Mapping interaction surface domains in lens crystallins

描述（由申请人提供）：透镜含有三种主要类型的结构蛋白：α-、β-和γ-晶体蛋白。先前的生化和光谱研究表明α-晶体蛋白和β-或γ-晶体蛋白之间的相互作用的可能性。我们最近使用哺乳动物双杂交系统的研究证实，晶体蛋白之间存在相互作用，并且这些相互作用被一些先天性白内障晶体蛋白突变所改变。双杂交系统不仅可以检测分子间的相互作用，而且还可以检测分子内的相互作用，并且在研究α-（α A-和α B）晶状体蛋白中特别有用。映射界面区域是双杂交系统的主要应用之一。α A-和α B-晶体蛋白是低聚物，其三维结构尚未确定，因为它们不能结晶用于X-射线衍射研究。在该提议中，假设亚基间反应结构域可以借助于位点特异性突变通过双杂交系统测定来定位，并且界面结构域主要由β链组成并且可以靶向突变。一旦识别出界面β链，就可以设计和合成模拟β链的小肽。肽与α A-或α B-晶状体蛋白之间的反应将防止形成二聚体和寡聚体，并且天然单体将可用于结构研究。第一个具体目标将是对已知结构的β-和γ-晶体蛋白进行初步研究;第二个具体目标将是绘制α A-和α B-晶体蛋白的界面结构域，然后研究肽-蛋白质相互作用。方法学包括克隆、亚克隆、定点突变、细胞培养、蛋白表达和纯化、小肽设计和合成以及双杂交系统分析。长期的目标是了解二聚体和寡聚体的机制，其结果可能有助于我们了解α-晶状体蛋白在正常透镜和白内障形成中的功能。

DESCRIPTION (provided by applicant): The lens contains three major types of structural protein: alpha-, beta-, and gamma-crystallins. Previous biochemical and spectroscopic studies indicate the possibility of interactions between alpha-crystallin and beta- or gamma-crystallin. Our recent studies using a mammalian two-hybrid system confirm that there are interactions among crystallins and that these interactions are altered by some congenital cataract crystallin mutations. The two-hybrid system can detect not only inter-molecular interactions but also intra-molecular interactions and is especially useful in studying alpha- (alphaA- and alphaB) crystallin. Mapping interface domains is one of the major applications of the two-hybrid system. alphaA- and alphaB-crystallins are oligomers whose three-dimensional structures have not been determined because of their inability to be crystallized for x-ray diffraction study. In this proposal, it is hypothesized that inter-subunit reaction domains can be mapped by the two-hybrid system assay with the aid of site-specific mutation, and that interface domains consist mainly of beta-strands and can be targeted for mutations. Once the interface beta-strands are identified, small peptides mimicking the beta-strands can be designed and synthesized. The reactions between peptides and alphaA- or alphaB-crystallin will prevent formation of dimers and oligomers, and native monomers will be available for structural studies. The first specific aim will be preliminary studies with known structural beta-and gamma-crystallins; the second specific aim will be mapping interface domains of alphaA- and alphaB-crystallins, followed by studying peptide-protein interactions. The methodology includes cloning, subcloning, site-specific mutagenesis, cell culture, protein expression and purification, small peptide design and synthesis, and two-hybrid system assay. The long-term objective is to understand the mechanisms of dimerization and oligomerization, and the results may help us to understand the function of alpha-crystallin in normal lens and in cataract formation.