Journal: The Journal of Biological Chemistry

Article Title: Stabilization of glucose-6-phosphate dehydrogenase oligomers enhances catalytic activity and stability of clinical variants

doi: 10.1016/j.jbc.2022.101610

Figure Lengend Snippet: Loss of structural NADP + binding affects G6PD oligomerization, activity, and stability. A , location of the K403 residue on the ß-sheet of the structural NADP + -binding site and its interaction with the NADP + phosphate group in G6PD WT (PDB 6E08 ). B , the SEC chromatogram for G6PD WT and G6PD K403Q in the absence of NADP + . C and D , the SEC chromatogram for G6PD WT and G6PD K403Q in the absence or presence of 10 μM NADP + (for B – D , n = 1 for each condition). E , the SEC chromatogram in the absence of NADP + , changing pH, ionic strength, and adding 10 mM G6P ( n = ≥2 for each mutant). F and G , P-Native PAGE Western blot and quantification ( n ≥ 3 for each mutant). H , Coomassie of SDS-PAGE ( n = 2 for each mutant), ( I ) activity ( n = 14 for each mutant), and ( J ) thermostability for G6PD WT and G6PD K403Q ( n = 3 for each mutant). For P-Native PAGE, differences between G6PD K403Q and G6PD WT were detected for each group of oligomeric species via four two-tailed unpaired t test s ( p value ≤ 0.0004 for each t test). Full Western blot and Coomassie images for F and H are provided in Figs. S10 and . G6PD, glucose-6-phosphate dehydrogenase.

Article Snippet: Chemicals: NADP + (Amresco – 0760), NADPH (Millipore Sigma – 10107824001), Glucose-6-phosphate (CHEM-IMPEX International – INC 00866), resazurin (ACROS Organics – AC189900050).

Techniques: Binding Assay, Activity Assay, Residue, Mutagenesis, Clear Native PAGE, Western Blot, SDS Page, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Stabilization of glucose-6-phosphate dehydrogenase oligomers enhances catalytic activity and stability of clinical variants

doi: 10.1016/j.jbc.2022.101610

Figure Lengend Snippet: The impact of loss of structural NADP + binding on G6PD oligomerization, activity, and stability in Class I variants at and distant from the structural NADP + -binding site. A , location of Class I mutations at the structural NADP + -binding site and their interactions with the structural NADP + in G6PD WT (PDB 6E08 ). For Class I mutations located at the structural NADP + -binding site, ( B ) the SEC chromatogram ( n = 2 for each mutant), ( C ) quantification of P-Native PAGE ( n ≥ 3 for each mutant), ( D ) and activity ( n ≥ 3 for each mutant). E , location of Class I mutations distant from the ß-sheet of the structural NADP + -binding site in G6PD WT (PDB 6E08 ). For Class I mutations distant from the structural NADP + -binding site, ( F ) the SEC chromatogram ( n = 2 for each mutant), ( G ) P-Native PAGE quantification ( n ≥ 3 for each mutant), ( H ) and activity ( n ≥ 3 for each mutant). For P-Native PAGE, differences between mutant and G6PD WT were detected for each group of oligomeric species via four one-way ANOVAs ( p value < 0.05 for each ANOVA). G6PD, glucose-6-phosphate dehydrogenase; SEC size-exclusion chromatography.

Article Snippet: Chemicals: NADP + (Amresco – 0760), NADPH (Millipore Sigma – 10107824001), Glucose-6-phosphate (CHEM-IMPEX International – INC 00866), resazurin (ACROS Organics – AC189900050).

Techniques: Binding Assay, Activity Assay, Mutagenesis, Clear Native PAGE, Size-exclusion Chromatography

Journal: The Journal of Biological Chemistry

Article Title: Stabilization of glucose-6-phosphate dehydrogenase oligomers enhances catalytic activity and stability of clinical variants

doi: 10.1016/j.jbc.2022.101610

Figure Lengend Snippet: Generation and characterization of synthetic G6PD mutants, either locked in the dimer or tetramer state or tetramer inhibited. Mutagenic strategy is as follows: A , for the dimer-locked (dL) mutant, a cysteine was added to the C-terminal tail; the C-terminal tails align at the dimer interface (PDB 2BH9 ). B and C , P-Native PAGE after crosslinking with 1% glutaraldehyde and SEC chromatogram for G6PD WT and G6PD WTdL mutant ( n = 1, n ≥ 2 for each mutant respectively). D , for the tetramer-locked (tL) mutant, a A277 was mutated to a cysteine, which is near C294 at the tetramer interface. Additionally, a salt bridge at the tetramer interface was disrupted to generate the tetramer impaired mutant, G6PD E347A (PDB 6E08 ). E , P-Native PAGE Western blot after cross-linking with 1% glutaraldehyde ( n = 3 for each mutant). F , the SEC chromatogram in a condition that dissociates the G6PD WT dimer and ( G ) a condition that promotes G6PD WT tetramerization ( n ≥ 2 for each mutant). H , P-Native PAGE Western blot quantification for synthetic variants ( n ≥ 3 for each mutant). Biochemical analysis of synthetic variants including ( I ) activity ( n = 3 for each mutant), ( J ) K m for G6P and NADP + ( n = 3 for each mutant), and ( K ) thermostability ( n = 3 for each mutant). For P-Native PAGE, differences between mutant and G6PD WT were detected for each group of oligomeric species via four one-way ANOVAs ( p value < 0.05 for each ANOVA). Full Western blot images for B and E are provided in Fig. S11 . G6PD, glucose-6-phosphate dehydrogenase; SEC size-exclusion chromatography.

Article Snippet: Chemicals: NADP + (Amresco – 0760), NADPH (Millipore Sigma – 10107824001), Glucose-6-phosphate (CHEM-IMPEX International – INC 00866), resazurin (ACROS Organics – AC189900050).

Techniques: Mutagenesis, Clear Native PAGE, Western Blot, Activity Assay, Size-exclusion Chromatography

Journal: The Journal of Biological Chemistry

Article Title: Stabilization of glucose-6-phosphate dehydrogenase oligomers enhances catalytic activity and stability of clinical variants

doi: 10.1016/j.jbc.2022.101610

Figure Lengend Snippet: Biochemical characterization of clinical variants locked in the dimer and/or tetrameric state. A , location of clinical variants displayed on the G6PD tetramer (PDB 6E08 ). B , P-Native PAGE quantification for clinical variants ( n ≥ 3 for each mutant). The residues for each clinical variant were displayed as spheres and color coded according to the percent activation over the nonlocked version (PDB 6E08 ). C , the dimer is displayed for dL variants and ( D ) the tetramer is displayed for dLtL variants ( n ≥ 3 for each mutant). The thermostability of G6PD WT compared to ( E ) G6PD K275N ( n = 3 for each mutant) and ( F ) G6PD Canton ( n = 3 for each mutant). For P-Native PAGE, differences between mutant and G6PD WT were detected for each group of oligomeric species via four one-way ANOVAs ( p value < 0. 05 for each ANOVA). G6PD, glucose-6-phosphate dehydrogenase.

Article Snippet: Chemicals: NADP + (Amresco – 0760), NADPH (Millipore Sigma – 10107824001), Glucose-6-phosphate (CHEM-IMPEX International – INC 00866), resazurin (ACROS Organics – AC189900050).

Techniques: Clear Native PAGE, Mutagenesis, Variant Assay, Activation Assay