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（申请人提供）：此应用程序是我们目前资助的项目的修订（家长补助金）题为纳米测定实时分子探测ABC转运蛋白（R 01 GM 076440，2006年5月1日至2011年4月30日），作为对“使RPG能够利用NCRR中心和类似中心的计划”的回应而提交（NOT-OD-09-058），旨在加强个人研究计划与NCRR中心的互动，并使中心能够获得最先进的仪器，从而推进生物医学研究。这一修订的理由如下：在分子水平上研究膜蛋白是当今生物学家的主要挑战之一。ATP结合盒（ABC）转运蛋白是从细菌到人类的生物体中发现的最大和最多样化的膜蛋白超家族之一。所有的ABC转运蛋白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，它们是细菌感染和癌症化疗中严重疾病和多药耐药性（MDR）的原因。尽管广泛的研究，ABC转运蛋白的分子机制仍然难以捉摸，许多问题仍然没有答案。在我们的母基金中，我们的目标是开发光稳定的（非光解和非闪烁）单纳米颗粒光学检测，并使用它们来模拟转运蛋白的底物，并作为探针来表征BmrA的功能机制，BmrA是一种属于ABC转运蛋白超家族的多药细菌转运蛋白。我们已经取得了重大进展，并已接近完成目标1和2的母基金，这是总结在初步研究和我们最近的论文。在这个修订后的申请，我们的目标是扩大我们原来提出的研究范围，使用高分辨率低温透射电子显微镜（cryo-TEM）、计算机重建和结构挖掘来解决BmrA的结构和分子机制，BmrA是属于ABC转运蛋白超家族的多药细菌转运蛋白，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，在http：//ncmi.bcm.tmc.edu/）合作。具体的研究目标如下所述：目标1：我们将重组纯化的BmrA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和光谱学，实时（ms-<s时间分辨率）确定它们的转运机制，并在NCMI使用cryo-TEM，以亚纳米（nm）分辨率确定BmrA的结构和BmrA中单个纳米颗粒的位置。我们将比较使用cryo TEM获得的结果与使用我们的单个纳米颗粒测定和光学显微镜获得的实时数据。这两种方法的结合将使我们能够使用单个纳米颗粒作为纳米标记物来追踪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。目标二：我们已经确定了BmrA在单个活细胞中的运输动力学实时使用我们的单纳米颗粒测定和单纳米颗粒显微镜和光谱学。我们将探索使用cryo-TEM来确定细胞中BmrA的结构（而不是脂质体）以及细胞中BmrA中单个纳米颗粒在亚nm分辨率下的位置的可能性，旨在进一步开发cryo-TEM，以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：新的纳米颗粒检测方法，用于使用光学显微镜实时测量膜转运途径和膜转运蛋白的机制，并作为纳米探针，用于使用Cryo-TEM追踪膜转运蛋白的分子机制; ABC转运蛋白对纳米颗粒进出活细胞的转运机制的新认识;以及对膜蛋白的组装和功能的新见解，以更好地理解ABC转运蛋白的作用机制，从而允许更有效的治疗。拟议的研究将加速科学研究，提供新的研究和就业机会，实现《复苏法》的目标。 公共卫生关系：本文描述的强大的成像工具和新的测定将提供对ABC膜转运蛋白的机制和功能的新见解。更好地理解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步骤，特别是对于那些涉及细菌和癌细胞多药耐药（MDR）的转运蛋白。因此，拟议的研究与健康有关。此外，膜转运在多种细胞和亚细胞途径中起主导作用，例如MDR、细胞信号传导和细胞-细胞通讯。因此，所提出的新的纳米分析和成像工具预计将成为非常有价值的工具，以解决与活细胞中的膜转运动力学相关的许多生物化学和生物医学问题，以及在亚纳米分辨率下解决膜转运蛋白的结构。结合膜转运蛋白的实时动力学测量和原子尺度的结构表征，将促进我们对膜转运蛋白和MDR的理解。