METALLOANTIBODY STRUCTURE AND ENGINEERING

我们在这里建议设计、构建和表征这两种功能 在结构上，新的金属抗体。来自七个国家的结果 关于金属场所的结构和化学作用的其他项目 折叠和组装、稳定性、调节和化学活性 金属蛋白将在实验和计算方面做出贡献 关于改进的金属抗体设计的信息。这种迭代方法 将使我们能够严格地测试和完善我们对 蛋白质中功能性金属部位的要求。 我们选择小鼠抗细胞色素c的单抗E8作为 为我们的重新设计实验搭建了脚手架。这种E8抗体结合它的 高亲和力的天然金属蛋白抗原(10-9-10-10M)， 为研究相互间的电子耦合提供了可能性 抗体和自然产生的血红素中的工程金属位置- 细胞色素c中的铁。E8抗体已被很好地表征 生物化学、免疫学和结构学：基因序列 E8可变区，以及两者的晶体结构 游离抗原结合片段(Fab)和Fab/细胞色素c复合体 在Getzoff实验室中被确定，以及一种分泌系统 表达野生型的转化细胞系中的突变抗体 与突变的E8重链配对的E8轻链已在 加斯科因实验室。 为了实现创造一种特定部位的锌蛋白酶的最终目标，我们有 将锌位点逐个移植到E8抗体的重链中- 定向诱变。除了蛋白分解活性外，设计目标 包括氧化还原反应和金属介导细胞色素c结合。 由此产生的定点突变金属抗体，我们的目标是 表征金属结合特性，测量抗原结合和 潜在的催化活性，并测定和分析三种- 三维晶体结构。这样做的长期目标是 建议是设计出科学上和医学上有用的 金属抗体协同结合了显著的结合和 具有强大功能和识别特性的抗体 金属蛋白的催化性能。

We propose here to design, construct, and characterize, both functionally and structurally, novel metalloantibodies. The results from the seven other projects on the structural and chemical roles of metal sites in the folding and assembly, stability, regulation, and chemical activity of metalloproteins will contribute experimental and computational information for improved metalloantibody design. This iterative approach will allow us to rigorously test and refine our understanding of the requirements for functional metal sites in proteins. We chose the murine monoclonal anti-cytochrome c antibody E8 as the scaffold for our redesign experiments. This E8 antibody binds its natural metalloprotein antigen with high affinity (10-9-10-10M), providing the potential for studies of electronic coupling between engineered metal sites in the antibody and the naturally occurring heme- Fe in cytochrome c. The E8 antibody has been well characterized biochemically, immunologically, and structurally: the gene sequences of the E8 variable domains, and the crystallographic structures of both the free antigen-binding fragment (Fab) and the Fab/cytochrome c complex have been determined in the Getzoff laboratory, and a system for secreting mutant antibodies in a transformed cell line that expresses the wild-type E8 light chain paired with mutant E8 heavy chains has been developed in the Gascoigne laboratory. Toward the ultimate goal of creating a site-specific Zn protease, we have transplanted a Zn site into the heavy chain of the E8 antibody by site- directed mutagenesis. Besides proteolytic activity, design targets include redox reactions and metal-mediated binding of cytochrome c. For the resulting site-directed mutant metalloantibodies, our aims are to characterize metal-binding properties, measure antigen-binding and potential catalytic activities, and determine and analyze three- dimensional crystallographic structures. The long term objective of this proposal is to engineer scientifically and medically useful metalloantibodies that synergistically combine the remarkable binding and recognition properties of antibodies with the powerful functional and catalytic properties of metalloproteins.