Structure, Function and Dynamics of G-Protein coupled Receptors

Understanding the function of membrane proteins is crucial to elucidate the molecular mechanisms by which transmembrane signaling based physiological processes,i. e., the interactions of extracellular ligands with membrane-bound receptors, are regulated. 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 fluorescence 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 specifically developed for the purpose of this study, to achieve a full prospect of time-resolved hydration effects 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 difference spectra and simultaneous static fluorescence recordings. The data reveal that lipids play a mediating role in transmitting hydration to the subsequent membrane protein response followed by water penetration into the receptor structure or into the sub-headgroup region in single membrane-spanning peptides carrying the conserved proton uptake site (monitored by the fluorescence emission 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.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Technische Universität Dresden

Year

2012

Author dc:creator

• Eichler, Stefanie

Contributors dc:contributor

• Fahmy, Karim
• Diez, Stefan
• Hildebrandt, Peter

• Membranprotein
• G-Protein gekoppelter Rezeptor
• Lipid
• Protonen
• Proteinstruktur
• FTIR
• Fluoreszenz
• membrane protein
• G-protein coupled receptor
• proton
• protein structure
• fluorescence