C -NLuc protein with ATP (blue solid triangle), no ATP (blue open triangle), or 1 nM NLuc without ATP (open red circle), or with ATP (solid red circle). Supplementary Data , support ( e − g ). RLU, relative light units; No., number; Cpd., compound. Source data are provided in a Source Data file.

    Journal: Nature Communications

    Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

    doi: 10.1038/s41467-025-59658-6

    Figure Lengend Snippet: a Conceptual framework for an SDR assay using terminal fusion protein of interest. The equilibrium between apo ( i ) and ligand-bound protein ( ii ) can be distinguished by the sensor ( iii ) activity, where a reddish color represents a dynamic apo protein relative to a more static blue ligand-bound protein. Created in BioRender. Inglese, J. (2025) https://BioRender.com/26yvpbw . b Key steps in an SDR assay. In this study the assay was performed in a 7 μL volume (4 μL target protein/ligand plus 3 μL detection reagent) permitting 1536-well plate quantitative high-throughput screening, qHTS (see Methods for full protocol). c SDR assay proof-of-concept using a C -terminal NLuc fusion to firefly luciferase (FLuc- C -NLuc, 1 nM), or d an N - or C -terminal fusion of HiBiT to FLuc (FLuc- N- or -C -HiBiT, 10 nM). The left axes indicate RLU generated by the enzyme activity of FLuc with increasing concentrations of FLuc inhibitor PTC124, for a C -terminal NLuc fusion ( c , black circles), or an N - (black circle) or C - (gray circle) HiBiT fusion ( d ) protein. The right axes indicate ligand-dependent SDR RLU, measured from NLuc ( c ) FLuc- C -NLuc (inverted blue triangle) or reconstituted NLuc ( d ) FLuc N - (blue triangle) or C - (blue square) HiBiT, with increasing PTC124 concentration in the presence (solid symbols) or absence (open symbols) of 10 µM ATP. Error bars are SD, n = 6 experiments. e qHTS waterfall plot illustrating concentration response curves (CRCs) obtained for a library of 1,343 compounds enriched for FLuc inhibitor chemotypes. Black CRCs from FLuc enzyme assay and blue CRCs from the 10 nM FLuc- C -HiBiT SDR assay (+ ATP condition). f Representative CRC from ( e ) for indicated clades, where symbols are activity from: SDR, plus ATP (blue solid square), SDR, no ATP (blue open square), and FLuc enzyme (black circle) assays. For full SAR around clade P see Supplementary Fig. . g Comparative SDR activity of clade representative compounds using the 1 nM FLuc- C -NLuc protein with ATP (blue solid triangle), no ATP (blue open triangle), or 1 nM NLuc without ATP (open red circle), or with ATP (solid red circle). Supplementary Data , support ( e − g ). RLU, relative light units; No., number; Cpd., compound. Source data are provided in a Source Data file.

    Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

    Techniques: Activity Assay, High Throughput Screening Assay, Luciferase, Generated, Concentration Assay, Enzymatic Assay

    a ABL1 kinase domain bound to PD166326 inhibitor (dark blue) and myristic acid (red) (PDB: 1OPK), Nagar B et al. b SDR output of imatinib (blue square) or ATP (blue diamond) binding to 0.5 nM (open symbol) or 10 nM (solid symbol) ABL1- N -HiBiT kinase domain or imatinib inhibition of kinase activity (black circle) using Kinase-Glo Plus reagent (KGP). Error bars represent the SD, n = 3 experiments (Imatinib SDR) or n = 4 experiments (ATP SDR and imatinib kinase activity). c Correlation plot comparing the ABL1 KGP assay pIC 50 vs pSDR 50 (10 nM enzyme without ATP) for all inhibitors identified from the 128-member kinase inhibitor library. Colored symbols reference compounds discussed in Results section. For underlying data see Supplementary Data , , and Supplementary Fig. . d , e SDR output for myristate site ligands asciminib (red solid triangle) and GNF-5 (red open triangle) compared to imatinib (blue square) and nilotinib (open square) for ABL1- C- or - N -HiBiT, respectively. f Effect of allosteric ligands on enzyme catalysis (imatinib, black circle, asciminib, solid red triangle, and GNF-5, open red triangle). Error bars for d , e , and f represent the SEM, n = 2 technical replicates. g Protein kinase A (PKA) bound to H-89 inhibitor (dark blue) (PDB: 1YDT), Engh RA et al. h PKA enzyme activity (black circle) and SDR assay (blue square) CRC for H-89. Error bars represent the SD, n = 3 experiments i Correlation plot comparing the PKA KGP assay pIC 50 to PKA SDR assay pSDR 50 (without ATP). H-89 (blue square) and staurosporine (staur., red circle) indicated. See Supplementary Data and Supplementary Fig. for underlying data and compound information. Supplementary Data – supports panels ( c , i ). Source data are provided in a Source Data file.

