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

大豆球蛋白是大豆种子的主要贮藏蛋白之一。利用蛋白质工程的方法，从分子水平上研究了大豆球蛋白的结构与食品功能性质的关系。根据大豆球蛋白的结构特征和在亚基水平上阐明的结构与功能性质之间的关系，对其进行了修饰。对修饰后的原球蛋白在大肠杆菌中表达的功能性质进行了检测。结果表明，C-末端的疏水性与乳化性密切相关，二硫键12-45在凝胶所需的二硫键交换反应的启动中起重要作用，游离巯基的数目和拓扑结构与凝胶密切相关，凝胶和乳化所需的结构不稳定因素是不同的。NORMAL、DELTAI、V4Met Gly12和Ser88的晶体对X射线有足够的衍射，可用于结晶学分析。正常、德尔泰、Clyl2和Ser88晶体属于四方晶系，晶胞尺寸为a=b=114.3-115.9 A和c=145.1-147.1 A的P4_1或P4_3空间群。V4Met晶体为单斜晶系，属于P2空间群，晶胞参数a=118.7 A，b=78.1A，c=109.9 A，β=119ﾟ。计算出这些晶体的原构体/不对称单元数约为3。这一值与原球蛋白是三聚体的事实相一致。这些数据表明，这些晶体适合于X射线结晶学在分子水平上阐明大豆球蛋白的结构与功能性质之间的关系。

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.