Mapping interaction surface domains in lens crystallins

描述(申请人提供)：晶状体含有三种主要类型的结构蛋白：α-、β-和伽马-晶体蛋白。先前的生化和光谱研究表明，α-晶体蛋白和β-或伽马-晶体蛋白之间可能存在相互作用。我们最近使用哺乳动物双杂交系统的研究证实了晶状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状体蛋白突变所改变。这种双杂交系统不仅可以检测分子间的相互作用，还可以检测分子内的相互作用，在研究α-(αA-和αB)晶体蛋白方面特别有用。映射接口域是双混合系统的主要应用之一。αA-和α-B-晶状体蛋白是一种低聚物，其三维结构尚未确定，因为它们不能结晶以进行X射线衍射研究。在这一建议中，假设亚基间反应结构域可以通过双杂交系统结合定点突变来定位，而界面结构域主要由β链组成，可以作为突变的靶点。一旦确定了界面的β-链，就可以设计和合成模拟该β-链的小肽。多肽与αA或αB晶体蛋白之间的反应将防止二聚体和低聚物的形成，天然单体将可用于结构研究。第一个特定目标将是对已知结构的β-和γ-晶体蛋白的初步研究；第二个特定目标将是绘制αA-和αB-晶体蛋白的界面结构域，然后研究多肽-蛋白质的相互作用。该方法包括克隆、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化和寡聚化的机制，其结果可能有助于我们理解α-晶状体蛋白在正常晶状体和白内障形成中的作用。

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.