Single-Molecule Scanning Tunneling Spectroscopy of Surface-Tethered Proteins

表面束缚蛋白的单分子扫描隧道光谱

基本信息

批准号：
7991338
负责人：
Shelley Ann Claridge
金额：
$ 5.05万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-12-17 至 2012-09-13
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7991338
关键词：
Azurin Behavior Binding Binding Proteins Biological Biological Models Biological Process Biological Sciences Caliber Cereals Chemicals Complex Copper Data Development Disease Drug Delivery Systems Electric Capacitance Electrons Ensure Film Frequencies G-Protein-Coupled Receptors Gold Image In Situ Inflammation Lateral Ligand Binding Ligands Lipid Bilayers Lipids Measurement Measures Mediating Medical Membrane Membrane Lipids Membrane Proteins Metalloproteins Metals Methodology Methods Modeling Nuclear Magnetic Resonance Oxidation-Reduction Pharmaceutical Preparations Process Property Proteins Pseudomonas aeruginosa Relative (related person)Resolution Respiration Sampling Scanning Scanning Probe Microscopes Scanning Tunneling Microscopy Science Serotonin Spectrum Analysis Structural Protein Structure Surface System Techniques Testing Thick Vacuum Variant Work alpha helix base biological systems design disease diagnosis drug development drug discovery experience imaging modality instrument instrumentation interest mutant nanometer nanoscale neurotransmission novel prevent protein structure quantum receptor research study serotonin receptor serotonin-binding protein single molecule small molecule spectroscopic imaging tool

项目摘要

DESCRIPTION (provided by applicant): The scanning tunneling microscope routinely provides atomic-resolution imaging and spectroscopic measurements of single molecules on surfaces. The primary obstacle preventing the full power of this capability from being applied to biological targets such as proteins is the limitation that, in general, electrons cannot tunnel through more than 2 nm of an insulating material. This restriction impacts entire classes of biologically interesting samples such as proteins (typically ~5 nm diameter) and lipid bilayers (~6 nm thickness). This proposal describes the development of instrumentation and methodologies expanding the sub- nanometer imaging capabilities of scanning tunneling microscopy (STM) to biologically important single molecules and biomolecule assemblies on surfaces, including proteins embedded in lipid membranes. In particular, this approach will allow direct observations and structural studies of G protein-coupled receptor (GPCR)-ligand complexes, which are the targets of over half of all commercially available drugs, and are not possible to visualize directly using other techniques. This work will utilize an alternating-current STM (AC-STM) that circumvents the conductivity requirement by measuring the tunneling current as an alternating bias is applied to the sample at frequencies from 0.5-20 GHz. The tunneling current has been shown to scale with surface polarizability or capacitance, providing spectroscopic as well as topographic information about molecules in the tunneling junction. This technique can be applied to make sub-nanometer topographic and spectroscopic measurements of biomolecules on surfaces. This proposal specifically aims to characterize two types of proteins on surfaces as representative examples of two classes of protein targets: 1) the copper redox protein P. aeruginosa azurin, as a model for the class of metalloproteins, 2) serotonin 5-HT1 receptor proteins specifically bound to their ligand on a surface, as models for GCPRs and other membrane-associated proteins, and as a demonstration of the ability to correlate structure with ligand-binding properties. Relevance: The ability to image the structures of single biomolecules such as proteins would have major implications for medical science ranging from disease diagnosis to drug discovery. This proposal describes the development of instrumentation and methodology that will enable structural information to be extracted from single molecules on surfaces. This capability will be applied to proteins embedded in lipid bilayers, which are the targets of over 50% of all modern drugs, and which are difficult to analyze by other means.

描述（由申请人提供）：扫描隧道显微镜通常提供表面上单分子的原子分辨率成像和光谱测量。阻止该能力的全部功能将其应用于蛋白质（例如蛋白质）的主要障碍是一种限制，通常，电子不能穿过超过2 nm的绝缘材料。这种限制会影响整个生物学上有趣的样品，例如蛋白质（通常直径约为5 nm）和脂质双层（厚度约为6 nm）。该提案描述了仪器和方法的发展，扩展了扫描隧道显微镜（STM）的亚纳米成像能力，以生物学上重要的单分子和生物分子组件在表面上，包括嵌入在脂质膜中的蛋白质。特别是，这种方法将允许直接观察和结构研究G蛋白偶联受体（GPCR） - 配合物，这些配合物是所有市售药物中一半以上的靶标，并且无法使用其他技术直接可视化。这项工作将利用交替电流STM（AC-STM），该STM（AC-STM）通过测量隧道电流作为交替偏置来规避电导率要求，以从0.5-20 GHz的频率应用于样品。隧道电流已显示出具有表面极化或电容的扩展，从而提供了有关隧道连接中分子的光谱和地形信息。该技术可以应用于对表面上生物分子的亚纳米地形和光谱测量。 This proposal specifically aims to characterize two types of proteins on surfaces as representative examples of two classes of protein targets: 1) the copper redox protein P. aeruginosa azurin, as a model for the class of metalloproteins, 2) serotonin 5-HT1 receptor proteins specifically bound to their ligand on a surface, as models for GCPRs and other membrane-associated proteins, and as a证明与配体结合特性相关的结构的能力。相关性：成像单个生物分子（例如蛋白质）结构的能力将对从疾病诊断到药物发现的医学科学产生重大影响。该建议描述了仪器和方法的发展，这些方法将使结构信息从表面上的单分子中提取。该能力将应用于嵌入脂质双层中的蛋白质，这是所有现代药物的50％以上的靶标，并且难以通过其他方式分析。