Characterization of a conformationally sensitive TOAC spin-labeled substance P

Characterization of a conformationally sensitive TOAC spin-labeled substance P

Autor Shafer, Aaron M. Google Scholar
Nakaie, Clovis R. Autor UNIFESP Google Scholar
Deupi, Xavier Google Scholar
Bennett, Vicki J. Google Scholar
Voss, John C. Google Scholar
Instituição Univ Calif Davis
Northeastern Ohio Univ Coll Med & Pharm
Universidade Federal de São Paulo (UNIFESP)
Univ Autonoma Barcelona
Resumo 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-tetramethyl-piperidino-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 similar to 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. (C) 2008 Elsevier Inc. All rights reserved.
Palavra-chave Substance P
TOAC spin label
Idioma Inglês
Financiador NIH
Ministerio de Educacion y Ciencia (Spain)
Número do financiamento NIH: NS25999
: C06 RR-12088-01
Data de publicação 2008-11-01
Publicado em Peptides. New York: Elsevier B.V., v. 29, n. 11, p. 1919-1929, 2008.
ISSN 0196-9781 (Sherpa/Romeo, fator de impacto)
Publicador Elsevier B.V.
Extensão 1919-1929
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000261023900009
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