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Updated: Jun 10, 2026
Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels
Published on: July 4, 2016
Electron paramagnetic resonance studies of functionally active, nitroxide spin-labeled peptide analogues of the
Ned Van Eps1, Lori L Anderson, Oleg G Kisselev
1Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-7008, USA.
Abstract:
The C-terminal tail of the transducin alpha subunit, Gtalpha(340-350), is known to bind and stabilize the active conformation of rhodopsin upon photoactivation (R*). Five spin-labeled analogues of Gtalpha(340-350) demonstrated native-like activity in their ability to bind and stabilize R*. The spin-label 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) was employed at interior sites within the peptide, whereas a Proxyl (3-carboxyl-2,2,5,5-tetramethyl-pyrrolidinyloxy) spin-label was employed at the amino terminus of the peptide. Upon binding to R*, the electron paramagnetic resonance spectrum of TOAC(343)-Gtalpha(340-350) revealed greater immobilization of the nitroxide when compared to that of the N-terminally modified Proxyl-Gtalpha(340-350) analogue. A doubly labeled Proxyl/TOAC(348)-Gtalpha(340-350) was examined by DEER spectrocopy to determine the distribution of distances between the two nitroxides in the peptides when in solution and when bound to R*. TOAC and Proxyl spin-labels in this GPCR-G-protein alpha-peptide system provide unique biophysical probes that can be used to explore the structure and conformational changes at the rhodopsin-G-protein interface.
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