Nanoassay for Realtime Molecular Probing ABC Transporter

用于实时分子探测 ABC 转运蛋白的纳米测定

• 批准号: 7231705
• 负责人: X. Nancy Xu
• 金额: $ 28.38万
• 依托单位: OLD DOMINION UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-10 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7231705
• 关键词: ATP Hydrolysis; ATP-Binding Cassette Transporters; ATP-binding cassette transport; Adrenoleukodystrophy; Antifungal Antibiotics; Bacteria; Binding; Biocompatible; Biological; Biological Assay; Cells; Charge; Chemotherapy-Oncologic Procedure; Complex; Coupling; Cystic Fibrosis; Dependence; Drug Kinetics; Family; Fluorescent Dyes; Freezing; Functional disorder; Fungal Drug Resistance; Gene Delivery; Genes; Genomics; Human; Image; Individual; Ions; Kinetics; Knowledge; Life; Lipids; Liposomes; Measurement; Mechanics; Membrane; Membrane Proteins; Membrane Transport Proteins; Modeling; Molecular; Molecular Conformation; Molecular Probes; Monitor; Multi-Drug Resistance; Nucleotides; Optics; Organism; Outcome; Pathway interactions; Peptides; Pharmaceutical Preparations; Pump; Range; Research; Research Personnel; Resistance; Resolution; Silver; Structure; Surface Properties; Time; Today; Toxic effect; Transmembrane Domain; Transmembrane Transport; Transmission Electron Microscopy; base; biomaterial compatibility; cancer cell; design; design and construction; insight; member; mutant; nanoassay; nanoparticle; programs; quantum; reconstitution; size; sugar; uptake

DESCRIPTION (provided by applicant): Study of membrane proteins at the molecular level is one of the major challenges for today's biologists. ATP-binding cassette (ABC) transporters are one of the largest and most diverse superfamily of membrane proteins found in all living organisms ranging from bacteria to human. All ABC transporters share a common structure organization, suggesting a similar mechanism of energy coupling. ABC transporters are medically relevant. For instance, they are responsible for severe sicknesses and multidrug resistance in bacteria and cancer chemotherapy. Despite extensive studies, the molecular mechanism of ABC transporters remains elusive and many questions remain unanswered. In this proposal, we aim to develop single nanoparticle optical assays for real-time characterizing the functioning mechanism of BmrA, a multidrug bacterial transporter belonging to the ABC transporter superfamily. The specific research aims are described below: Aim 1: We will develop the optimum nanoparticle probes for tracing the transport mechanisms and kinetics of BrmA (WT) and their mutants in real-time at the single transporter resolution with high temporal and spatial information, aiming to determine individual steps of the catalytic cycle directly responsible for substrate translocation. Combining with high-resolution transmission electron microscopy (TEM) imaging, we aim to depict the transport pathways and verify proposed models. Aim 2: We aim to use the optimum nanoparticle probes developed in Aim 1 to trace the real-time transport mechanism and kinetics of drugs and genes via the ABC transporter. In addition, we will determine the nanoparticles that can escape the extrusion mechanisms of ABC transporter, and use them as carriers to deliver genes and drugs into living cells, aiming to explore the effective delivery means for better therapy. Aim 3: We will study the biocompatibility of nanoparticles at the cellular and genomic level, aiming to design biocompatible nanoparticles for molecular study of the ABC transports in living cells in real-time. The outcomes include: new nanoparticle assays for real-time measurement of membrane transport pathways and mechanisms of membrane transporters; better understanding of fundamental mechanism of ABC membrane transport; and effective means for the delivery of genes and drugs into living cells.

描述（由申请人提供）：分子水平上的膜蛋白的研究是当今生物学家的主要挑战之一。 ATP结合盒（ABC）转运蛋白是从细菌到人类的所有活生物体中发现的最大，最多样化的膜蛋白的超家族之一。所有ABC转运蛋白共享一个共同的结构组织，提出了类似的能量耦合机制。 ABC转运蛋白与医学相关。例如，它们负责严重的疾病和细菌和癌症化学疗法的多药耐药性。尽管进行了广泛的研究，但ABC转运蛋白的分子机制仍然难以捉摸，许多问题仍未得到解决。在此提案中，我们旨在开发单个纳米颗粒光学测定，以实时表征BMRA的功能机理，BMRA是一种属于ABC Transporter超级家族的多药细菌转运蛋白。如下所述：目标1：我们将开发最佳的纳米颗粒探针，以实时在单个转运蛋白分辨率下实时实时和空间信息，以实时和空间信息实时实时和它们的突变体，旨在确定催化周期的单个步骤直接负责基质转移的催化周期。与高分辨率透射电子显微镜（TEM）成像结合使用，我们的目标是描绘传输途径并验证提出的模型。目标2：我们旨在使用AIM 1中开发的最佳纳米粒子探针通过ABC转运蛋白追踪药物和基因的实时传输机制和动力学。此外，我们将确定可以逃脱ABC转运蛋白的挤出机制的纳米颗粒，并将其用作载体将基因和药物输送到活细胞中，旨在探索有效的递送手段，以提供更好的治疗。 AIM 3：我们将研究细胞和基因组水平上纳米颗粒的生物相容性，旨在设计生物相容性的纳米颗粒，以实时对活细胞中ABC转运的分子研究。结果包括：用于实时测量膜转运途径和膜转运蛋白机理的新纳米颗粒测定；更好地了解ABC膜运输的基本机制；以及将基因和药物输送到活细胞中的有效手段。