UV Plasmon-Enhanced Chiroptical Spectroscopy of Membrane-Binding Proteins

摘要 圆二色谱(CD)和拉曼光学活性(ROA)是手性光谱，它提供了有价值的 无水缓冲溶液中天然条件下生物分子和药物的结构信息 需要特殊的样品制备或结晶。这两种方法在探索时是相辅相成的 分子电子跃迁和分子振动跃迁的圆二向色性。一种组合 两种方法中的一种特别适合于研究膜结合蛋白的结构， 仍然很难用其他生物物理表征工具来表征。尽管从理论上讲， CD/ROA表征有可能提供膜结合的重要结构信息 对于蛋白质，在实践中，这两种光谱的低灵敏度使得这种潜力很难实现。一个 对高样品浓度和长采集时间的需要限制了Cd和Cd的更广泛使用 特别是ROA光谱作为表征膜结合蛋白的工具。这个项目的目的是 通过发展等离子体增强CD克服CD和ROA光谱仪的灵敏度限制 (PECD)和表面增强型ROA(SEROA)光谱仪，它们使用等离子体纳米天线，这是 具有特定电场(E)和磁场(H)特性以及定义的相的工程纳米结构 属性，以增强信号强度。为了最大限度地增强信号，天线基板将 开发了在紫外线(UV)中的等离子体共振，使电磁共振可以重叠 利用生物靶分子的分子电子共振，促进了强信号强度 CD和ROA都有。由于本提案的重点是开发PECD和SEROA作为 膜结合蛋白，所提出的天线的另一个重要设计组件是组装 等离子体纳米天线表面的脂膜，为膜结合提供结合部位 蛋白质。这种方法丰富了CD和ROA信号的电磁热点中的感兴趣蛋白质 增强是最高的，并允许对其中的蛋白质结构进行光谱表征 膜结合型。开发的等离子体增强光谱仪将使重要的新见解成为可能 膜结合蛋白的结构和手性，例如作为脂质成分的函数，以及 将有助于极大地提高对蛋白质-膜相互作用的理解。这样做的具体目的是 申请对象为： 目的1：建立膜结合蛋白的等离子体增强紫外圆二色谱 目的2：建立膜结合蛋白的等离子体增强拉曼光谱(ROA) 目的3：用于膜结合特性表征的电子CD/ROA组合样机 蛋白质

Summary Circular dichroism (CD) and Raman optical activity (ROA) are chiroptical spectroscopies that provide valuable structural information about biomolecules and pharmaceuticals under native conditions in aqueous buffer without the need for special sample preparation or crystallization. The two methods are complementary as they probe the circular dichroism of molecular electronic and molecular vibrational transitions, respectively. A combination of the two methods is particularly well suited for investigating the structure of membrane binding proteins, which remain very difficult to characterize with other biophysical characterization tools. Although in theory a combined CD / ROA characterization has the potential for providing important structural information of membrane binding proteins, in practice the weak sensitivities of the two spectroscopies makes it difficult to realize this potential. A need for high sample concentrations and long acquisition times has limited a more widespread use of CD and in particular ROA spectroscopy as tool for characterizing membrane binding proteins. This project intends to overcome the sensitivity limitations of CD and ROA spectroscopies by developing plasmon-enhanced CD (PECD) and surface-enhanced ROA (SEROA) spectroscopies that utilize plasmonic nanoantennas, which are engineered nanostructures with specific electric (E) and magnetic (H) field properties as well as defined phase properties, to enhance signal intensities. To maximize the signal enhancement, antenna substrates will be developed with plasmon resonances in the ultraviolet (UV) so that the electromagnetic resonances can overlap with the molecular electronic resonances of biological target molecules, facilitating strong signal intensities for both CD and ROA. As this proposal focuses on developing PECD and SEROA as characterization tool for membrane binding proteins, another important design component of the proposed antennas is the assembly of a lipid membrane on the surface of the plasmonic nanoantennas to provide binding sites for membrane binding proteins. This approach enriches the proteins of interest in electromagnetic hot spots where CD and ROA signal enhancements are highest and allows for a spectroscopic characterization of the protein structure in its membrane-bound form. The developed plasmon-enhanced spectroscopies will enable important new insights into the structure and chirality of membrane-binding proteins, for instance as function of lipid compositions, and will contribute to a greatly improved understanding of protein-membrane interactions. The specific aims of this application are to: Aim 1: Develop a Plasmon-Enhanced Ultraviolet CD Spectroscopy for Membrane Binding Proteins Aim 2: Develop Plasmon Enhanced Raman Optical Activity (ROA) Spectroscopy for Membrane Binding Proteins Aim 3: Prototype Combined Electronic CD / ROA Instrument for the Characterization of Membrane Binding Proteins