title: Structure-based biophysical analysis of the interaction of rhodopsin with G protein and arrestin.
abstract: In this chapter, we describe a set of complementary techniques that we use to study the activation of rhodopsin, a G protein-coupled receptor (GPCR), and its functional interactions with G protein and arrestin. The protein reagents used for these studies come from native disc membranes or heterologous expression, and G protein and arrestin are often replaced with less complex synthetic peptides derived from key interaction sites of these binding partners (BPs). We first report on our approach to protein X-ray crystallography and describe how protein crystals from native membranes are obtained. The crystal structures provide invaluable resolution, but other techniques are required to assess the dynamic equilibria characteristic for active GPCRs. The simplest approach is "Extra Meta II," which uses UV/Vis absorption spectroscopy to monitor the equilibrium of photoactivated states. Site-specific information about the BPs (e.g., arrestin) is added by fluorescence techniques employing mutants labeled with reporter groups. All functional changes in both the receptor and interacting proteins or peptides are seen with highest precision using Fourier transform infrared (FTIR) difference spectroscopy. In our approach, the lack of site-specific information in FTIR is overcome by parallel molecular dynamics simulations, which are employed to interpret the results and to extend the timescale down to the range of conformational substates.
authors: Institut für Medizinische Physik und Biophysik (CC2), Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors: Martha E
authors: ME
authors: Sommer
authors: Institut für Medizinische Physik und Biophysik (CC2), Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors: Matthias
authors: M
authors: Elgeti
authors: Institut für Medizinische Physik und Biophysik (CC2), Charité-Universitätsmedizin Berlin, Berlin, Germany; AG ProteInformatics.
authors: Peter W
authors: PW
authors: Hildebrand
authors: Institut für Medizinische Physik und Biophysik (CC2), Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors: Michal
authors: M
authors: Szczepek
authors: Institut für Medizinische Physik und Biophysik (CC2), Charité-Universitätsmedizin Berlin, Berlin, Germany; Zentrum für Biophysik und Bioinformatik, Humboldt-Universität zu Berlin, Berlin, Germany. Electronic address: kph@charite.de.
authors: Klaus Peter
authors: KP
authors: Hofmann
authors: Institut für Medizinische Physik und Biophysik (CC2), Charité-Universitätsmedizin Berlin, Berlin, Germany; AG Protein X-ray Crystallography & Signal Transduction.
authors: Patrick
authors: P
authors: Scheerer
mesh_terms: D000818:Animals; D019393:Arrestin / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D018360:Crystallography, X-Ray / Q000379:methods; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods*; D012243:Rhodopsin / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
chemical_list: D019393:Arrestin; D012243:Rhodopsin; D019204:GTP-Binding Proteins
keywords: Absorption spectroscopy; Arrestin; FTIR difference spectroscopy; G protein; G protein-coupled receptor; Molecular dynamics; Protein crystallography; Rhodopsin; Site-directed fluorescence spectroscopy