Date of this Version
Makayla Gill. 2022. Using Proximity Induced Approaches to Label Membrane Proteins. Undergraduate Honors Thesis. University of Nebraska - Lincoln.
Cell-to-cell communication has been a primary focus of chemical biology research for decades, and transmembrane proteins, a protein that is integral to the membrane and spans across the entire surface of the cell, play a crucial role. Fully understanding the ligand-receptor interactions of transmembrane proteins that occur in living systems can open the door to understanding a wide range of biochemical events that are required for regular cell function as well as disease. While we hope to establish a more global application of this strategy, we chose neurotensin and the neurotensin receptor 1 (NTS1) as the initial bioactive peptide model system. Deciding on NTS and NTS1 was because of neurotensin’s ease of synthesis and well-established crystal structure with the NTS1 receptor. We started by utilizing solid-phase peptide synthesis to synthesize neurotensin derivatives designed to test the success and limitations of our proposed reaction. The 4-aminophenylalanine of the neurotensin peptide analog was diazotized, transfected into cells, underwent in vitro azo coupling, and were exposed to primary and secondary antibodies via SDS-PAGE and western blotting. Our results indicate that the proposed azo coupling chemical reaction using the rationally designed neurotensin analog can be used to label NTSR1 in a selective manner.