    Journal: Nature Communications

    Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

    doi: 10.1038/s41467-025-59658-6

    Figure Lengend Snippet: a ABL1 kinase domain bound to PD166326 inhibitor (dark blue) and myristic acid (red) (PDB: 1OPK), Nagar B et al. b SDR output of imatinib (blue square) or ATP (blue diamond) binding to 0.5 nM (open symbol) or 10 nM (solid symbol) ABL1- N -HiBiT kinase domain or imatinib inhibition of kinase activity (black circle) using Kinase-Glo Plus reagent (KGP). Error bars represent the SD, n = 3 experiments (Imatinib SDR) or n = 4 experiments (ATP SDR and imatinib kinase activity). c Correlation plot comparing the ABL1 KGP assay pIC 50 vs pSDR 50 (10 nM enzyme without ATP) for all inhibitors identified from the 128-member kinase inhibitor library. Colored symbols reference compounds discussed in Results section. For underlying data see Supplementary Data , , and Supplementary Fig. . d , e SDR output for myristate site ligands asciminib (red solid triangle) and GNF-5 (red open triangle) compared to imatinib (blue square) and nilotinib (open square) for ABL1- C- or - N -HiBiT, respectively. f Effect of allosteric ligands on enzyme catalysis (imatinib, black circle, asciminib, solid red triangle, and GNF-5, open red triangle). Error bars for d , e , and f represent the SEM, n = 2 technical replicates. g Protein kinase A (PKA) bound to H-89 inhibitor (dark blue) (PDB: 1YDT), Engh RA et al. h PKA enzyme activity (black circle) and SDR assay (blue square) CRC for H-89. Error bars represent the SD, n = 3 experiments i Correlation plot comparing the PKA KGP assay pIC 50 to PKA SDR assay pSDR 50 (without ATP). H-89 (blue square) and staurosporine (staur., red circle) indicated. See Supplementary Data and Supplementary Fig. for underlying data and compound information. Supplementary Data – supports panels ( c , i ). Source data are provided in a Source Data file.

    Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

    Techniques: Binding Assay, Inhibition, Activity Assay

    a Crystal structures of the apo (PDB 5KGL) and ipglycermide Ce-2d bound (PDB 5KGN) iPGM from Yu H et al. Metal ions are identified as purple spheres. b Saturation binding of Ce-2 to 1 nM (black circle) 0.5 nM (gray circle) or 0.1 nM iPGM (open black circle). c Relative detection sensitivity of the SDR assay (blue circle) compared to a functional couple-enzyme assay (gray/black square) for ipglycermide Ce-2 binding C. elegans iPGM. Error bars for ( b , c ) represent the SEM of 2 experiments. d Correlation plot comparing the binding potencies for ipglycermide analogs from either an FP-based competition binding (10 nM iPGM) or SDR assay (0.5 nM iPGM- C -HiBiT) format. The FP assay uses a fluorescein-labeled Ce-2d analog (Ce-2d-FL). e Correlation plot comparing the binding potencies for ipglycermide analogs between B. malayi and C. elegans iPGM orthologs using the SDR assay. Ipglycermides are identified by symbols to the right. Error bars for ( d , e ) represent the SD, n = 3 experiments. RLU, relative light units; FP, fluorescence polarization; Ce, C. elegans ; Bm, B. malayi . Source data are provided in a Source Data file.

