LIGAND GATED TRANSPORT THROUGH FEPA

描述(摘自申请者的摘要)：细菌精制铁 从环境中清除铁的螯合剂，包括它们的人类和 动物寄主。铁的获得是致病性的决定因素。这样的一个 铁络合物，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是一种配体门控的孔蛋白， 在铁结合肠杆菌蛋白触发其转运过程中 跨膜孔。这种高亲和力的多特定、多组分的能量 依赖转运过程是原核膜的一种范式 生物化学。基于FePA的晶体结构，拟议的研究将 利用分子生物学、生化和生物物理方法研究 肠上皮铁摄取的机制。这些实验将解决两个问题 运输事件、绑定和内化的各个阶段。克雷巴医生会 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，包含改变到 N-末端顶环的任一个外环中的残基 球状域。他将以类似的方式描述配基内化 通过对其中保守的靶残基进行突变而产生的反应 革兰氏阴性细菌配基门控蛋白。感兴趣的突变蛋白质， 那些配体结合或配体内化表型受损的人，将 最后用X射线衍射法进行结晶和研究，他将表演 Fepa配基过程中构象变化的生物物理分析 运输。

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.