Single-Molecule Scanning Tunneling Spectroscopy of Surface-Tethered Proteins

描述（由申请人提供）：扫描隧道显微镜常规提供表面上单个分子的原子分辨率成像和光谱测量。阻止这种能力的全部功率应用于诸如蛋白质的生物靶的主要障碍是限制，即通常电子不能隧穿超过2nm的绝缘材料。这一限制影响了整个生物学上感兴趣的样品类别，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，将扫描隧道显微镜（STM）的亚纳米成像能力扩展到生物学上重要的单分子和表面上的生物分子组装体，包括嵌入脂质膜中的蛋白质。特别是，这种方法将允许直接观察和G蛋白偶联受体（GPCR）-配体复合物的结构研究，这是超过一半的所有市售药物的目标，并且不可能直接使用其他技术可视化。这项工作将利用一个交流STM（AC-STM），通过测量隧道电流的电导率的要求，作为一个交变偏置施加到样品在0.5-20 GHz的频率。隧穿电流已被证明与表面极化率或电容成比例，提供有关隧穿结中分子的光谱和形貌信息。该技术可应用于对表面上的生物分子进行亚纳米形貌和光谱测量。该提案特别旨在表征表面上的两种类型的蛋白质，作为两类蛋白质靶标的代表性示例：1）铜氧化还原蛋白铜绿假单胞菌天青蛋白（P. aeruginosa azurin），作为金属蛋白类的模型，2）与表面上的配体特异性结合的血清素5-HT 1受体蛋白，作为GCPR和其它膜相关蛋白的模型，并作为将结构与配体结合性质相关联的能力的证明。相关性：对蛋白质等单个生物分子的结构进行成像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。该提案描述了仪器和方法的发展，这将使结构信息从表面上的单分子中提取。这种能力将应用于嵌入脂质双层中的蛋白质，这些蛋白质是所有现代药物的50%以上的目标，并且难以通过其他手段进行分析。

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.