LIGAND GATED TRANSPORT THROUGH FEPA

通过 FEPA 进行配体门控运输

• 批准号: 6386250
• 负责人: PHILLIP E KLEBBA
• 金额: $ 25.86万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386250
• 关键词: X ray crystallography; adsorption; bacterial proteins; bacterial toxins; binding sites; conformation; flow cytometry; fluorescence spectrometry; gram negative bacteria; iron; laboratory mouse; laboratory rabbit; membrane permeability; membrane proteins; membrane transport proteins; molecular cloning; nucleic acid sequence; polymerase chain reaction; pore forming protein; protein purification; protein transport; receptor binding; siderophores; site directed mutagenesis; tissue /cell culture

DESCRIPTION (Adapted from the Applicant's Abstract): Bacteria elaborate iron chelators that scavenge iron from the environment, including their human and animal hosts. Iron acquisition is a determinant of pathogenicity. One such iron chelate, the siderophore enterobactin, enters gram-negative bacteria through the FepA protein of the outer membrane. FepA is a ligand-gated porin, in that binding of ferric enterobactin triggers transport through its transmembrane pore. This high affinity multispecific, multicomponent, energy dependent transport process is a paradigm of prokaryotic membrane biochemistry. Based on the FepA crystal structure, the proposed research will use molecular biological, biochemical, and biophysical methods to investigate the mechanism of ferric enterobactin uptake. The experiments will address two stages of the transport event, binding and internalization. Dr. Klebba will study the specificity of the initial recognition event by binding experiments on both wild type FepA and site-directed mutants, containing alterations to residues in either the external loops of the top loops of the N-terminal globular domain. He will similarly characterize the ligand internalization reaction by mutagenesis of target residues that are conserved among other Gram-negative bacterial ligand-gated porins. Mutant proteins of interest, those with impaired ligand binding or ligand internalization phenotypes, will be crystallized and studied by X-ray diffraction Finally, he will perform biophysical analyses of conformation changes that occur in FepA during ligand transport.

描述（改编自申请人的摘要）：细菌精细的铁 从环境中清除铁的螯合剂，包括其人和 动物宿主。铁采集是致病性的决定因素。 一个这样的 铁螯合物，铁载体肠actin，进入革兰氏阴性细菌 通过外膜的FEPA蛋白。 FEPA是配体门控孔， 在铁肠霉素的结合中 跨膜孔。 这种高亲和力多特异性，多组分，能量 依赖运输过程是原核生物膜的范式 生物化学。 基于FEPA晶体结构，拟议的研究将 使用分子生物学，生化和生物物理方法研究 铁摄起蛋白摄取的机制。 实验将解决两个 运输事件，绑定和内在化的阶段。 克莱巴·威尔博士 通过结合实验研究初始识别事件的特异性 在野生型FEPA和位于现场的突变体上，包含对 N端顶环的任何一个外回路中的残留物 球状域。 他将同样描述配体内在化 通过诱变的诱变反应在其他目标残基中保守的反应 革兰氏阴性细菌配体门控孔蛋白。 感兴趣的突变蛋白， 那些患有配体结合或配体内在化表型受损的人将 最终通过X射线衍射结晶和研究，他将执行 配体期间FEPA中发生的构象变化的生物物理分析 运输。