Targeted TERS Investigations of Ligand-Receptor Binding

 描述（由申请方提供）：本提案的目的是鉴定与细胞膜中药物靶向和化学信号传导相关的分子相互作用。市场上超过一半的药物靶向膜蛋白，这类分子在完整膜中获得分子细节挑战现有方法。我们将使用纳米粒子的等离子体特性，使化学特异性光谱研究的配体受体结合在完整的细胞膜。这些研究将展示一种新的方法来探测受体的化学残基，结合肽拮抗剂，并提供深入了解分子相互作用，调节参与信号传导和药物靶向的膜蛋白。 我们的方法结合了尖端增强拉曼散射（TERS），表面等离子体共振（SPR）光谱，和单粒子跟踪，以表征化学相互作用，调节膜受体的结合。TERS将确定细胞膜中存在的与配体结合相关的化学残留物。在我们实验室获得的初步结果表明，配体功能化的纳米粒子选择性地增强参与配体结合的受体的光谱信号。可以使用SPR测量从固定在表面上的蛋白质确定配体亲和力。可以使用单粒子跟踪在细胞中执行补充测量。通过利用能够实时跟踪纳米颗粒的技术，我们将表征与活细胞上的受体结合的单个配体功能化纳米颗粒的配体结合和膜组织。 该建议的具体目标是：1）将原位研究的配体亲和力与受体的分子身份相关联，所述受体与肽结合至 通过TERS和SPR的结合，整合素受体。 2)使用靶向TERS探索整合素受体和肽配体之间的体外蛋白质受体结合相互作用。 3)通过将单个纳米颗粒跟踪研究与TERS研究相结合，将体外结合亲和力与化学相互作用相关联。我们提出的技术和平台将解决从完整细胞膜中结合受体蛋白的配体中获得化学信息的挑战。这些研究将为调节信号通路的分子相互作用以及这些相互作用中的异常如何与疾病和治疗相关提供新的见解。

 DESCRIPTION (provided by applicant): The goal of this proposal is to identify molecular interactions relevant to drug targeting and chemical signaling in cell membranes. More than half the drugs on the market target membrane proteins, a class of molecules where obtaining molecular detail in intact membranes challenges existing methods. We will use the plasmonic properties of nanoparticles to enable chemical-specific spectroscopic studies of ligand-receptor binding in intact cellular membranes. These investigations will demonstrate a new approach to probe the receptor's chemical residues that bind peptide antagonists and provide insight into molecular interactions that regulate the membrane proteins involved in signaling and drug targeting. Our approach combines tip enhanced Raman scattering (TERS), surface plasmon resonance (SPR) spectroscopy, and single particle tracking to characterize chemical interactions that regulate binding to membrane receptors. TERS will determine the chemical residues present in cell membranes that are associated with ligand binding. Initial results obtained in our lab indicate that ligand-functionalized nanoparticles selectively enhance the spectroscopic signals of the receptor involved in ligand binding. The ligand affinity can be determined from proteins immobilized on surfaces using SPR measurements. Complementary measurements can be performed in cells using single particle tracking. By taking advantage of technology that enables tracking of nanoparticles in real-time, we will characterize both ligand binding and membrane organization of individual ligand-functionalized nanoparticles bound to receptors on living cells. The specific aims of this proposal are: 1) Correlate in situ studies ligand affinity with the molecular identity of the receptor associated with peptide binding to the integrin receptors by combing TERS and SPR. 2) Explore protein receptor-binding interactions in vitro between integrin receptors and peptide ligands using targeted TERS. 3) Correlate in vitro binding affinity with chemical interaction by combining single nanoparticle tracking studies with TERS studies. The technology and platform we propose will address the challenge of obtaining chemical information from ligands binding to receptor proteins in intact cell membranes. These studies will provide new insights into the molecular interactions that regulate signaling pathways and how anomalies in these interactions are associated with disease and treatment.