    Journal: Nature Communications

    Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

    doi: 10.1038/s41467-025-59658-6

    Figure Lengend Snippet: a Crystal structures of the apo (PDB 5KGL) and ipglycermide Ce-2d bound (PDB 5KGN) iPGM from Yu H et al. Metal ions are identified as purple spheres. b Saturation binding of Ce-2 to 1 nM (black circle) 0.5 nM (gray circle) or 0.1 nM iPGM (open black circle). c Relative detection sensitivity of the SDR assay (blue circle) compared to a functional couple-enzyme assay (gray/black square) for ipglycermide Ce-2 binding C. elegans iPGM. Error bars for ( b , c ) represent the SEM of 2 experiments. d Correlation plot comparing the binding potencies for ipglycermide analogs from either an FP-based competition binding (10 nM iPGM) or SDR assay (0.5 nM iPGM- C -HiBiT) format. The FP assay uses a fluorescein-labeled Ce-2d analog (Ce-2d-FL). e Correlation plot comparing the binding potencies for ipglycermide analogs between B. malayi and C. elegans iPGM orthologs using the SDR assay. Ipglycermides are identified by symbols to the right. Error bars for ( d , e ) represent the SD, n = 3 experiments. RLU, relative light units; FP, fluorescence polarization; Ce, C. elegans ; Bm, B. malayi . Source data are provided in a Source Data file.

    Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

    Techniques: Binding Assay, Functional Assay, Enzymatic Assay, FP Assay, Labeling, Fluorescence

    a DNA ligase crystal structures illustrating the relative positions of the N - and C -termini, DNA and nucleotide binding sites for the E. coli ligase (MW 75 kDa) complexed with NAD + (PDB 5TT5) from Unciuleac MC et al., with NAD + and DNA (PDB 2OWO) from Nandakumar J et al., and bacteriophage T7 ligase (MW 41.1 kDa) complexed with ATP (1A0I) from Subramanya HS et al. The structure of NAD + is shown. b Agarose gel electrophoresis of Hin dIII-digested λDNA repair by ligases from E. coli and bacteriophage T7 containing N - or C -terminal HiBiT α-peptide. Ligase concentrations used: 1, NEB E. coli ligase, 0.5 U/μL; 2, E. coli N -HiBiT, 1 µM; 3, E. coli C -HiBiT, 100 nM; 4, NEB T7 ligase, 150 U/ μL; 5, T7 N -HiBiT, 100 nM; 6, T7 C -HiBiT, 1 μM. Control ligases were from NEB and used as directed. Gel is representative of 2 replicates. Concentration-response curves obtained for the SDR assay for 0.5 nM E. coli Lig- N -HiBiT (solid circle), 1 nM E. coli Lig- C -HiBiT (open circle) and 1 nM T7 Lig- N / C -HiBiT (solid/open squares, respectively). c , d a 22-mer dsDNA oligo; e , f NAD + ; g , h ATP. Error bars are the SD, n = 3 experiments. Supplementary Table supports panels ( c − h ). kb, kilobase; Lig, ligase. The uncropped gel and source data are provided in a Source Data file.

    Journal: Nature Communications

    Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

    doi: 10.1038/s41467-025-59658-6

    Figure Lengend Snippet: a DNA ligase crystal structures illustrating the relative positions of the N - and C -termini, DNA and nucleotide binding sites for the E. coli ligase (MW 75 kDa) complexed with NAD + (PDB 5TT5) from Unciuleac MC et al., with NAD + and DNA (PDB 2OWO) from Nandakumar J et al., and bacteriophage T7 ligase (MW 41.1 kDa) complexed with ATP (1A0I) from Subramanya HS et al. The structure of NAD + is shown. b Agarose gel electrophoresis of Hin dIII-digested λDNA repair by ligases from E. coli and bacteriophage T7 containing N - or C -terminal HiBiT α-peptide. Ligase concentrations used: 1, NEB E. coli ligase, 0.5 U/μL; 2, E. coli N -HiBiT, 1 µM; 3, E. coli C -HiBiT, 100 nM; 4, NEB T7 ligase, 150 U/ μL; 5, T7 N -HiBiT, 100 nM; 6, T7 C -HiBiT, 1 μM. Control ligases were from NEB and used as directed. Gel is representative of 2 replicates. Concentration-response curves obtained for the SDR assay for 0.5 nM E. coli Lig- N -HiBiT (solid circle), 1 nM E. coli Lig- C -HiBiT (open circle) and 1 nM T7 Lig- N / C -HiBiT (solid/open squares, respectively). c , d a 22-mer dsDNA oligo; e , f NAD + ; g , h ATP. Error bars are the SD, n = 3 experiments. Supplementary Table supports panels ( c − h ). kb, kilobase; Lig, ligase. The uncropped gel and source data are provided in a Source Data file.

    Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

    Techniques: Binding Assay, Agarose Gel Electrophoresis, Control, Concentration Assay

    a DHFR bound to folate (red molecular surface) and NADP + (blue molecular surface) (PDB 4M6K) from Bhabha G et al. with N - and C -termini indicated. b Co-factor dependent SDR saturation binding curves for methotrexate (MTX) binding to 5 nM (solid symbol) and 0.5 nM (open symbol) DHFR- C -HiBiT in the presence (square) or absence (circle) of saturating NADPH. c SDR assay concentration response curves for MTX binding to 0.5 nM DHFR- C -HiBiT for various [NADPH]. Data normalized to 32.5 μM MTX response. d Correlation analysis of antifolate chemotherapeutic affinities as determined by a functional DHFR- C -HiBiT assay (100 nM, 75 μM NADPH) versus SDR assay using DHFR- C -HiBiT (0.5 nM, 5 μM NADPH), respectively. e SDR assay concentration response curves for MTX binding in 1:100 cellular lysate from DHFR C -terminus HiBiT gene edited HEK293 cells for indicated NADPH concentrations. Data normalized to vehicle control. f Correlation analysis of antifolate affinities determined by the SDR assay for a 1:100 lysate (~ 0.2 nM enzyme) without added NADPH or with 1 μM NADPH, respectively. The antifolates tested were methotrexate (M), γ-fluoromethotrexate (F), pralatrexate (Pr), aminopterin (A), trimetrexate (T), and pemetrexed (Pm). Error bars represent the SEM, n = 2 technical replicates, representative of 2 ( b ) or 3 ( c ) experiments, or SD, n = 3 experiments ( d , e , and f ). g Aligned minimal energy solution NMR conformer structures of apo (cyan, PDB: 2L28) and holo MTX-bound (raspberry, PDB: 1AO8) L. casei DHFR illustrating the conformational shift induced by the association of MTX (green). h Aligned solution NMR conformer ensemble structures of apo (cyan, 25 conformers, PDB: 2L28) and holo MTX-bound (raspberry, 21 conformers, PDB: 1AO8) L. casei DHFR illustrating the structural dynamics shift in DHFR upon MTX (green) binding as shown by the coalescence of conformers in the ensemble. RLU, relative light units. Source data are provided in a Source Data file.

    Journal: Nature Communications

    Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

    doi: 10.1038/s41467-025-59658-6

    Figure Lengend Snippet: a DHFR bound to folate (red molecular surface) and NADP + (blue molecular surface) (PDB 4M6K) from Bhabha G et al. with N - and C -termini indicated. b Co-factor dependent SDR saturation binding curves for methotrexate (MTX) binding to 5 nM (solid symbol) and 0.5 nM (open symbol) DHFR- C -HiBiT in the presence (square) or absence (circle) of saturating NADPH. c SDR assay concentration response curves for MTX binding to 0.5 nM DHFR- C -HiBiT for various [NADPH]. Data normalized to 32.5 μM MTX response. d Correlation analysis of antifolate chemotherapeutic affinities as determined by a functional DHFR- C -HiBiT assay (100 nM, 75 μM NADPH) versus SDR assay using DHFR- C -HiBiT (0.5 nM, 5 μM NADPH), respectively. e SDR assay concentration response curves for MTX binding in 1:100 cellular lysate from DHFR C -terminus HiBiT gene edited HEK293 cells for indicated NADPH concentrations. Data normalized to vehicle control. f Correlation analysis of antifolate affinities determined by the SDR assay for a 1:100 lysate (~ 0.2 nM enzyme) without added NADPH or with 1 μM NADPH, respectively. The antifolates tested were methotrexate (M), γ-fluoromethotrexate (F), pralatrexate (Pr), aminopterin (A), trimetrexate (T), and pemetrexed (Pm). Error bars represent the SEM, n = 2 technical replicates, representative of 2 ( b ) or 3 ( c ) experiments, or SD, n = 3 experiments ( d , e , and f ). g Aligned minimal energy solution NMR conformer structures of apo (cyan, PDB: 2L28) and holo MTX-bound (raspberry, PDB: 1AO8) L. casei DHFR illustrating the conformational shift induced by the association of MTX (green). h Aligned solution NMR conformer ensemble structures of apo (cyan, 25 conformers, PDB: 2L28) and holo MTX-bound (raspberry, 21 conformers, PDB: 1AO8) L. casei DHFR illustrating the structural dynamics shift in DHFR upon MTX (green) binding as shown by the coalescence of conformers in the ensemble. RLU, relative light units. Source data are provided in a Source Data file.

    Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

    Techniques: Binding Assay, Concentration Assay, Functional Assay, Control