alpha1-purothionin-protein--wheat and dimyristoylphosphatidylglycerol

Two-dimensional infrared correlation spectroscopy has been used to investigate the structure of beta-purothionin, a small basic protein found in the endosperm of wheat seeds, in the absence and presence of dimyristoylphosphatidylglycerol (DMPG) membranes. To generate the two-dimensional synchronous and asynchronous maps, hydrogen-deuterium exchange of the protein amide protons has been used as an external perturbation. This method has allowed us to separate the different secondary structure elements and side chain contributions in the regions of amide I, II, and II' bands to determine that the relative order of deuteration of the beta-purothionin protons is as follows: turns, asparagines, and lysines > unordered structure and tyrosine > beta-sheet > alpha-helices and arginines. The results also indicate that the protein undergoes significant changes both in secondary structure and in deuteration in the presence of DMPG bilayers. The helical content of beta-purothionin is higher in the presence of the lipid, and the relative order of deuteration is as follows: lysines and arginines > asparagines and beta-sheet > unordered structure and alpha-helices. The inversion in the deuteration order of the arginine residues is assigned to a change of the degree of association of the protein in the membrane. In addition, the results reveal that the part of the protein containing the tyrosine residue interacts with the lipid membrane. Our results combined with those previously published suggest that the toxicity of beta-purothionin is more associated with the formation of functional channels in cell membranes rather than with a lytic phenomenon.

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Deuterium; Kinetics; Models, Molecular; Phosphatidylglycerols; Plant Proteins; Protein Conformation; Protein Structure, Secondary; Seeds; Spectrophotometry, Infrared; Toxins, Biological; Triticum

The interaction of beta-purothionin, a small basic and antimicrobial protein from the endosperm of wheat seeds, with multilamellar vesicles of dimyristoylphosphatidylglycerol (DMPG) was investigated by (31)P solid-state NMR and infrared spectroscopy. NMR was used to study the organization and dynamics of DMPG in the absence and presence of beta-purothionin. The results indicate that beta-purothionin does not induce the formation of nonlamellar phases in DMPG. Two-dimensional exchange spectroscopy shows that beta-purothionin decreases the lateral diffusion of DMPG in the fluid phase. Infrared spectroscopy was used to investigate the perturbations, induced by beta-purothionin, of the polar and nonpolar regions of the phospholipid bilayers. At low concentration of beta-purothionin, the temperature of the gel-to-fluid phase transition of DMPG increases from 24 degrees C to ~33 degrees C, in agreement with the formation of electrostatic interactions between the cationic protein and the anionic phospholipid. At higher protein concentration, the lipid transition is slightly shifted toward lower temperature and a second transition is observed below 20 degrees C, suggesting an insertion of the protein in the hydrophobic core of the lipid bilayer. The results also suggest that the presence of beta-purothionin significantly modifies the lipid packing at the surface of the bilayer to increase the accessibility of water molecules in the interfacial region. Finally, orientation measurements indicate that the alpha-helices and the beta-sheet of beta-purothionin have tilt angles of ~60 degrees and 30 degrees, respectively, relative to the normal of the ATR crystal.

Topics: Antimicrobial Cationic Peptides; Biophysical Phenomena; Biophysics; Diffusion; Lipid Bilayers; Magnetic Resonance Spectroscopy; Models, Biological; Phosphatidylglycerols; Plant Proteins; Protein Structure, Secondary; Spectrophotometry, Infrared; Spectroscopy, Fourier Transform Infrared; Temperature; Triticum