Dynamics and Oligomerization Tune Seven-Transmembrane Protein Function: Studies Based on Proteorhodopsin

Abstract

Seven-transmembrane (7TM) proteins have diverse and important functions, ranging from signaling receptors to ion pumps. They share a reversible switching property, epitomized by the solar-powered microbial proton pump Proteorhodopsin (PR), which uses light energy to facilitate transport by a conformational “switch”. Here, we use PR as a model to capture the elusive details of activation and oligomerization necessary for the function of physiologically important membrane proteins.We have preliminary spectroscopic evidence that suggests altered photocycle kinetics and shifted pKa values for hexameric and monomeric forms of detergent-solubilized PR. These findings prove that PR function is tuned by self-association, and we apply magnetic resonance together with functional assays of proton transport to further understand dynamics changes that occur upon oligomerization. Our unique magnetic resonance techniques of electron paramagnetic resonance (EPR) and dynamic nuclear polarization (DNP) provide insight into the protein segment mobility and local hydration water dynamics of an amino acid residue spin-labeled with nitroxide-based radicals.Using these methods, we have found that PR's third cytoplasmic (E-F) loop is a short α-helical segment that experiences conformational change upon photoactivation. This structure is a common motif to the non-homologous G-protein coupled receptor bovine rhodopsin (Rh), where it is a docking point for a signal G-protein. Towards understanding how function hinges on dynamics, we developed a PR-Rh chimera by replacing the E-F loop of PR with the corresponding loop of Rh. The chimera successfully expresses and maintains optical properties. We evaluate its capability to activate the G-protein transducin, and apply EPR and DNP to obtain unique information about the biophysics of receptor/G-protein interactions. By controlling the oligomeric form of the PR-Rh chimera, we measure any changes in G-protein activation caused by varying the amount of receptor-receptor interactions.