Structure, Function and Dynamics of G-Protein coupled Receptors

Understanding the function of membrane proteins is crucial to elucidate the molecular mechanisms by which physiological processes are regulated by transmembrane signaling based on the interaction of extracellular ligands with membrane-bound receptors. In this work, synthetic transmembrane segments derived from the visual photoreceptor rhodopsin, the full length system rhodopsin and mutants of opsin are used to study physical processes that underlie the function of this prototypical class-A G protein-coupled receptor. The dependency of membrane protein hydration and protein-lipid interactions on side chain charge neutralization is addressed by uorescence spectroscopy on synthetic transmembrane segments in detergent and lipidic environment constituting transmembrane segments of rhodopsin in the membrane. Results from spectroscopic
studies allow us to construct a structural and thermodynamical model of coupled protonation of the conserved ERY motif in transmembrane helix 3 of rhodopsin and of helix restructuring in the micro-domain formed at the protein/lipid water phase boundary. Furthermore, synthesized peptides and full length systems were studied by time resolved FTIR-Fluorescence Cross Correlation Hydration Modulation, a technique specically developed for the purpose of this study, to achieve a full prospect of time-resolved hydration eects on lipidic and proteinogenic groups, as well as their interactions. Multi-spectral experiments and time-dependent analyses based on 2D correlation where established to analyze large data sets obtained from time-resolved FTIR dierence spectra and simultaneous static fluorescence recordings. The data reveal a sequential process where water H-bond formation to the lipid carbonyl precedes transmembrane protein conformational changes which are eventually followed by the intrusion of water into the protein interior as monitored by the fluorescence of hydrophobic buried tryptophan. Our results support the assumption of the critical role of the lipid/water interface in membrane protein function and they prove in particular the important influence of electrostatics, i.e., side chain charges at the phase boundary, and hydration on that function.