MULTIDOMAIN METALLOPROTEINS

Cys2His2类型的锌指蛋白由28- 30个氨基酸结构域，每个结构域都与一个锌离子结合。看起来， 几乎所有这些蛋白质都以一个序列与双链DNA结合 特定的时尚。这是一个非常大和重要的蛋白质家族。它是 据估计，人类基因组可能编码多达1000个这样的蛋白质 家庭，其中许多似乎在发展中发挥作用。此外， 编码这个家族蛋白质的基因的变化与 患有几种不同类型的癌症。从结晶学和其他 研究表明，每个结构域似乎都与DNA的三个碱基对接触，并 这些三联体亚基是直接毗邻的。这种模块化结构 提示这些蛋白质可用于序列设计。 特定的DNA结合域一旦与氨基酸之间的关系 蛋白质结构域序列及其亚位点的核苷酸序列 已经建立起来了。其中一些关系已经被开发出来 通过以往的研究，特别是针对G/C丰富的结合部位。穿过 突变和结合研究，这些关系将扩展到 包括尽可能多的结合位点。此外，这一程度 从特定的蛋白质-核苷酸接触中获得的区分将 要下定决心。这种歧视在不同的地点差别很大 并且可以与结构相互作用的性质相关联。这些 识别规则有两种使用方式。首先，这些允许预测 来自蛋白质序列的结合位点。这些预测将被检验为 结合部位是由额外的蛋白质决定的。第二，这些规则 可用于设计新的序列特异性DNA结合蛋白。这些 设计的蛋白质，将基于固定的结构框架 随着与DNA相互作用相关的四个关键残基的变化， 将在DNA结合特异性方面进行表征，总体上 亲和力和结构。可以为各种应用程序构思 这些设计的序列特异性结合蛋白。

Zinc finger proteins of the Cys2His2 type consists of tandem arrays of 28- 30 amino acid domains, each of which binds a zinc ion. It appears that almost all of these proteins bind double stranded DNA in a sequence specific fashion. This is a very large and important protein family. It is estimated that the human genome may encode up to 1000 proteins of this family, many of which appear to play a role in development. Moreover, changes in genes encoding proteins of this family have been associated with several different types of cancer. From crystallographic and other studies, it appears that each domain contacts three base pairs of DNA and these triplet subsites are directly abutted. This modular construction suggests that these proteins could be used for the design of sequence specific DNA binding domains once the relationships between the amino acid sequence of the protein domain and the nucleotide sequence of its subsite have been established. Some of these relationships have been developed through previous studies, particular for G/C rich binding sites. Through mutational and binding studies, these relationships will be extended to include as many binding sites as possible. Furthermore, the degree of discrimination available from particular protein-nucleotide contacts will be determined. This discrimination varies considerably from site to site and can be correlated with the nature of the structural interaction. These recognition rules used in two ways. First, these allow the prediction to binding sites from protein sequence. These predictions will be tested as binding sites are determined from additional proteins. Second, these rules can be used to design novel sequence specific DNA binding proteins. These designed proteins, which will be based on a fixed structural framework with variation of the four key residues associated with DNA interactions, will be characterized in terms of DNA binding specificity, overall affinity, and structure. A variety of applications can be conceived for these designed sequence specific binding proteins.