METALLOPROTEIN STRUCTURES WITH THREE FOLDS

本提案的总体目标是提高对金属蛋白中具有重要生物学意义的过渡金属离子结合和活性的结构基础的理解，并通过工程、表征和确定包含移植金属结合位点的位点导向突变蛋白的x射线结构来验证这一理解。并产生设计的金属蛋白，模仿或扩展原始金属结合位点的金属结合和催化性能。我们将采用基于递归结构的设计，将已知结构的含铜和含锌金属蛋白中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，我们将从已知的高分辨率抗体，光活性黄色蛋白（PYP）和绿色荧光蛋白（GFP）的晶体结构开始。通过将相同的金属离子模板与不同的蛋白质支架以及在单个支架内的不同位置之间使用，我们将测试蛋白质框架在确定金属位点几何形状以及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环境方面的作用。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、三维结构测定和分析后，我们将利用我们的研究结果和其他项目的研究结果对原有设计进行改进和优化，或提出新的设计建议。其他项目对金属位点在金属蛋白折叠和组装中的结构和化学作用、稳定性、调控和化学活性的研究结果将为改进我们的金属蛋白设计提供实验和计算信息。这种相互作用的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需求的理解。本提案的长期目标是设计科学和医学上有用的金属蛋白，将抗体的特异性或PYP和GRP的天然报告基团与蛋白质结合金属离子的显着功能和催化特性协同结合。

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.