Determination of the Secondary Structure and Conformation of Puroindolines by Infrared and Raman Spectroscopy

Abstract

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.

A Puroindoline Mutigene Family Exhibits Sequence Diversity in Wheat and is Associated with Yield-Related Traits

“…Puroindoline proteins possess a characteristic tryptophan-rich domain and cysteine backbone; isoforms occur in the starchy endosperm of the Triticeae. Their secondary structure, determined by infrared and Raman spectroscopies, is comprised of approximately 30% -helices, 30% -sheets, and 40% unordered structure at pH 7.4 in solution (Bihan et al 1996). Puroindoline genes are present throughout the Triticeae tribe of the Poaceae (Gramineae), including wheat (Triticum sp.…”

NMR Structure of PW2 Bound to SDS Micelles

“…proteins are able to bind to polar phospholipids, increasing the permeability of model membranes (10,11,34,35) as observed for the Trp-rich antimicrobial peptides indolicidin and tritrpticin (5,9).…”

“…The function of puroindolines in vivo is still unknown, but their in vitro antifungal and antibacterial activities together with the localization of the proteins in the aleurone of the seeds suggest that they play an important role in the protection of the seed (11,35). Structural studies revealed that puroindolines and Ace-AMP1 present tryptophan residues that are exposed to the solvent.…”

“…Their structures were solved in solution in the presence of micellar interfaces (8,9). Other Trp-rich antimicrobial peptides have been described in the literature: ACE-AMP1 (10) is a lipid transfer protein, and puroindolins A and B are wheat plant defensins stabilized by disulfide bridges (11).…”

Determination of Food Quality by Using Spectroscopic Methods

“…For instance, the technique permits to determine the conformation of a non-specific wheat phospholipid transfer protein, and to study the role of disulphide bridges in the stabilization of the α-helical structure. In addition, Raman microspectroscopy was used to determine the secondary structure and conformation of puroindolines, lipid-binding protein of wheat [64].…”