title: Characterization of a conformationally sensitive TOAC spin-labeled substance P.
abstract: To probe the binding of a peptide agonist to a G-protein coupled receptor in native membranes, the spin-labeled amino acid analogue 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidino-1-oxyl (TOAC) was substituted at either position 4 or 9 within the substance P peptide (RPKPQQFFGLM-NH2), a potent agonist of the neurokinin-1 receptor. The affinity of the 4-TOAC analog is comparable to the native peptide while the affinity of the 9-TOAC derivative is approximately 250-fold lower. Both peptides activate receptor signaling, though the potency of the 9-TOAC peptide is substantially lower. The utility of these modified ligands for reporting conformational dynamics during the neurokinin-1 receptor activation was explored using EPR spectroscopy, which can determine the real-time dynamics of the TOAC nitroxides in solution. While the binding of both the 4-TOAC substance P and 9-TOAC substance P peptides to isolated cell membranes containing the neurokinin-1 receptor is detected, a bound signal for the 9-TOAC peptide is only obtained under conditions that maintain the receptor in its high-affinity binding state. In contrast, 4-TOAC substance P binding is observed by solution EPR under both low- and high-affinity receptor states, with evidence of a more strongly immobilized peptide in the presence of GDP. In addition, to better understand the conformational consequences of TOAC substitution into substance P as it relates to receptor binding and activation, atomistic models for both the 4- and 9-TOAC versions of the peptide were constructed, and the molecular dynamics calculated via simulated annealing to explore the influence of the TOAC substitutions on backbone structure.
authors.affiliation: Department of Biochemistry & Molecular Medicine, University of California, Davis, CA 95616, United States.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Shafer
authors.forename: Clovis R
authors.initials: CR
authors.lastname: Nakaie
authors.forename: Xavier
authors.initials: X
authors.lastname: Deupi
authors.forename: Vicki J
authors.initials: VJ
authors.lastname: Bennett
authors.forename: John C
authors.initials: JC
authors.lastname: Voss
mesh_terms: D000818:Animals; D016466:CHO Cells; D003198:Computer Simulation; D006224:Cricetinae; D003412:Cricetulus; D004578:Electron Spin Resonance Spectroscopy; D018040:Receptors, Neurokinin-1 / Q000187:drug effects / Q000378:metabolism; D013373:Substance P / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000187:drug effects
chemical_list: D018040:Receptors, Neurokinin-1; D013373:Substance P