Determination of the secondary structure and conformation of puroindolines by infrared and Raman spectroscopy.

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% alpha-helices, 30% beta-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 beta-sheet content by about 15% at the expense of the alpha-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-gauche-gauche conformation, and the unique tyrosine residue present in both puroindolines is hydrogen-bonded to water. Raman spectra have been recorded in both H2O and D2O 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. Near-ultraviolet 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.