Elucidation of structure-function relationships of soybean glycinin by protein engineering

甘氨酸是大豆种子的主要贮藏蛋白之一。利用蛋白质工程技术，从分子水平研究了甘氨酸的结构与食品功能特性之间的关系。根据甘氨酸的结构特点，在亚基水平上阐明结构与功能特性之间的关系，对其进行修饰。测定了在大肠杆菌中表达的改性前甘氨酸的功能特性。结果表明，c端区域的疏水性与乳化密切相关，二硫键12-45在凝胶化所需的二硫交换反应的启动中起重要作用，游离巯基残基的数量和拓扑结构与凝胶化密切相关，凝胶化和乳化所需的结构不稳定性因素各不相同。正常和修饰的前甘氨酸（DELTAI, DELTAV8, IV+4Met, V+4Met, Gly12, Ser88）在大肠杆菌中表达。正常晶体、DELTAI晶体、V+4Met晶体、Gly12晶体和Ser88晶体的x射线衍射足以进行晶体学分析。Normal、DELTAI、Clyl2和Ser88晶体属于四边形体系，空间群P4_1或P4_3，晶胞尺寸a=b=114.3-115.9 a, c=145.1-147.1 a。V+ 4Met晶体为单斜晶，属于空间群P2，晶胞参数a=118.7 a, b=78.1 a, c=109.9 a, β =119偶联。这些晶体的原构象数/不对称单位约为3。这个值与前甘氨酸是三聚体的事实是一致的。这些数据表明，这些晶体适合于x射线晶体学在分子水平上阐明甘氨酸的结构和功能特性之间的关系。

Glycinin is one of the dominant storage proteins of soybean seeds. The relationships between the structure and the food functional properties of glycinin were investigated at the molecular level by means of protein engineering. Glycinin was modified on the basis of its structural characteristics and the relationships between the structure and the functional properties elucidated at the subunit level. The functional properties of the modified proglycinins expressed in E.coli were measured. The results suggest that the hydrophobicity of the C-terminal region is closely related to emulsification, that the disulfide bond 12-45 plays an important role in the initiation of the disulfide exchange reaction required for gelation, that the number and the topology of free sulfhydryl residues are closely related to gelation, and that the factors of the structural instability desired for gelation and emulsification are different each other.Crystals of the normal and modified proglycinins (DELTAI, DELTAV8, IV+4Met, V+4Met, Gly12, Ser88) expressed in E.coli have been grown. The crystals of normal, DELTAI, V+4Met Gly12 and Ser88 diffracted X-rays sufficiently for crystallographic analysis. Normal, DELTAI, Clyl2 and Ser88 crystals belong to the tetragonal system, space group P4_1 or P4_3 with unit cell dimensions a=b=114.3-115.9 A and c=145.1-147.1 A.V+ 4Met crystals are monoclinic, belonging to space group P2 with unit cell parameters a=118.7 A, b=78.1 A, c=109.9 A and beta=119ﾟ. The number of protomers/asymmetric unit of these crystals is calculated to be around 3. This value is consistent with the fact that proglycinins are trimers. These data indicate that these crystals examined here are suitable for X-ray crystallography to elucidate the relationships between the structure and the functional properties of glycinin at molecular level.