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Fluorescence Anisotropy-Based Assay for Characterization of Ligand Binding Dynamics to GPCRs: The Case of Cy3B-Labeled Ligands Binding to MC Receptors in Budded Baculoviruses. | LitMetric

AI Article Synopsis

  • - Fluorescence methods, especially fluorescence anisotropy (FA) assays, have gained importance in studying ligand binding to G protein-coupled receptors (GPCRs), allowing real-time monitoring of interactions.
  • - Using budded baculoviruses to display GPCRs on their surfaces helps achieve the necessary receptor concentration and minimizes autofluorescence for accurate readings in the FA assay.
  • - The data generated can be challenging to analyze due to proprietary formats, but the proposed Aparecium software allows for better data management and integration, facilitating the screening of new compounds and modeling ligand binding dynamics for GPCRs like the melanocortin 4 receptor.

Article Abstract

During the past decade, fluorescence methods have become valuable tools for characterizing ligand binding to G protein-coupled receptors (GPCRs). However, only a few of the assays enable studying wild-type receptors and monitor the ligand binding in real time. One of the approaches that is inherently suitable for this purpose is the fluorescence anisotropy (FA) assay. In the FA assay, the change of ligand's rotational freedom connected with its binding to the receptor can be monitored with a conventional fluorescence plate reader equipped with suitable optical filters. To achieve the high receptor concentration required for the assay and the low autofluorescence levels essential for reliable results, budded baculoviruses that display GPCRs on their surfaces can be used. The monitoring process generates a substantial amount of kinetic data, which is usually stored as a proprietary file format limiting the flexibility of data analysis. To solve this problem, we propose the use of the data curation software Aparecium ( http://gpcr.ut.ee/aparecium.html ), which integrates experimental data with metadata in a Minimum Information for Data Analysis in Systems Biology (MIDAS) format. Aparecium enables data export to different software packages for fitting to suitable kinetic or equilibrium models. A combination of the FA assay with the novel data analysis strategy is suitable for screening new active compounds, but also for modeling complex systems of ligand binding to GPCRs. We present the proposed approach using different fluorescent probes and assay types to characterize ligand binding to melanocortin 4 (MC) receptor.

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Source
http://dx.doi.org/10.1007/978-1-0716-1221-7_8DOI Listing

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