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: 7842889
• 负责人: X. Nancy Xu
• 金额: $ 43.04万
• 依托单位: OLD DOMINION UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7842889
• 关键词: ATP Hydrolysis; ATP-Binding Cassette Transporters; Address; Antibiotics; Arts; Bacteria; Bacterial Infections; Binding; Biochemical; Biological; Biological Assay; Biomedical Research; Blinking; Cancerous; Cell Communication; Cells; Cellular Membrane; Chemotherapy-Oncologic Procedure; Complex; Computers; Coupling; Cystic Fibrosis; Data; Development; Environment; Family; Functional disorder; Funding; Fungal Drug Resistance; Health; Human; Image; Imaging Device; Individual; Ions; Kinetics; Knowledge; Life; Lipids; Liposomes; Location; Measurement; Mechanics; Membrane; Membrane Proteins; Membrane Transport Proteins; Microscopy; Mining; Molecular; Molecular Probes; Molecular Structure; Multi-Drug Resistance; National Center for Research Resources; Nucleotides; Occupations; One-Step dentin bonding system; Optics; Organism; Outcome; Paper; Pathway interactions; Peptides; Play; Principal Investigator; Pump; Recovery; Research; Resistance; Resolution; Resources; Role; Signal Transduction; Spectrum Analysis; Structure; Therapeutic; Time; Transmembrane Domain; Transmembrane Transport; Transmission Electron Microscopy; base; cancer cell; effective therapy; insight; instrumentation; member; millisecond; mutant; nanoassay; nanometer; nanoparticle; novel; parent grant; programs; protein structure; public health relevance; reconstitution; reconstruction; response; structural biology; sugar; time use; tool; uptake

DESCRIPTION (provided by applicant): This application is a revision of our current funded project (parent grant) entitled Nanoassay for Realtime Molecular Probing of ABC Transporters (R01 GM076440, 05/01/2006-04/30/2011), submitted in response to Enabling RPGs to Leverage NCRR Center and Center-like Programs (NOT-OD-09-058) issued by NCRR, aiming to enhance the interaction of individual research programs with the NCRR center and enabling access to state-of-the-art instrumentation available at the center and thereby advance biomedical research. The justifications of this revision are given below: The 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 super families of membrane proteins found in living organisms ranging from bacteria to human. All ABC transporters share a common structural organization, suggesting a similar mechanism of energy coupling. ABC transporters are medically relevant. For instance, they are responsible for severe sicknesses and multidrug resistance (MDR) in bacterial infections and cancer chemotherapy. Despite extensive studies, the molecular mechanism of ABC transporters remains elusive and many questions remain unanswered. In our parent grant, we aim to develop photostable (non-photodecomposition and non-blinking) single nanoparticle optical assays, and to use them to mimic the substrates of the transporters, and as the probes to characterize the functional mechanism of BmrA, a multidrug bacterial transporter belonging to the ABC transporter super family. We have made significant progress, and have nearly completed Aims 1 and 2 in the parent grant, which is summarized in preliminary studies and our recent papers. In this revised application, we aim to expand our original proposed research scope to use high resolution cryo transmission-electron-microscopy (cryo-TEM), computer reconstruction and structure mining to solve the structure and molecular mechanism(s) of BmrA, a multidrug bacterial transporter belonging to the ABC transporter super family, by collaborating with the National Center for Macromolecular Imaging (NCMI) (a NCRR designated Biomedical Research Resource for Structural Biology, at http://ncmi.bcm.tmc.edu/). The specific research aims are described below: Aim 1: We will reconstitute purified BmrA into liposomes and use the single nanoparticle assay developed in our lab to determine their transport mechanism in real-time (ms-<s temporal resolution) using our single nanoparticle microscopy and spectroscopy, and use cryo-TEM at NCMI to determine the structure of BmrA and locations of single nanoparticles in BmrA at sub-nanometer (nm) resolution. We will compare the results acquired using cryo TEM with real-time data acquired using our single nanoparticle assay and optical microscopy. The combination of both approaches will enable us to use single nanoparticles as nm markers to trace the molecular mechanism of the ABC transporter, aiming to better understand how the ABC pump functions and its related molecular mechanisms. Aim 2: We have determined the transport kinetics of BmrA in single living cells in real-time using our single nanoparticle assay and single nanoparticle microscopy and spectroscopy. We will explore the possibility of using cryo-TEM to determine the structures of BmrA in cells (instead of liposomes) and the location of single nanoparticles in BmrAs in cells at sub-nm resolution, aiming to further develop cryo-TEM to be well suited to probe protein structures in its native environments (cellular membranes). The outcomes of the proposed research include: new nanoparticle assays for real-time measurement of membrane transport pathways and mechanisms of membrane transporters using optical microscopy, and as nm probes for tracing molecular mechanisms of membrane transporter using Cryo-TEM; new knowledge of transport mechanisms of nanoparticles in and out of living cells by ABC transporters; and new insights into the assembly and functioning of membrane proteins for better understanding of mechanisms of actions of ABC transporters, thus permitting more effective therapies. The proposed research will accelerate scientific research and offer new research and job opportunities, achieving the objectives of the Recovery Act. PUBLIC HEALTH RELEVANCE: The powerful imaging tools and novel assays described herein will offer new insights into the mechanisms and functions of ABC membrane transporters. Better understanding of the molecular mechanisms and functions of ABC transporters is essential step for one to develop more effective therapeutic treatments, notably for those transporters involved in multi-drug resistance (MDR) of bacteria and cancerous cells. Thus, the proposed research is health relevant. Furthermore, membrane transport plays a leading role in a wide spectrum of cellular and subcellular pathways, such as MDR, cellular signaling and cell-cell communication. Therefore, the proposed new nanoassay and imaging tools are expected to become extremely valuable tools to address numerous biochemical and biomedical problems associated with kinetics of membrane transports in living cells, as well as solving structure of membrane transporters at sub-nm resolution. Combination of real-time kinetic measurements with atomic scale structure characterization of membrane transporters will advance our understanding of membrane transporters and MDR.

