Conformational properties of a fragment (amino acids 92-100) of the first extracellular loop of the AT1 receptor of angiotensin II in solution and in the presence of model membranes

Author(s)

Roberto Kopke Salinas

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2000-01-07

Examining board members

Shirley Schreier; Elizabeth Pinheiro Gomes Areas; Laerte de Oliveira

• Advisor: Shirley Schreier

Abstract

In this work the conformational properties of a peptide (YRWPFGNHL-NH2) whose sequence is present in the first extra-cellular loop of the angiotensin II AT1 receptor were examined. Circular dichroism (CD) and fluorescence spectra were obtained in aqueous solution (as a function of pH and temperature), in the presence of a secondary structure inducing solvent (trifluoroethanol, TFE), and in the presence of negatively charged (sodium dodecyl sulfate, SDS) or zwitterionic (N-hexadecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, HPS and lysophosphatidylcholine, liso-PC) micelles, and of bilayers containing a zwitterionic phospholipid (l-palmitoyl-2-oleoyl phosphatidylcholine, POPC) or a mixture of POPC and a negatively charged phospholipid (l-palmitoyl-2-oleoyl phosphatidic acid, POPA). Studies of titration calorimetry allowed the analysis of the binding thermodynamics. CD spectra indicated a flexible structure in aqueous solution, which was modulated by pH and temperature. In the presence of TFE, micelles and of POPC:POPA vesicles, the peptide acquired secondary structure, which is suggestive of a β turn. The intrinsic fluorescence spectra (W3) showed a blue-shift of the maximum emission wavelenght (λmax) in the presence of micelles and of POPC:POPA vesicles. An increase in fluorescence intensity was also observed, except in the case of SDS. These results indicated that the peptide interacted with the aggregates. No fluorescence changes were observed in the presence of POPC. Fluorescence anisotropy measurements showed that, when bound to the aggregates, the peptide becomes more immobilized. Fluorescence quenching studies using water soluble and membrane-bound quenchers suggested that W3 is located close to the water-bilayer interface. The results showed that the peptide binds to negativelly charged and zwitterionic micelles, while in the case of bilayers (which are more tightly packed) the binding depends on the presence of negative charges. Titration calorimetry showed that ΔH of peptide binding to the vesicles is negative, which is compatible with the ocurrence of eletrostatic interactions. Qualitative and quantitative differences in binding of the peptide to HPS and liso-PC micelles were observed. In order to examine whether these differences were due to molecular packing, electron paramagnetic resonance spectra of spin labels intercalated in both systems were obtained. Differences were observed, mainly in the polar head group region, that could be responsible for the different behaviour displayed by the peptide in both aggregates. The results obtained for the first extra-cellular loop of the AT1 receptor indicated that its conformation can be modulated by pH and by the polarity of the medium, and that it can interact with the membrane through hydrophobic and electrostatic interactions, and also that this interaction depends on the molecular packing of the lipid phase. These results are in aggrement with the idea that the GPCRs extra-membrane domains are located at the water-membrane interface. Conformational changes induced by the medium, as well as by the interaction between extra-cellular segments and the membrane or by ligand binding, could play a role in the molecular mechanism of signal transduction. (AU)