Spin-labeled extracellular loop from a seven-transmembrane helix receptor: Studies in solution and interaction with model membranes

T. A. Pertinhez, C. R. Nakaie, A. C M Paiva, S. Schreier

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

A spin-labeled pentadecapeptide was synthesized containing 2,2,6,6- tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) as the N- terminal amino acid and residues 253-266 (EYWSTFGNLHHISL) of the mas oncogene receptor, a membrane-bound protein from the G-protein coupled receptors family. According to predictions, this protein folds into seven transmembrane helices connected by three extra- and three intracellular loops, and the peptide encompasses part of the third extracellular loop and part of the seventh helix. Electron paramagnetic resonance (EPR) spectra of the spin- labeled peptide (TOAC-14) were obtained in aqueous solution as a function of pH and temperature, in a secondary structure-inducing solvent [trifluoroethanol (TFE)], and in the presence of detergent micelles and phospholipid bilayers. The charged and uncharged amino groups of TOAC and TOAC-14 yielded spectra with different isotropic hyperfine splittings (a(N)). The slow exchange between protonated and unprotonated forms in the EPR time scale gave rise to composite spectra weighted by the Henderson-Hasselbalch equation. Plots of a(N) vs pH allowed the determination of the amino group pK values (8.4 and 4.5, for TOAC and TOAC-14, respectively). A small change in a(N) centered at pH 6.5 was ascribed to the titration of the histidines. Values of calcidated rotational correlation times were indicative of a pH- induced conformational change. A conformational change was also observed in TFE. TOAC-14 bound to micelles irrespective of peptide and detergent head group charge. In contrast, the peptide bound to phospholipid bilayers only when both carried opposite charges. The slow exchange (in the EPR time scale) between membrane-bound and free TOAC-14 allowed the calculation of the peptide's partition coefficient. The spectral line shapes were affected by aggregate size and degree of packing of the constituent molecules. It is proposed that pH, polarity, and lipid environment can affect the conformation of water-exposed regions of membrane-bound receptors, thereby playing a role in the mechanism of signal transduction.

Original languageEnglish
Pages (from-to)821-829
Number of pages9
JournalBiopolymers
Volume42
Issue number7
Publication statusPublished - Dec 1997

Fingerprint

Carboxylic acids
Membranes
Peptides
Electron Spin Resonance Spectroscopy
Trifluoroethanol
Paramagnetic resonance
Phospholipids
Detergents
Micelles
Proteins
Signal transduction
2,2,6,6-tetramethylpiperidine-N-oxide-4-amino-4-carboxylic acid
G-Protein-Coupled Receptors
Titration
Oncogenes
Histidine
Lipids
Conformations
Amino acids
Signal Transduction

Keywords

  • Electron paramagnetic resonance
  • Model membrane
  • Peptide
  • Receptor
  • Spin label

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biophysics

Cite this

Spin-labeled extracellular loop from a seven-transmembrane helix receptor : Studies in solution and interaction with model membranes. / Pertinhez, T. A.; Nakaie, C. R.; Paiva, A. C M; Schreier, S.

In: Biopolymers, Vol. 42, No. 7, 12.1997, p. 821-829.

Research output: Contribution to journalArticle

Pertinhez, T. A. ; Nakaie, C. R. ; Paiva, A. C M ; Schreier, S. / Spin-labeled extracellular loop from a seven-transmembrane helix receptor : Studies in solution and interaction with model membranes. In: Biopolymers. 1997 ; Vol. 42, No. 7. pp. 821-829.
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N2 - A spin-labeled pentadecapeptide was synthesized containing 2,2,6,6- tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) as the N- terminal amino acid and residues 253-266 (EYWSTFGNLHHISL) of the mas oncogene receptor, a membrane-bound protein from the G-protein coupled receptors family. According to predictions, this protein folds into seven transmembrane helices connected by three extra- and three intracellular loops, and the peptide encompasses part of the third extracellular loop and part of the seventh helix. Electron paramagnetic resonance (EPR) spectra of the spin- labeled peptide (TOAC-14) were obtained in aqueous solution as a function of pH and temperature, in a secondary structure-inducing solvent [trifluoroethanol (TFE)], and in the presence of detergent micelles and phospholipid bilayers. The charged and uncharged amino groups of TOAC and TOAC-14 yielded spectra with different isotropic hyperfine splittings (a(N)). The slow exchange between protonated and unprotonated forms in the EPR time scale gave rise to composite spectra weighted by the Henderson-Hasselbalch equation. Plots of a(N) vs pH allowed the determination of the amino group pK values (8.4 and 4.5, for TOAC and TOAC-14, respectively). A small change in a(N) centered at pH 6.5 was ascribed to the titration of the histidines. Values of calcidated rotational correlation times were indicative of a pH- induced conformational change. A conformational change was also observed in TFE. TOAC-14 bound to micelles irrespective of peptide and detergent head group charge. In contrast, the peptide bound to phospholipid bilayers only when both carried opposite charges. The slow exchange (in the EPR time scale) between membrane-bound and free TOAC-14 allowed the calculation of the peptide's partition coefficient. The spectral line shapes were affected by aggregate size and degree of packing of the constituent molecules. It is proposed that pH, polarity, and lipid environment can affect the conformation of water-exposed regions of membrane-bound receptors, thereby playing a role in the mechanism of signal transduction.

AB - A spin-labeled pentadecapeptide was synthesized containing 2,2,6,6- tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) as the N- terminal amino acid and residues 253-266 (EYWSTFGNLHHISL) of the mas oncogene receptor, a membrane-bound protein from the G-protein coupled receptors family. According to predictions, this protein folds into seven transmembrane helices connected by three extra- and three intracellular loops, and the peptide encompasses part of the third extracellular loop and part of the seventh helix. Electron paramagnetic resonance (EPR) spectra of the spin- labeled peptide (TOAC-14) were obtained in aqueous solution as a function of pH and temperature, in a secondary structure-inducing solvent [trifluoroethanol (TFE)], and in the presence of detergent micelles and phospholipid bilayers. The charged and uncharged amino groups of TOAC and TOAC-14 yielded spectra with different isotropic hyperfine splittings (a(N)). The slow exchange between protonated and unprotonated forms in the EPR time scale gave rise to composite spectra weighted by the Henderson-Hasselbalch equation. Plots of a(N) vs pH allowed the determination of the amino group pK values (8.4 and 4.5, for TOAC and TOAC-14, respectively). A small change in a(N) centered at pH 6.5 was ascribed to the titration of the histidines. Values of calcidated rotational correlation times were indicative of a pH- induced conformational change. A conformational change was also observed in TFE. TOAC-14 bound to micelles irrespective of peptide and detergent head group charge. In contrast, the peptide bound to phospholipid bilayers only when both carried opposite charges. The slow exchange (in the EPR time scale) between membrane-bound and free TOAC-14 allowed the calculation of the peptide's partition coefficient. The spectral line shapes were affected by aggregate size and degree of packing of the constituent molecules. It is proposed that pH, polarity, and lipid environment can affect the conformation of water-exposed regions of membrane-bound receptors, thereby playing a role in the mechanism of signal transduction.

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