Veiksina Santa, Kopanchuk Sergei, Mazina Olga, Link Reet, Lille Anne, Rinken Ago
Institute of Chemistry, University of Tartu, Ravila 14a, 50411, Tartu, Estonia.
Methods Mol Biol. 2015;1272:37-50. doi: 10.1007/978-1-4939-2336-6_3.
Despite the availability of numerous conceptually different approaches for the characterization of ligand-receptor interactions, there remains a great requirement for complementary methods that are suitable for kinetic studies, especially for the characterization of membrane protein systems and G protein-coupled receptors (GPCRs) in particular. One of the potential approaches that inherently fits well for this purpose is fluorescence anisotropy (FA), a method that allows continuous monitoring of ligand binding processes and characterization of ligand binding dynamics. However, significant changes in FA signal of fluorescently labeled ligands can be detected only if the ratio of bound to free fluorescent ligand portions is altered, which means that receptor and ligand concentrations have to be comparable. As most of the GPCRs are normally present at relatively low concentrations in native tissues and conventional receptor preparations from overexpressed systems often generate high background levels due to significant autofluorescence, receptor preparations with sufficiently high receptor concentrations have become a critical requirement for successful FA assay performance. We propose that budded baculoviruses that display GPCRs on their surfaces can be used as a receptor source in FA assays. Here, we describe the experimental setup of this homogeneous budded baculovirus/FA-based assay system for investigation of receptor-ligand interactions and a novel strategy for FA kinetic data analysis that is taking into account the effect of nonspecific interactions and the depletion of the fluorescent ligand during the binding reaction. The developed budded baculovirus/FA-based assay system brings the experimental data to a level that could solve complex models of ligand-receptor interactions and become a valuable tool for the screening of pharmacologically active compounds. Melanocortin 4 (MC4) receptors and the fluorescent ligand Cy3B-NDP-α-MSH were used as the model system.
尽管有许多概念上不同的方法可用于表征配体 - 受体相互作用，但对于适用于动力学研究的互补方法仍有很大需求，特别是对于膜蛋白系统的表征，尤其是G蛋白偶联受体（GPCRs）。荧光各向异性（FA）是一种潜在的方法，从本质上讲非常适合此目的，该方法允许连续监测配体结合过程并表征配体结合动力学。然而，只有当结合的荧光配体部分与游离荧光配体部分的比例发生变化时，才能检测到荧光标记配体的FA信号的显著变化，这意味着受体和配体浓度必须相当。由于大多数GPCRs在天然组织中的浓度通常相对较低，并且来自过表达系统的传统受体制剂由于显著的自发荧光常常产生高背景水平，因此具有足够高受体浓度的受体制剂已成为成功进行FA测定的关键要求。我们提出，在其表面展示GPCRs的出芽杆状病毒可作为FA测定中的受体来源。在此，我们描述了这种基于出芽杆状病毒/FA的均相测定系统的实验设置，用于研究受体 - 配体相互作用，以及一种用于FA动力学数据分析的新策略，该策略考虑了非特异性相互作用的影响以及结合反应过程中荧光配体的消耗。所开发的基于出芽杆状病毒/FA的测定系统将实验数据提升到可以解决配体 - 受体相互作用复杂模型的水平，并成为筛选药理活性化合物的有价值工具。黑皮质素4（MC4）受体和荧光配体Cy3B - NDP -α - MSH被用作模型系统。