描述（由申请人提供）：本申请是我们当前资助项目（母基金）的修订版，题为“ABC 转运蛋白实时分子探测纳米测定”（R01 GM076440，05/01/2006-04/30/2011），作为回应提交使 RPG 能够利用 NCRR 中心和类似中心的程序 (NOT-OD-09-058) NCRR 旨在加强各个研究项目与 NCRR 中心的互动，并能够使用该中心最先进的仪器，从而推进生物医学研究。此次修订的理由如下： 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) 转运蛋白是从细菌到人类等活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，这表明能量耦合机制相似。 ABC 转运蛋白具有医学相关性。例如，它们会导致细菌感染和癌症化疗中的严重疾病和多重耐药性（MDR）。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。在我们的母基金中，我们的目标是开发光稳定（非光分解和非闪烁）单纳米粒子光学测定，并使用它们来模拟转运蛋白的底物，并作为探针来表征 BmrA（一种多药）的功能机制。细菌转运蛋白属于 ABC 转运蛋白超家族。我们已经取得了重大进展，几乎完成了家长补助金的目标 1 和 2，这在初步研究和我们最近的论文中进行了总结。在这个修订后的申请中，我们的目标是扩大我们最初提出的研究范围，使用高分辨率冷冻透射电子显微镜（cryo-TEM）、计算机重建和结构挖掘来解决 BmrA（一种多药）的结构和分子机制。属于 ABC 转运蛋白超家族的细菌转运蛋白，通过与国家大分子成像中心 (NCMI)（NCRR 指定的结构生物学生物医学研究资源，位于http://ncmi.bcm.tmc.edu/）。具体研究目标如下所述： 目标 1：我们将纯化的 BmrA 重组到脂质体中，并使用我们实验室开发的单纳米颗粒测定法，使用我们的单纳米颗粒显微镜实时（ms-<s 时间分辨率）确定其转运机制和光谱学，并使用 NCMI 的冷冻 TEM 以亚纳米 (nm) 分辨率确定 BmrA 的结构以及 BmrA 中单个纳米颗粒的位置。我们将使用冷冻 TEM 获得的结果与使用我们的单纳米粒子测定和光学显微镜获得的实时数据进行比较。两种方法的结合将使我们能够使用单个纳米粒子作为纳米标记来追踪ABC转运蛋白的分子机制，旨在更好地了解ABC泵的功能及其相关的分子机制。目标 2：我们使用单纳米颗粒测定以及单纳米颗粒显微镜和光谱学实时确定了 BmrA 在单个活细胞中的转运动力学。我们将探索使用冷冻透射电镜以亚纳米分辨率确定细胞中 BmrA 结构（而不​​是脂质体）以及细胞中 BmrA 中单个纳米粒子的位置的可能性，旨在进一步开发冷冻透射电镜，使其非常适合探测其天然环境（细胞膜）中的蛋白质结构。拟议研究的成果包括：使用光学显微镜实时测量膜转运途径和膜转运蛋白机制的新纳米粒子测定，以及使用冷冻透射电镜作为纳米探针追踪膜转运蛋白的分子机制；关于 ABC 转运蛋白进出活细胞的纳米粒子转运机制的新知识；以及对膜蛋白组装和功能的新见解，以更好地了解 ABC 转运蛋白的作用机制，从而实现更有效的治疗。拟议的研究将加速科学研究并提供新的研究和就业机会，从而实现《复苏法案》的目标。 公共健康相关性：本文描述的强大成像工具和新颖测定将为 ABC 膜转运蛋白的机制和功能提供新的见解。更好地了解 ABC 转运蛋白的分子机制和功能是开发更有效的治疗方法的重要一步，特别是对于那些涉及细菌和癌细胞多药耐药性 (MDR) 的转运蛋白。因此，拟议的研究与健康相关。此外，膜运输在多种细胞和亚细胞途径中发挥着主导作用，例如 MDR、细胞信号传导和细胞间通讯。因此，所提出的新纳米测定和成像工具有望成为极其有价值的工具，以解决与活细胞膜转运动力学相关的众多生化和生物医学问题，以及以亚纳米分辨率解决膜转运蛋白的结构。实时动力学测量与膜转运蛋白的原子尺度结构表征相结合将增进我们对膜转运蛋白和 MDR 的理解。