Trifluoroethanol and binding to model membranes stabilize a predicted turn in a peptide corresponding to the first extracellular loop of the angiotensin II AT1A receptor

• Published in: Biopolymers Vol. 65; no. 1; pp. 21 - 31
• Main Authors: Salinas, Roberto K., Shida, Cláudio S., Pertinhez, Thelma A., Spisni, Alberto, Nakaie, Clóvis R., Paiva, Antonio C. M., Schreier, Shirley
• Format: Journal Article
• Language: English
• Published: New York Wiley Subscription Services, Inc., A Wiley Company 05.10.2002
• Subjects: extracellular loop; fluorescence; G-protein coupled receptor; NMR; peptide-membrane interaction; trifluoroethanol
• DOI: 10.1002/bip.10209
• ISSN: 0006-3525

Homology modeling of the angiotensin II AT1A receptor based on rhodopsin′s crystal structure has assigned the 92–100 (YRWPFGNHL) sequence of the receptor to its first extracellular loop. Solution and membrane‐bound conformational properties of a peptide containing this sequence (EL1) were examined by CD, fluorescence, and 1H‐NMR. CD spectra in aqueous solution revealed an equilibrium between less organized and folded conformers. NMR spectra indicated the coexistence of trans and cis isomers of the Trp3–Pro4 bond. A positive band at 226 nm in the CD spectra suggested aromatic ring stacking, modulated by EL1's ionization degree. CD spectra showed that trifluoroethanol (TFE), or binding to detergent micelles and phospholipid bilayers, shifted the equilibrium toward conformers with higher secondary structure content. Different media gave rise to spectra suggestive of different β‐turns. Chemical shift changes in the NMR spectra corroborated the stabilization of different conformations. Thus, environments of lower polarity or binding to interfaces probably favored the formation of hydrogen bonds, stabilizing β‐turns, predicted for this sequence in the whole receptor. Increases in Trp3 fluorescence intensity and anisotropy, blue shifts of the maximum emission wavelength, and pK changes also evinced the interaction between EL1 and model membranes. Binding was seen to depend on both hydrophobic and electrostatic interactions, as well as lipid phase packing. Studies with water‐soluble and membrane‐bound fluorescence quenchers demonstrated that Trp3 is located close to the water–membrane interface. The results are discussed with regard to possible implications in receptor folding and function. © 2002 Wiley Periodicals, Inc. Biopolymers 65: 21–31, 2002