ACCELERATING IRON CHELATION FROM SERUM TRANSFERRIN

加速血清转铁蛋白的铁螯合

• 批准号: 6390952
• 负责人: WESLEY R HARRIS
• 金额: $ 23.55万
• 依托单位: UNIVERSITY OF MISSOURI-ST. LOUIS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6390952
• 关键词: allosteric site; anions; binding sites; chelating agents; chelation therapy; chemical kinetics; computer simulation; conformation; fluorescence spectrometry; fluorescent dye /probe; iron metabolism; iron storage disorder; ligands; metal complex; molecular dynamics; mutant; protein binding; protein structure function; recombinant proteins; site directed mutagenesis; transferrin

DESCRIPTION (adapted from the application) Serum transferrin is the protein that transports iron through blood. The protein consists of two similar lobes, with a single iron binding site located at the base of a cleft within each lobe. Under conditions of iron overload, transferrin becomes saturated with iron. However, the rate of iron exchange with low molecular weight ligands tends to be very slow, which severely restricts the ability of therapeutic iron chelating agents to target this readily accessible iron pool. The rate of iron release from transferrin shows a complex dependence on both the ligand concentration and the concentration of inorganic salts in the buffer. For many ligands it appears that the maximum rate of iron release is limited by a slow protein conformational change, but this process is not well understood. Some ligands appear to be able to avoid this limitation either by taking advantage of a separate, first-order pathway that somehow avoids the conformational gating, or by binding at a poorly characterized allosteric anion-binding site that accelerates the rate of the conformational change. The primary objectives of this study are to determine the details of the mechanism of iron exchange between transferrin and low molecular weight ligands and to use this information to design new agents for accelerating iron removal from transferrin under physiological conditions. The proposed work will emphasize studies on recombinant transferrin half-molecules, Tf/2N and Tf/2C, as well as a series of site directed mutants of these molecules. The kinetics of iron release will be followed by visible and fluorescence spectroscopies. Kinetic studies on the native and mutant proteins will characterize the pathways available for iron release. Both computation studies and site directed mutagenesis will be used to locate and characterize the allosteric anion binding site and to determine how anion binding at this site is able to accelerate iron release. New compounds will be synthesized that will be designed to avoid the limitations of the protein conformational change either by accelerate iron release through a separate, first-order pathway or by targeting the allosteric anion binding site to accelerate the rate of the gating conformational change. This is a collaborative project involving faculty with expertise in molecular biology, solution kinetics, organic synthesis, and computational chemistry.

描述（改编自应用程序） 血清转铁蛋白是通过血液运输铁的蛋白质。这 蛋白质由两个相似的叶组成，具有单个铁结合位点 位于每个叶内裂口的底部。在铁过载的情况下， 转铁蛋白被铁饱和。但铁交换率 与低分子量配体的反应往往非常慢，这严重 限制了治疗性铁螯合剂靶向此的能力 易于使用的铁池。 转铁蛋白释放铁的速率显示出对两者的复杂依赖性 配体浓度和无机盐浓度 缓冲。对于许多配体来说，铁释放的最大速率似乎是 受到缓慢的蛋白质构象变化的限制，但这个过程并不顺利 明白了。一些配体似乎能够通过以下方式避免这种限制 利用一个单独的一阶路径，以某种方式避免 构象门控，或通过结合在特征较差的变构上 加速构象变化速率的阴离子结合位点。这 这项研究的主要目标是确定该机制的细节 转铁蛋白和低分子量配体之间的铁交换 利用这些信息设计新的试剂来加速除铁 生理条件下的转铁蛋白。 拟议的工作将重点研究重组转铁蛋白 半分子、Tf/2N 和 Tf/2C 以及一系列定点突变体 这些分子。铁释放的动力学将跟随可见的 和荧光光谱。天然和突变体的动力学研究 蛋白质将表征铁释放的可用途径。两个都 计算研究和定点诱变将用于定位和 表征变构阴离子结合位点并确定阴离子如何 在此位点的结合能够加速铁的释放。新化合物将 合成的目的是为了避免蛋白质的局限性 通过单独的加速铁释放来实现构象变化， 一级途径或通过靶向变构阴离子结合位点 加速门控构象变化的速度。这是一个 涉及具有分子生物学专业知识的教师的合作项目， 溶液动力学、有机合成和计算化学。