The conformation of puroindoline-a and -b, two basic lipid-binding proteins isolated from wheat seedlings, has been studied for the first time by infrared and Raman spectroscopy. The infrared results show that puroindoline-a and -b have similar secondary structure composed of approximately 30% R-helices, 30% -sheets, and 40% unordered structure at pH 7. The conformation of both puroindolines is significantly pH-dependent. The reduction of the disulfide bridges leads to a decrease of the solubility of puroindolines in water and to an increase of the -sheet content by about 15% at the expense of the R-helix content. Raman spectroscopy confirms the structure similarity between the two puroindolines with little differences in the side chains' environment. All the disulfide bridges are in a gauche-gauchegauche conformation, and the unique tyrosine residue present in both puroindolines is hydrogen-bonded to water. Raman spectra have been recorded in both H 2 O and D 2 O media, thus providing additional information concerning the accessibility of certain residues to water. We have also observed that puroindoline-a tends to form some aggregates under acidic and high ionic strength conditions. Nearultraviolet circular dichroism measurements suggest that the tryptophan-rich domain is involved in this aggregate formation. Finally, on the basis of a combined infrared and sequence conformational analysis, we propose a secondary structure assignment for both puroindolines. The results show that puroindolines exhibit a similar folding pattern with plant nonspecific lipid-transfer protein and some amylase-protease inhibitors. These proteins could form a homogeneous structural family of plant proteins involved in the defense against pathogens that are probably derived from a common "helicoidal" protein ancestor.