Electron paramagnetic resonance studies of functionally active, nitroxide spin-labeled peptide analogues of the C-terminus of a G-protein α subunit

The C-terminal tail of the transducin α subunit, Gtα(340-350), is known to bind and stabilize the active conformation of rhodopsin upon photoactivation (R*). Five spin-labeled analogues of Gtα(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 TOAC³⁴³-Gtα(340- 350) revealed greater immobilization of the nitroxide when compared to that of the N-terminally modified Proxyl-Gtα(340-350) analogue. A doubly labeled Proxyl/TOAC³⁴⁸-Gtα(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 α-peptide system provide unique biophysical probes that can be used to explore the structure and conformational changes at the rhodopsin-G-protein interface.