Targeted TERS Investigations of Ligand-Receptor Binding

 描述(由申请人提供)：本提案的目标是确定与细胞膜中的药物靶向和化学信号相关的分子相互作用。市场上超过一半的药物针对膜蛋白，这是一类在完整膜上获得分子细节的分子，挑战了现有的方法。我们将利用纳米粒子的等离子体性质，对完整细胞膜中的配体-受体结合进行特定的化学光谱研究。这些研究将展示一种新的方法来探测与多肽拮抗剂结合的受体的化学残基，并提供对调节参与信号传递和药物靶向的膜蛋白的分子相互作用的洞察。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)光谱和单粒子跟踪来表征调节与膜受体结合的化学相互作用。TERS将确定与配体结合相关的细胞膜中存在的化学残留物。我们实验室获得的初步结果表明，配体功能化的纳米颗粒选择性地增强了参与配体结合的受体的光谱信号。通过SPR测量，可以从固定在表面的蛋白质中确定配体的亲和力。可以使用单粒子跟踪在细胞中进行互补测量。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。这一建议的具体目的是：1)将原位研究的配体亲和力与与多肽结合的受体的分子同一性相关联 整合素受体通过梳子和SPR。2)利用靶向TERs研究整合素受体与多肽配体之间的体外蛋白质受体结合作用。3)将单纳米粒子跟踪研究与TERS研究相结合，将体外结合亲和力与化学相互作用联系起来。我们提出的技术和平台将解决从与完整细胞膜上的受体蛋白结合的配体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联提供新的见解。

 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.