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: 7477324
• 负责人: X. Nancy Xu
• 金额: $ 25.27万
• 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/7477324
• 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）：在分子水平上研究膜蛋白是当今生物学家面临的主要挑战之一。atp结合盒（ABC）转运蛋白是在从细菌到人类的所有生物中发现的最大和最多样化的膜蛋白超家族之一。所有的ABC转运体都有一个共同的结构组织，这表明它们具有相似的能量耦合机制。ABC转运蛋白与医学相关。例如，它们是导致严重疾病和细菌耐多药以及癌症化疗的原因。尽管进行了广泛的研究，但ABC转运体的分子机制仍然难以捉摸，许多问题仍未得到解答。在这项提议中，我们的目标是开发单纳米颗粒光学检测，实时表征BmrA的功能机制，BmrA是一种多药细菌转运体，属于ABC转运体超家族。具体的研究目标如下：目标1：我们将开发最佳的纳米颗粒探针，用于追踪BrmA （WT）及其突变体的运输机制和动力学，实时追踪具有高时空信息的单转运体分辨率，旨在确定直接负责底物易位的催化循环的各个步骤。结合高分辨率透射电子显微镜（TEM）成像，我们旨在描述运输途径并验证所提出的模型。目标2：我们的目标是使用在目标1中开发的最佳纳米颗粒探针来追踪药物和基因通过ABC转运体的实时转运机制和动力学。此外，我们将确定能够逃避ABC转运蛋白挤压机制的纳米颗粒，并将其作为载体将基因和药物递送到活细胞中，旨在探索有效的递送手段，以获得更好的治疗效果。目标3：我们将在细胞和基因组水平上研究纳米颗粒的生物相容性，旨在设计生物相容性纳米颗粒，用于实时研究活细胞中ABC转运的分子。这些成果包括：用于实时测量膜转运途径和膜转运体机制的新型纳米颗粒分析；进一步了解ABC膜运输的基本机制；以及将基因和药物送入活细胞的有效手段。