METALLOPROTEIN STRUCTURES WITH THREE FOLDS

三折叠金属蛋白结构

基本信息

批准号：
6455801
负责人：
ELIZABETH D GETZOFF
金额：
$ 8.4万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-06-01 至 2003-05-31
项目状态：
已结题

项目摘要

The overall goals of this proposal are to improve understanding of the structural basis for the binding and activity of biologically important transition metal ions in metalloproteins, to test this understanding by engineering, characterizing, and determining x-ray structures for site- directed mutant proteins that incorporate transplanted metal-binding sites, and to produce designed metalloproteins that mimic or extend the metal-binding and catalytic properties of the original metal-binding site. We will use recursive structure-based design to transplant metal- ions templates from Cu- and Zn-containing metalloproteins of known structure into three different protein structural frameworks. For protein scaffolds, we will start from the known, high resolution, crystallographic structures of antibodies, photoactive yellow protein (PYP) and green fluorescent protein (GFP). By using the same metal ion templates with different protein scaffolds and among different locations within a single scaffold, we will test the roles of the protein framework in determining metal-site geometry and providing the environment needed for the affinity, specificity, and activity of metal- binding sites in proteins. After design and construction, biochemical and spectroscopic characterization, and 3-dimensional structure determination and analysis of these metalloprotein mutants, we will apply our results and those from other projects to improve and optimize the original designs, or to suggest new designs. The results from the 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 to improve our metalloprotein designs. This interactive approach will allow us to rigorously test and refine our understanding of the requirements for functional metal sites in proteins. The long term objective of this proposal is to engineer scientifically and medically useful metalloproteins that synergistically combined the specificity of antibodies or the natural reporter groups of PYP and GRP, with the remarkable functional and catalytic properties of protein-bound metal ions.

该提案的总体目标是提高对金属蛋白中生物学上重要的过渡金属离子的结合和活性的结构基础的理解，通过工程设计、表征和确定定点突变蛋白的 X 射线结构来测试这种理解。整合移植的金属结合位点，并产生设计的金属蛋白，模拟或扩展原始金属结合位点的金属结合和催化特性。我们将使用基于递归结构的设计将已知结构的含铜和锌金属蛋白的金属离子模板移植到三种不同的蛋白质结构框架中。对于蛋白质支架，我们将从抗体、光活性黄色蛋白（PYP）和绿色荧光蛋白（GFP）的已知高分辨率晶体结构开始。通过使用具有不同蛋白质支架以及单个支架内不同位置的相同金属离子模板，我们将测试蛋白质框架在确定金属位点几何形状并提供金属亲和力、特异性和活性所需的环境中的作用- 蛋白质中的结合位点。在对这些金属蛋白突变体进行设计和构建、生化和光谱表征以及三维结构测定和分析之后，我们将应用我们的结果和其他项目的结果来改进和优化原始设计，或提出新的设计。其他关于金属位点在金属蛋白的折叠和组装、稳定性、调节和化学活性中的结构和化学作用的项目的结果将为改进我们的金属蛋白设计提供实验和计算信息。这种交互式方法将使我们能够严格测试和完善我们对蛋白质中功能性金属位点要求的理解。该提案的长期目标是设计科学和医学上有用的金属蛋白，将抗体或 PYP 和 GRP 天然报告基团的特异性与蛋白质结合金属离子的显着功能和催化特性协同结合。