Role of Membrane Lipids and Hydration in G-Protein–Coupled Receptor Signaling
PublisherThe University of Arizona.
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EmbargoRelease after 15-Jun-2019
AbstractMy thesis is focused on role of soft matter (membrane lipids plus water) in G-protein coupled receptor (GPCR) activation. Notably GPCRs are one of the hot topics that have attracted considerable attention in pharmacology and drug companies. About 50% of the modern drugs available in the market target GPCRs. Although GPCRS are of immense importance in pharmacology, critical information is missing about their activation mechanism. The major reason is that extensive studies have not been done in native conditions. However GPCRs are integral membrane protein and the role of membrane lipids and water is crucial in rhodopsin signaling, which has not been investigated considerably. Here we used rhodopsin, a canonical GPCR, to study the role of soft-matter (membrane lipids and water) in rhodopsin signaling. We hypothesize that rhodopsin activation involves an ensemble of states with an increase in hydrated protein volume. The opening of the protein paves the way for G-protein binding and initiation of rhodopsin signaling. Since rhodopsin is an integral membrane protein, the properties of membrane lipids could modulate rhodopsin activation. We used a novel charge-induced directed-reconstitution method to recombine rhodopsin spontaneously with artificial membranes and polymerosomes. UV-visible spectroscopy was used to characterize the light activation of the protein. We report a novel allosteric mode of rhodopsin activation, where electrostatic interaction between positively charged membrane head-groups and negatively charged Glu134 disrupts the ERY motif of the second ionic lock, and leads to constitutive activation of the protein. Our results are in agreement with the molecular dynamics simulations analysis, and are further supported by results using the ATR-FTIR spectroscopy technique. In addition we have studied the role of water in rhodopsin function using different size osmolytes. We observed a surprising size-reversal effect on the rhodopsin activation mechanism. Small osmolytes stabilize the active state whereas large osmolytes favor the inactive state. It is proposed that small osmolytes can penetrate the protein core and interact with the protein binding cleft. By contrast large osmolytes cannot access the protein binding cleft, and exerts an osmotic pressure on the protein, leading to expulsion of water and stabilizing the inactive state. Based on osmotic stress studies and transducin peptide binding assays, we propose a hydration-dehydration cycle that explains the rapid amplification rate and high fidelity in rhodopsin signaling. Our studies lay the foundation to understanding role of soft matter in rhodopsin-like GPCR activation, and pave the way for developing new drugs in the pharmaceutical industry.
Degree ProgramGraduate College