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: 7260773
• 负责人: X. Nancy Xu
• 金额: $ 3.12万
• 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/7260773
• 关键词: biomaterial compatibility; biomaterial development /preparation; chemical kinetics; conformation; drug delivery systems; gene delivery system; gene mutation; membrane transport proteins; molecular probes; nanotechnology; optical tomography; protein structure function; protein transport; surface property; technology /technique development; tissue /cell culture; transmission electron microscopy

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研究是当今生物学家面临的主要挑战之一。 ATP 结合盒 (ABC) 转运蛋白是从细菌到人类的所有生物体中发现的最大、最多样化的膜蛋白超家族之一。所有 ABC 转运蛋白都有一个共同的结构组织，这表明能量耦合的机制相似。 ABC 转运蛋白具有医学相关性。例如，它们是导致严重疾病以及细菌和癌症化疗中的多重耐药性的原因。尽管进行了广泛的研究，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答。在本提案中，我们的目标是开发单纳米粒子光学测定法，用于实时表征 BmrA 的功能机制，BmrA 是一种属于 ABC 转运蛋白超家族的多药物细菌转运蛋白。具体研究目标如下： 目标 1：我们将开发最佳的纳米粒子探针，用于在具有高时空信息的单一转运蛋白分辨率下实时追踪 BrmA (WT) 及其突变体的转运机制和动力学，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，我们的目标是描述传输路径并验证所提出的模型。目标 2：我们的目标是使用目标 1 中开发的最佳纳米粒子探针来追踪药物和基因通过 ABC 转运蛋白的实时转运机制和动力学。此外，我们还将确定能够逃脱ABC转运蛋白挤压机制的纳米颗粒，并利用它们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实现更好的治疗。目标3：我们将在细胞和基因组水平上研究纳米粒子的生物相容性，旨在设计生物相容性纳米粒子，用于实时活细胞中ABC运输的分子研究。结果包括：用于实时测量膜转运途径和膜转运蛋白机制的新纳米颗粒测定法；更好地理解ABC膜运输的基本机制；以及将基因和药物递送到活细胞中的有效方法。