Journal: eLife

Article Title: Unfolding and identification of membrane proteins in situ

doi: 10.7554/eLife.77427

Figure Lengend Snippet: ( A ) Superimposition of 83 force-distance (F-D) curves of Bacteriorhodopsin (the red line is the worm-like chain curve with persistence length = 0.4 nm and contour length = 85 nm). ( B, C, D ) Transformation of the ordinates of the points in ( A ) to the contour length space ( L c ) with persistence length of 0.3 nm in ( B ), 0.4 in ( C ), and 0.5 in ( D ). Solid red lines indicate the best fit of the points belonging to the last unfolding peak: 0.4 nm of persistence length is the correct/optimal value because in the contour length space, the force points representing the unfolding of the polymer with a constant contour length must lay on a vertical line. ( D ) Global histogram of contour length of ( C ).

Article Snippet: Peptide, recombinant protein , Hs Bacteriorhodopsin (bR) , Cube-Biotech , Cat#28903 , .

Techniques: Transformation Assay, Polymer

Journal: eLife

Article Title: Unfolding and identification of membrane proteins in situ

doi: 10.7554/eLife.77427

Figure Lengend Snippet: ( A ) Scheme of the structure of the three purified proteins used in the in vitro preliminary step: Channelrhodopsin (ChR1), SthK, and Bacteriorhodopsin (bR) (cylinders represent α-helices). ( B ) Superimposition of 101 unfolding curves (density plot) of the full unfolding of ChR1 from the N-terminus. ( C ) Density plot of the full unfolding of Sthk from the C-terminus. ( D ) Density plot of the full unfolding of bR. ( E ) Protein profile, that is, the histogram of the maximal contour length ( L c max ) of all the force-distance (F-D) curves in the clusters (black line), and of all the F-D curves collected from the sample (gray line, not in scale), from samples with mixtures of bR, ChR1, and SthK with a relative abundance of 1:1:1, 1:7:20, and 1:0:1 (bR-SthK only). Arrows indicate where the F-D curves of ( B ), ( C ), and ( D ) accumulate in the histogram. The peak at ~90 nm indicated by bR+short also contains the shorter clusters of ChR1 and SthK shown in , which also appear in the mixtures.

Article Snippet: Peptide, recombinant protein , Hs Bacteriorhodopsin (bR) , Cube-Biotech , Cat#28903 , .

Techniques: Purification, In Vitro

Journal: eLife

Article Title: Unfolding and identification of membrane proteins in situ

doi: 10.7554/eLife.77427

Figure Lengend Snippet: ( A ) Scheme of the structure of the three purified proteins used in the in vitro preliminary step: Channelrhodopsin (ChR1), SthK, and Bacteriorhodopsin (bR) (cylinders represent α-helices). ( B ) Geometrical length of the amino acid chains constituting the whole protein, the C- and N-terminals and of the length of the whole protein minus the C- and N-terminal. The codes in red represent the name and the respective unfolding cluster. ( C ) Clusters obtained from a dataset of n =18,868 traces from a sample with ChR1. The histogram bars on the right of each density plot represent the Bayesian assignment according to our procedure (described in Methods). [N] and [C] stand for ‘protein unfolded from N-terminus/C-terminus’. Below each density plot the corresponding global histogram of contour length showing the most likely position for the unfolding peaks to occur: the circles with numbers are then reported in ( A ) to show the position of the preferential unfolding position in the structure. ( D ) Clusters obtained from a dataset of n =115,155 traces from a sample with SthK. ( E ) Clusters obtained from a dataset of n =35,599 traces from a sample with bR.

Article Snippet: Peptide, recombinant protein , Hs Bacteriorhodopsin (bR) , Cube-Biotech , Cat#28903 , .

Techniques: Purification, In Vitro

Journal: eLife

Article Title: Unfolding and identification of membrane proteins in situ

doi: 10.7554/eLife.77427

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Hs Bacteriorhodopsin (bR) , Cube-Biotech , Cat#28903 , .

Techniques: Plasmid Preparation, Modification, Transfection, Construct, Sequencing, Labeling, Isolation, Recombinant, Software, Staining