Journal of Peptide Science J. Pept. Sci. 2007; 13: 575–580 Published online 29 June 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/psc.875 Temperature-dependent variations of ligand-receptor contact points in hAT ‡ 1 J. ARSENAULT, M. P. LA HAYE RENAUD, M. CL ´ EMENT, D. FILLION, G. GUILLEMETTE, R. LEDUC, P. LAVIGNE and E. ESCHER* D´ epartement de pharmacologie, Facult ´ e de M ´ edecine et des Sciences de la Sant ´ e, Universit ´ e de Sherbrooke, 3001, 12 i ` eme Avenue Nord, Sherbrooke, Quebec, J1H 5N4, Canada Received 12 April 2007; Accepted 12 April 2007 Abstract: Photoaffinity labelling is regularly used to investigate proteins, including peptidergic G protein-coupled receptors (GPCR). To this purpose benzophenone photolabels have been widely used to identify many contact residues in ligand-binding pockets. The three-dimensional binding environment of the human angiotensin II type 1 receptor hAT 1 has been determined using an iterative methionine mutagenesis strategy based on the photochemical properties and preferential incorporation of benzophenone onto methionine. This has led to the construction of a ligand-bound receptor structure. The present study investigated the effect of temperature on the accessibility of some of these contact points. The hAT 1 receptor and two representative Met mutants (H256M-hAT 1 and F293M-hAT 1 ) from the iterative mutagenesis study were photolabelled with the benzophenone- ligand 125 I-[Sar 1 , Bpa 8 ]AngII at temperatures ranging from −15 ° C to 37 ° C. Labelled receptors were partially purified and digested with cyanogen bromide to identify the contact points or segments. There were no changes in receptor contacts or labelling in the 7th transmembrane domains (TMD) of hAT 1 and F293M-hAT 1 across the temperature range. However, a temperature-dependent change in the ligand-receptor contact of H256M-hAT 1 was observed. At −15 ° C, H256M labelling was identical to that of hAT 1 , indicating that the interaction was specific to the 7th TMD. Significant labelling changes were observed at higher temperatures and at 37 ° C labelling occurred almost exclusively at mutated residue H256M-hAT 1 in the 6th TMD. Simultaneous competitive labelling of different areas of this target protein indicated that the ligand-receptor structure became increasingly fluctual at physiological temperatures, while a more compact, low mobility, and low energy conformation prevailed at low temperatures. Copyright  2007 European Peptide Society and John Wiley & Sons, Ltd. Keywords: GPCR; angiotensin II receptor; hAT 1 ; photoaffinity labelling; ligand contact points; thermodynamics INTRODUCTION Over 50% of all pharmaceuticals target G protein- coupled receptors (GPCRs). GPCRs, like many other membrane proteins, are quite refractory to structural characterisation using traditional methods such as nuclear magnetic resonance, electron microscopy, and X-ray crystallography [1–4]. In addition, these methods mainly provide a static picture and thus cannot be used to investigate the microconformational variations within a given protein population that are so crucial to understanding their physiological functions. The need to better understand tertiary structures and the fluctuations inside such proteins is primordial in order to develop a rational approach to designing drugs with the appropriate activities. Structural analysis by photoaffinity labelling is one of the biochemical methods that has been used for over 40 years to investigate ligand-receptor interactions * Correspondence to: E. Escher, D´ epartement de pharmacologie, Facult´ e de M´ edecine et des Sciences de la Sant´ e, Universit´ e de Sherbrooke, 3001, 12 i ` eme Avenue Nord, Sherbrooke, Quebec, J1H 5N4, Canada; e-mail: Emanuel.Escher@USherbrooke.ca ‡ This article is published as part of the special issue 2 nd International Congress on Natural Peptides to Drugs, April 18–21, 2006, Zermatt, Switzerland. and the in situ localisation of receptors [5–9]. In brief, a biologically relevant ligand containing a photoactivable ligand such as azide, diazirine, or benzophenone is first reversibly bound to its target. Photolysis of the ligand-receptor complex produces a radical intermediate (nitrene, carbene, or keto radical), which forms a covalent bond between the ligand and the receptor [5,9–12]. In the case of receptors, this method has been applied mainly to peptidergic receptors and their ligands, since binding of the photolabile moiety of a peptide causes only minor structural changes to the ligand, thus assuring native- like ligand binding. If sufficient ligand-receptor contact points can be obtained from such receptor labelling experiments, structural elements can be deduced by applying homology modelling approaches to the structural constraints of experimentally determined contact points [13]. In addition, the preferential binding of benzophenone to methionine has been exploited to develop the methionine proximity assay (MPA), an iterative Met-mutagenesis strategy that has been applied to the human angiotensin II type 1 receptor (hAT 1 ) [14]. Its interaction with angiotensin II (AngII) has been extensively studied in our laboratory [8,13–16]. A key feature of this interaction is the Copyright  2007 European Peptide Society and John Wiley & Sons, Ltd.