Journal: eLife

Article Title: A terminal selector prevents a Hox transcriptional switch to safeguard motor neuron identity throughout life

doi: 10.7554/eLife.50065

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pcDNA 3.1(+)- C-Flag (Plasmid) , Genscript , pcDNA 3.1(+) , C-terminus Flag-tagged UNC-3.

Techniques: CRISPR, Magnetic Beads, Recombinant, Plasmid Preparation, Cloning, Purification, PCR Cloning, Software

Journal: eLife

Article Title: The metal cofactor zinc and interacting membranes modulate SOD1 conformation-aggregation landscape in an in vitro ALS model

doi: 10.7554/eLife.61453

Figure Lengend Snippet: Binding constants (K a ,M −1 ) of the protein variants with model DPPC SUVs as obtained from the FCS study for WT SOD1 and all the mutants.

Article Snippet: Recombinant DNA reagent , G37R (plasmid) , Genscript , , .

Techniques: Binding Assay

Journal: eLife

Article Title: The metal cofactor zinc and interacting membranes modulate SOD1 conformation-aggregation landscape in an in vitro ALS model

doi: 10.7554/eLife.61453

Figure Lengend Snippet: SOD1 disease mutants, their corresponding distance parameters in terms of the distances of mutational stress points from the Zn and Cu center, and the membrane binding energies of the disease mutants.

Article Snippet: Recombinant DNA reagent , G37R (plasmid) , Genscript , , .

Techniques: Membrane, Binding Assay

Journal: eLife

Article Title: The metal cofactor zinc and interacting membranes modulate SOD1 conformation-aggregation landscape in an in vitro ALS model

doi: 10.7554/eLife.61453

Figure Lengend Snippet: ( a ) Computational validation. The distances of the disease mutation sites from the Zn (red) and Cu (blue) co-factor were plotted against the binding energy towards membrane, which were computed. The binding energy decreased linearly as the distance between Zn and mutation site increased (red curve), while there was no significant change for Cu (blue curve). The distance information for these mutants was calculated from their solved structures. ( b ) The ALS disease severity in terms of average survival time after diagnosis has been plotted against the distance parameters of the mutation points from Zn centre for the disease mutants.( c ) percentage populations of membrane bound alexa-labeled protein variants which were obtained from the two component diffusion model fitting of the FCS data for G37R and I113T were plotted against the concentrations of DPPC SUVs added to evaluate the binding affinities of the protein variants towards membranes. ( d ) Fibril formation kinetics. ThT fluorescence spectra of G37R and I113T were plotted in the absence and presence of DPPC SUVs. The 3D AFM images of I113T in absence ( e ) and presence ( f ) of membranes. Error bar indicates the standard deviation for the triplicate experiments. Figure 5—source data 1. Conformational characterization, membrane binding, aggregation and toxicity of ALS disease mutants.

Article Snippet: Recombinant DNA reagent , G37R (plasmid) , Genscript , , .

Techniques: Biomarker Discovery, Mutagenesis, Binding Assay, Membrane, Labeling, Diffusion-based Assay, Fluorescence, Standard Deviation

Journal: eLife

Article Title: The metal cofactor zinc and interacting membranes modulate SOD1 conformation-aggregation landscape in an in vitro ALS model

doi: 10.7554/eLife.61453

Figure Lengend Snippet: ( a )–( d ) Deconvoluted FTIR spectral signatures that correspond to the carbonyl vibrational frequency of amide-I region (1600–1700 cm −1 ) of I113T and G37R mutant both in the absence and in the presence of lipid membranes. ( e ) Percentages of secondary conformations of G37R and I113T disease mutants both in the absence and in the presence of DPPC SUVs. Error bar indicates the standard deviation for the triplicate experiments.

Article Snippet: Recombinant DNA reagent , G37R (plasmid) , Genscript , , .

Techniques: Mutagenesis, Standard Deviation

Journal: eLife

Article Title: The metal cofactor zinc and interacting membranes modulate SOD1 conformation-aggregation landscape in an in vitro ALS model

doi: 10.7554/eLife.61453

Figure Lengend Snippet: ( a ) Residue-wise vibrational entropy changes for G37R and I113T; ( b ) the structure of G37R indicating the increase in the structural rigidity (blue color indicates the region becomes rigid due to the mutation); ( c ) structural flexibility increases in I113T mutant (red color indicates the region becomes more flexible due to mutation).

Article Snippet: Recombinant DNA reagent , G37R (plasmid) , Genscript , , .

Techniques: Residue, Mutagenesis

Journal: eLife

Article Title: The metal cofactor zinc and interacting membranes modulate SOD1 conformation-aggregation landscape in an in vitro ALS model

doi: 10.7554/eLife.61453

Figure Lengend Snippet: AFM topographic images of the aggregates of G37R that was formed in the absence ( a ) and presence ( b ) of membrane.

Article Snippet: Recombinant DNA reagent , G37R (plasmid) , Genscript , , .

Techniques: Membrane

Journal: eLife

Article Title: The metal cofactor zinc and interacting membranes modulate SOD1 conformation-aggregation landscape in an in vitro ALS model

doi: 10.7554/eLife.61453

Figure Lengend Snippet: ( a ) Calcein release percentage when dye entrapped small unilamellar vesicles (SUVs) were treated with the aggregates of I113T and G37R, which were formed in the absence and presence of membranes. ( b )–( e ) The Gaussian fittings of the CH 2 wagging band region of planar DPPC membrane after the treatments of different disease mutant aggregates. ( f ) Populations of nonplanar conformations in membrane bilayer that is perturbed by the aggregates of I113T and G37R, which are formed both in the absence and in the presence of membranes. Here n.s. denotes nonsignificant, whereas ** is significant with p-values<0.01 and *** is highly significant with p-values<0.001. I113T agg and I113T aggm indicate the aggregates of I113T protein which are formed in the absence and presence of DPPC SUVs. G37R agg and G37R aggm indicate the aggregates of G37R protein that are formed in the absence and presence of DPPC SUVs. Error bar indicates the standard deviations of the values obtained from experiments those were performed triplicate.

Article Snippet: Recombinant DNA reagent , G37R (plasmid) , Genscript , , .

Techniques: Membrane, Mutagenesis

Journal: eLife

Article Title: The metal cofactor zinc and interacting membranes modulate SOD1 conformation-aggregation landscape in an in vitro ALS model

doi: 10.7554/eLife.61453

Figure Lengend Snippet: ( a ) Plot of fraction bound of labeled proteins against the concentration of DPPC SUVs to estimate the binding constants of G37R, G85R, and I113T (labeled with Alexa-488 Maleimide) using FCS experiments. AFM micrographs for ( b ) G37R, ( c ) G85R, and ( d ) I113T in the presence of DPPC SUVs.

Article Snippet: Recombinant DNA reagent , G37R (plasmid) , Genscript , , .

Techniques: Labeling, Concentration Assay, Binding Assay

Journal: eLife

Article Title: The metal cofactor zinc and interacting membranes modulate SOD1 conformation-aggregation landscape in an in vitro ALS model

doi: 10.7554/eLife.61453

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , G37R (plasmid) , Genscript , , .

Techniques: Recombinant, Plasmid Preparation, Mutagenesis, Sequencing, MTT Cell Proliferation, Software