Biochemistry /structure comparison of transferrin recept

转铁蛋白受体的生物化学/结构比较

• 批准号: 6591513
• 负责人: Pamela J Bjorkman
• 金额: $ 3.9万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6591513
• 关键词: X ray spectrometry; analytical ultracentrifugation; atomic absorption spectrometry; binding sites; human tissue; iron metabolism; mass spectrometry; protein protein interaction; protein sequence; protein structure function; receptor binding; receptor expression; site directed mutagenesis; surface plasmon resonance; tissue /cell culture; transferrin receptor

We are interested in characterizing the structures and biochemical properties of proteins involved in iron metabolism, with the assumption that biochemical and 3D structural information can be used to guide design of biological experiments to understand their function. We previously solved the crystal structures of HFE, a type I membrane protein that binds to transferrin receptor 1 (TfR1) and is mutated in the iron overload disease hereditary hemochromatosis, and an HFE/TfR1 complex. We now wish to combine our expertise in structural biology and biochemistry with the skills of Drs. Enns and Blackburn in cell biology and metalloprotein spectroscopy respectively, to study different aspects of metallobiology, with the goal of shedding new light on the function of a newly discovered transferrin receptor (TfR2). We have already demonstrated that a soluble form of TfR2 binds iron loaded transferrin (FeTf), but not HFE, at the pH of the cell surface. We now seek to further characterize the TfR2 interaction with Tf in order to compare and contrast the binding properties of TfR2 with those of TfR1. The aims of this project are to: (1) Examine the binding profiles of apo and diferric Tf to TfR2 compared to TfR1, with an emphasis on characterizing the pH dependence of the interactions in order to predict which intracellular compartments contain TfR2-Tf complexes, (2) Define the binding site on TfR2 for Tf by site directed mutagenesis and by a cross-linking, proteolysis and mass spectrometry approach (in collaboration with Dr. Enns), (3) Conduct structural studies of TfR2, the Tf-TfR2 complex and other interesting proteins coming out of these projects in collaboration with all of the investigators participating in this program project.

我们有兴趣表征参与铁代谢的蛋白质的结构和生化特性，并假设生化和3D结构信息可用于指导生物学实验的设计以了解其功能。我们先前求解了HFE的晶体结构，HFE是一种与转铁蛋白受体1（TFR1）结合的I型膜蛋白，并在铁超载疾病遗传性血糖沉着症和HFE/TFR1复合物中突变。现在，我们希望将结构生物学和生物化学方面的专业知识与DR的技能相结合。细胞生物学和金属蛋白光谱法中的ENN和Blackburn研究肠生生物学的不同方面，目的是对新发现的转铁蛋白受体（TFR2）的功能进行新的启示。我们已经证明，TFR2的可溶形式在细胞表面的pH值下结合了铁载蛋白（FETF），而不是HFE。现在，我们试图进一步表征TFR2与TF的相互作用，以将TFR2的结合特性与TFR1的结合特性进行比较。该项目的目的是：（1）与TFR1相比，检查APO和DIDERRIC TF与TFR2的结合曲线，重点是表征相互作用的pH依赖性，以预测哪些细胞内隔室包含TFR2-TF复合物，（2），（2） 通过现场定向诱变，以及通过交联，蛋白水解和质谱方法（与ENNS博士合作），（3）TFR2的结构研究，TF-TFR2复合物和其他有趣的蛋白质与研究人员一起参与该计划的项目，将结合位点定义为TF2的结合位点。