title: Electron paramagnetic resonance studies of functionally active, nitroxide spin-labeled peptide analogues of the C-terminus of a G-protein alpha subunit.
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.
authors.affiliation: Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-7008, USA.
authors.forename: Ned
authors.initials: N
authors.lastname: Van Eps
authors.forename: Lori L
authors.initials: LL
authors.lastname: Anderson
authors.forename: Oleg G
authors.initials: OG
authors.lastname: Kisselev
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Baranski
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
authors.forename: Garland R
authors.initials: GR
authors.lastname: Marshall
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry / Q000378:metabolism; D013113:Spin Labels
chemical_list: D044385:GTP-Binding Protein alpha Subunits; D010455:Peptides; D013113:Spin Labels; D012243:Rhodopsin