Figure 6.

Hydrogen–deuterium exchange patterns for lysenin^WT and lysenin^V88C/Y131C in aqueous solution. (A,B) Plots show the hydrogen–deuterium exchange pattern of amide protons (y axis) for lysenin^WT (black), lysenin^V88C/Y131C without DTT (blue), and lysenin^V88C/Y131C with 10 mM DTT (red) in aqueous solution (top panels). Samples were incubated with deuterium buffer for 10 s (A) or 20 min (B) Peptides are represented with horizontal bars indicating their lengths and positions in the lysenin amino acid sequence (horizontal axis). Regions organized into β strands and the 3/10 helix of the N-terminal and C-terminal domains are indicated at the top of the graph. Y-axis error bars indicate standard deviations calculated from at least three independent experiments. The HDX data are represented with color-coding on schematic representations of the monomeric crystallographic structure of lysenin (bottom panels). Color coding: red regions: 80–100% deuteration; yellow regions: 40–80% deuteration; blue regions: 0–40% deuteration; grey regions: fragments not covered by the data. (C,D) Subtraction plots of the HDX patterns were generated to better visualize changes in the exchange patterns of lysenin^V88C/Y131C vs. lysenin^WT in aqueous solution. Data were obtained by subtracting the fraction of hydrogen–deuterium exchange of lysenin^WT from that of lysenin^V88C/Y131C without DTT (blue) or lysenin^V88C/Y131C with 10 mM DTT (red) after 10 s (C) or 20 min (D) deuteration in aqueous solution. Positive and negative values, respectively, indicate regions that were destabilized and stabilized in the lysenin mutant. Error bars were calculated as the square root of the sum of variances of the subtracted numbers.