Date of this Version
Lesiak, L. 2020. Synthesis and Evaluation of Nebraska Red Derivatives for Protein Labeling Studies. Undergraduate Honors Thesis. University of Nebraska-Lincoln.
G protein-coupled receptors (GPCRs) play a vital role in cell signaling, and imaging the internalization of these proteins will allow for better understand of protein trafficking within the cell, as well as identification of potential drug targets. However, there is a need for further development of fluorescent protein labels that are able to effectively monitor GPCR internalization in the near-infrared (NIR) range. We recently described a series of NIR fluorophores in which a phosphinate functionality is inserted at the bridging position of the xanthene scaffold, called the Nebraska Red (NR) dye series. These dyes are attractive reagents for the imaging of protein localization, such as the internalization of GPCRs, because of their remarkable photostability and brightness. Herein, we report a NR-based system for protein imaging using HaloTag ligands for imaging membrane proteins in living cells, including a spirolactonized derivative that enables no-wash imaging. We use these probes to image the human orexin type 2 receptor, an established target for the treatment of insomnia, and we demonstrate the noncovalent association of fetal bovine serum (FBS) with the spirolactonized probe that causes quenching of unbound dye. Furthermore, we demonstrate the utility of NR-based HaloTag ligands for real-time monitoring of orexin internalization upon agonist stimulation.