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

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1997-12-01
Autores
Pertinhez, T. A.
Nakaie, C. R.
Paiva, ACM
Schreier, S.
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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 parr of the third extracellular loop and parr 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 calculated 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. (C) 1997 John Wiley & Sons, Inc.
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Biopolymers. New York: John Wiley & Sons Inc, v. 42, n. 7, p. 821-829, 1997.