Analysis of the dynamics and interactions of an inhibitory Gα subunit and thermostabilized neurotensin receptor variants by NMR spectroscopy

One of the most important classes of membrane proteins are G-Protein-coupled-receptors (GPCRs). GPCRs represent a big part of the protein coding genome and are responsible for many biochemical processes, ranging from cancer to neurology. Due to their big role in physiology and pathophysiology, many drugs target GPCRs, to date approximately 1/3 of all FDA approved drugs. In order to understand these processes better and to develop drugs targeting those signaling pathways, it is from biggest importance to get a deeper understanding of the structure and dynamics of GPCRs and their corresponding G-proteins. In this work, I used evolutionary stabilized Neurotensin 1 Receptor (NTR1) variants with different signaling capacity as a model system for the investigating the dynamics and interactions of a GPCR by NMR spectroscopy. The reason for this choice is an easy and effective production of this GPCR in E. coli, which is needed for isotope labeling for nuclear magnetic resonance (NMR) spectroscopy experiments. On the G-protein side, either the heterotrimeric G-protein extracted from insect cells or the alpha subunit (Gα) produced in E.coli was used. The key goal was to understand the effects of the dynamics of GPCRs and G-proteins on their interaction with each other. The first goal was to study the role of the nucleotide state of Gα on its structure and dynamics and therefor on its interaction with an activated GPCR. Thereby I showed that Gα in an apo state has the highest affinity to an activated GPCR as well as an open conformation enhancing GTP binding. In line with these findings, GTP-bound Gα shows no significant affinity to an activated GPCR with a tightly closed conformation. I used NMR experiments for the investigation of labeled Gα in this project. The second part of my work was focused on the GPCR side. Therefore, different labeling strategies like 13C methyl sulfide or selective isoleucine, leucine, valine and alanine (ILVA) labeling were used in order to facilitate NMR experiments to detect allosteric structural changes and dynamics during signaling. I could show that GPCR activation affects the whole GPCR with an allosteric mechanism, resulting in a helix 6 rearrangement on the cytosolic side. This is essential for the interaction of a GPCR with G-proteins. Furthermore, I could verify the importance of the ionic lock motif GPCR signaling. In summary, I was able to obtain a deeper understanding of the interaction between GPCRs and G-proteins, with a focus on each binding partner. Nonetheless, future experiments are required to further characterize GPCR plasticity and determine the allosteric structural changes that are required to transfer the signal induced by ligand binding through the membrane to activate a bound G-protein.