PMC

Stereoviews showing alignment of Ara h 1 (blue) with (A) β-conclycinin (green) and (B) Ara h 3 (gray). For β-conclycinin and Ara h 3 structures with PDB codes 1uij and 3c3v, respectively, were used.

Gastric (phase 1) and duodenal (phase 2) digestion of nAra h 1, rAra h 1 and rsAra h 1. Three micrograms of each sample taken at the indicated times were analyzed by 16% SDS-PAGE under reducing conditions and detected by Coomassie staining.

IgE binding to folded and unfolded, native and recombinant Ara h 1. A, SDS-PAGE and Western blot analysis of patient sera (1–6) IgE binding to linear native (n), full length recombinant (r), and recombinant shortened version of Ara h 1 (rs). B, spot blot of patient sera-IgE binding to folded n, r, and rsAra h 1 according to the template shown in the bottom left box.

Structure of rsAra h 1. A, ribbon diagram of the Ara h 1 core fragment presented in two different orientations. Numbers indicate the ends of fragments that could be traced. α-Helices are shown in red, β-strands in yellow, and loop regions are shown in green. B, molecular surface of Ara h 1 with hydrophobic residues are shown in olive. Orientation of the molecules is the same as for the ribbon diagrams. Figures from the right side of the panel show molecules as seen from the center of trimer.

Trimer formed by rsAra h 1. Ara h 1 trimer shown in three different orientations corresponding to 0°, 90°, and 180° rotation along the horizontal axis. Part A of the panel shows the trimer in ribbon representation with one protein molecule in the same orientation and color schema as in A, and two other molecules are shown in blue and gray. B, molecular surface of Ara h 1 trimer.

Summary of SAXS data. A, experimental scattering data of each version of Ara h 1 (in gray, black axes) along with the fits of the most probable bead models (in green) and the particle distance distribution functions (in blue, blue axes). Aiii, in addition, shows the theoretical scattering curves of the four trimer (g) and the three trimer (h) SASREF models. B, Kratky plot with experimental points for each version. Where i, k, and m represent rsAra h 1, nAra h 1, and rAra h 1, respectively, along with the corresponding trend lines (j,l,n) determined with the smooth bezier function in GNUplot. C, representation of the most probable bead models of rsAra h 1 (in blue) and nAra h 1 (in gray mesh). The dimensions in Angstroms of each are given in blue and black, respectively. The bottom figure was obtained by rotating the top figure 90 degrees around the specified axis.

Two-dimensional projection of the CLANS clustering results. Proteins are indicated by dots. Lines indicate sequence similarity detectable with BLAST and are colored by a spectrum of gray shades according to the BLAST p value (black: p value < 10⁻²²⁵, light gray: p value < 10⁻⁵). Sequences corresponding to structures in the PDB are indicated by blue dots, sequences of known allergens are indicated by red dots. Peanut allergens Ara h 1 and Ara h 3 are marked with arrows. Because the sequences were downloaded as they are in the nr database, sequences of separate, nonidentical chains in PDB deposits are represented separately. For example, Jack Bean canavalin structures were obtained by crystallization of the proteolytic product of the protein, and both domains are represented by separate sequences, which is why they moved away from the central cluster containing the full-length protein.

Composite of the antibody binding studies and sequence conservation mapped on rsAra h 1 structure. A, sequence conservation between different allergens belonging to the vicilin group (Ana o 1, Ara h 1, Cor a 11, Gly m 5, Jug n 2, Jug r 2, Len c 1, Lup an 1, Pis s 1, and Ses i 3) are mapped onto the molecular surface of the rsAra h 1 trimer. Invariant residues are shown in red, and conserved residues are shown in blue. B–D, molecular surface of rsAra h 1 trimer is depicted with peptides that were shown to interact with human antibodies. B, epitopes identified by Burks et al. (): each epitope is shown using a different color. Immunodominant epitopes are shown in shades of purple and blue, and other epitopes in shades of green. C, epitopes identified by Cong et al. (): immunodominant epitopes are colored using different shades of gray, and other epitopes in shades of red, orange, and yellow. D, amino acids from an IgM binding epitope as reported by Shinmoto et al. () are shown in green. Only residues present in the rsAra h 1 structure are labeled.