Richard Julie-Andrée, Kelly Isabelle, Marion Didier, Auger Michèle, Pézolet Michel
Département de chimie, Centre de Recherche en Sciences et Ingénierie des Macromolécules, Université Laval, Québec, Canada G1K 7P4.
Biochemistry. 2005 Jan 11;44(1):52-61. doi: 10.1021/bi048443t.
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.
二维红外相关光谱已被用于研究β-硫堇蛋白的结构，β-硫堇蛋白是一种存在于小麦种子胚乳中的小分子碱性蛋白质，研究是在不存在和存在二肉豆蔻酰磷脂酰甘油（DMPG）膜的情况下进行的。为了生成二维同步和异步图谱，蛋白质酰胺质子的氢-氘交换已被用作外部扰动。这种方法使我们能够区分酰胺I、II和II'带区域中不同的二级结构元件和侧链贡献，从而确定β-硫堇蛋白质子的氘化相对顺序如下：转角、天冬酰胺和赖氨酸>无规结构和酪氨酸>β-折叠>α-螺旋和精氨酸。结果还表明，在存在DMPG双层膜的情况下，该蛋白质在二级结构和氘化方面都发生了显著变化。在脂质存在的情况下，β-硫堇蛋白的螺旋含量更高，氘化的相对顺序如下：赖氨酸和精氨酸>天冬酰胺和β-折叠>无规结构和α-螺旋。精氨酸残基氘化顺序的反转归因于蛋白质在膜中缔合程度的变化。此外，结果表明，蛋白质中含有酪氨酸残基的部分与脂质膜相互作用。我们的结果与先前发表的结果相结合表明，β-硫堇蛋白的毒性更多地与细胞膜中功能通道的形成有关，而不是与溶解现象有关。
