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.

该建议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离子的结构基础的理解，以通过工程，表征和确定X射线结构来测试现场的X射线结构，以构建了X射线蛋白，这些突变蛋白可以将金属蛋白或养育金属构成的金属蛋白构成金属蛋白质和养育金属构成的金属蛋白，并养成金属型物质，并养成了金属构造和养育金属的纽带。我们将使用基于递归结构的设计将已知结构的含有Cu和Zn的金属蛋白从三种不同的蛋白质结构框架中移植金属离子模板。对于蛋白质支架，我们将从已知的高分辨率，抗体的晶体学结构，光活性黄蛋白（PYP）和绿色荧光蛋白（GFP）开始。通过使用具有不同蛋白质支架的相同金属离子模板以及单个支架内的不同位置之间，我们将测试蛋白质框架在确定金属位点几何形状方面的作用，并为蛋白质中金属结合位点的亲和力，特异性和活性提供所需的环境。在设计和构造，生化和光谱表征以及对这些金属蛋白突变体的三维结构确定和分析后，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。其他项目的结果是关于金属位点在折叠和组装，金属蛋白的稳定性，调节和化学活性中的结构和化学作用的结果，将促进实验和计算信息，以改善我们的金属蛋白设计。这种互动方法将使我们能够严格测试和完善我们对蛋白质功能金属位点的要求的理解。该提案的长期目标是在科学和医学上有用的金属蛋白进行工程，以协同结合抗体或PYP和GRP的天然报告基团的特异性，并与蛋白质结合金属离子的显着功能和催化性能相结合。