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 pro teins isolated from wheat seedlings, has been studied fur the first ti me by infrared and Raman spectroscopy. The infrared results show that puroindoline-a and -b have similar secondary structure composed of app roximately 30% alpha-helices, 30% beta-sheets, and 40% unordered struc ture at pH 7. The conformation of both puroindolines is significantly pH-dependent. The reduction of the disulfide bridges leads to a decrea se of tile 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' envi ronment. All the disulfide bridges are in a gauche-gauche-gauche confo rmation, 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 t he accessibility of certain residues to water. We have also observed t hat puroindoline-a tends to form some aggregates under acidic and high ionic strength conditions. Near-ultraviolet circular dichroism measur ements suggest that the tryptophan-rich domain is involved in this agg regate formation. Finally, on the basis of a combined infrared and seq uence conformational analysis, we propose a secondary structure assign ment for both puroindolines. The results show that puroindolines exhib it a similar folding pattern with plant nonspecific lipid-transfer pro tein and some amylase-protease inhibitors. These proteins could form a homogeneous structural family of plant proteins involved in the defen se against pathogens that are probably derived from a common ''helicoi dal'' protein ancestor.