Trifluoroethanol and binding to model membranes stabilize a predicted turn in a peptide corresponding to the first extracellular loop of the angiotensin II AT(1A) receptor.
Homology modeling of the angiotensin II AT(1A) 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 (1)H-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 Trp(3)-Pro(4) 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 beta-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 beta-turns, predicted for this sequence in the whole receptor. Increases in Trp(3) 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 Trp(3) is located close to the water-membrane interface. The results are discussed with regard to possible implications in receptor folding and function.
Salinas
Roberto K
RK
Department of Biochemistry, Institute of Chemistry, University of São Paulo, CP 26077, 05513-970, São Paulo, Brazil.
Shida
Cláudio S
CS
Pertinhez
Thelma A
TA
Spisni
Alberto
A
Nakaie
Clóvis R
CR
Paiva
Antonio C M
AC
Schreier
Shirley
S
D002942:Circular Dichroism; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008954:Models, Biological; D017510:Protein Folding; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000737:chemistry / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D014270:Trifluoroethanol / Q000737:chemistry / Q000378:metabolism*
D008823:Micelles; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D012967:Sodium Dodecyl Sulfate; D014270:Trifluoroethanol