LIGAND GATED TRANSPORT THROUGH FEPA

通过 FEPA 进行配体门控传输

• 批准号: 6892013
• 负责人: PHILLIP E KLEBBA
• 金额: $ 9.26万
• 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 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6892013
• 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）：细菌精制铁 从环境（包括人类和环境）中清除铁的螯合剂 动物宿主。铁的获取是致病性的决定因素。 这样的一位 铁螯合物，即铁载体肠杆菌素，进入革兰氏阴性细菌 通过外膜的FepA蛋白。 FepA ​​是一种配体门控孔蛋白， 因为肠杆菌素铁的结合触发了通过其转运 跨膜孔。 这种高亲和力的多特异性、多组分、能量 依赖性转运过程是原核细胞膜的一个范例 生物化学。 基于FepA晶体结构，拟议的研究将 使用分子生物学、生物化学和生物物理方法进行研究 肠杆菌素铁摄取机制。 实验将解决两个问题 运输事件、结合和内化的阶段。 克莱巴博士将 通过结合实验研究初始识别事件的特异性 在野生型 FepA ​​和定点突变体上，包含对 N-末端顶部环的外部环中的残基 球状域。 他将类似地描述配体内化 通过诱变目标残基进行反应，这些残基在其他残基中是保守的 革兰氏阴性细菌配体门控孔蛋白。 感兴趣的突变蛋白， 那些配体结合或配体内化表型受损的人，将 进行结晶并通过X射线衍射进行研究最后，他将进行 FepA ​​配体过程中发生的构象变化的生物物理分析 运输。