Abstract / Summary
Abstract Sola l 4.02 is a major tomato allergen of the PR-10 family and a Bet v 1 homolog implicated in pollen–food cross-reactivity, yet its molecular properties remain poorly defined. Here, we report the recombinant production and its comprehensive structural and functional characterization. Sequence and structural analysis support a canonical Bet v 1-like fold including the conserved hydrophobic ligand binding site cavity typical of PR-10 proteins. Biophysical analyses showed that recombinant Sola l 4.02 is a compact, monomeric, and well-folded protein in solution. Circular dichroism measurements revealed moderate thermal stability, with a melting temperature of 57.5 °C and partial recovery of the native conformation after cooling, indicating partially reversible unfolding. The protein remained structurally stable between pH 5.5 and 9.0, whereas mildly acidic conditions promoted partial destabilization. To probe ligand recognition, we tested selected phenolic compounds by microscale thermophoresis. Sola l 4.02 bound quercetin-3-glucoside, naringenin, and daidzein with low micromolar affinities, while caffeic acid showed only weak interaction and hydroxycinnamic acid displayed no detectable binding. Limited proteolysis assays further demonstrated that ligand binding modulates protein susceptibility to digestion. In the presence of bound ligands, Sola l 4.02 displayed enhanced resistance to both trypsin and α-chymotrypsin, with naringenin and daidzein providing the strongest effect. These results identify Sola l 4.02 as a folded, ligand-binding PR-10 allergen whose proteolytic stability is influenced by small-molecule association, providing new insights into the relationship between structural dynamics, ligand occupancy, and the molecular behavior of tomato food allergens.