DESCRIPTION (provided by applicant): Our investigation aims to establish how the atomic and membrane-level events operate to trigger visual signal transduction by rhodopsin in a unified multi-scale framework, with broad implications for biological signaling. The high impact of understanding G-protein-coupled receptor (GPCR) activation is well appreciated. Yet numerous gaps in our understanding remain, both with rhodopsin, as well as other Family A GPCRs for which rhodopsin is a highly significant prototype. Here we plan to resolve the long sought, critical mechanistic features by an innovative approach that combines magnetic resonance (solid-state 2H and 13C NMR), Fourier transform infrared (FTIR), and electronic (UV-visible) spectroscopy. Our novel hypothesis for rhodopsin activation is formulated in terms of factors that drive a progression of transient conformational substates through an active ensemble: release of retinal strain, retinal-specific protein dynamics, hydration changes, pH catalysis, dynamical G-protein coupling, and membrane stress due to the polyunsaturated lipid composition. (1) Application of our novel solid-state 2H and 13C NMR technology will reveal how the release of conformational strain through retinal isomerization and relaxation unlocks the active rhodopsin (Meta-II) state. The angular and distance restraints from 2H and 13C solid-state NMR will illuminate the dynamical structure of retinal through its progression of active sub-states toward the active Meta-II form. (2) Changes in local retinal dynamics as studied by 2H and 13C NMR relaxation studies will be correlated with rhodopsin's activating motions by combining the results of spectroscopy with molecular dynamics (MD) simulations. Local retinal mobility will be related to large-scale protein dynamics involving fluctuations of the transmembrane helices. Notably, this work will address ambiguous X-ray structural data with results obtained at more physiological temperatures. Our investigation will decide the question of why active Meta-II rhodopsin and the ligand-free Opsin* apoprotein have similar X-ray structures, yet completely different activities. (3) Next we plan to investigate the specific role f pH and hydration throughout the active ensemble in relation to rhodopsin's interaction with transducin. We plan to combine our spectroscopic methods (UV-visible, FTIR, site-directed spin-labeling) with osmotic pressure studies to investigate changes in water-mediated H-bonding networks, together with a dramatic water influx due to transmembrane helical movements in rhodopsin activation. (4) Additional research will uncover the influences of polyunsaturated lipids on rhodopsin through modulation of membrane curvature stress, and how lipid composition biases the distribution of states in the active ensemble mechanism. Our plan encapsulates a new multi-scale view of how rhodopsin initiates visual signal transduction, tying together mechanical, environmental, temporal, and structural factors. Understanding how these factors interoperate across various length and time scales is fundamental to understanding of how rhodopsin achieves the extreme high fidelity required for visual signaling. Our robust and novel approach will provide important insights that are transferable to the broader class of GPCRs in biology and pharmacology.

PUBLIC HEALTH RELEVANCE: Rhodopsin is a prototype for membrane proteins called G-protein-coupled receptors (GPCRs) that are implicated in biological signaling, and constitute the targets of 30-50% of all known pharmaceuticals (J. P. Overington et al. (2006) Nat. Rev. Drug. Disc. 5, 993-996). Because the majority of pharmaceuticals target GPCRs, unraveling the mechanism GPCR function and activation is clearly of great interest for medicine and the society at large. Our approach uses solid-state NMR spectroscopy in combination with molecular dynamics simulations to uncover the molecular motions that underlie triggering of the visual process. We will discover how rhodopsin achieves the extreme high fidelity required for visual signaling. The methodological developments will set the foundation for future investigations of rhodopsin mutants in debilitating visual diseases, such as retinitis pigmentosa. Our robust and novel approach will give us important insights that are transferable to the broader class of GPCRs in human biology and pharmacology. Our studies will test new hypotheses about the ways that rhodopsin becomes activated by light, yielding the interaction with the signaling protein transducin. Human hereditary blindness can be understood through structural studies of rhodopsin. Extending the approach to other GPCRs can stimulate ligand-based drug discovery. More than a hundred mutants of human rhodopsin have been identified in patients afflicted with eye diseases, such as macular degeneration, congenital night blindness, and retinitis pigmentosa (A. Rattner et al. (1999) Ann. Rev. Gen. 33, 89-131). Losses of receptor-G protein binding can contribute to the dysfunctions of essential fatty deficiency, including losses in cognitive skills and learning, spatial and odor discrimination, and visual acuity (N. Salem et al. (2001) Lipids 36, 945-959). Furthermore, about 10% of individuals 65-75 years of age have macular degeneration, which increases to 30% for patients 75-85 years of age. Here the loss of central vision profoundly affects the inability to read texts or distinguish faces-for elderly people this can be devastating. Such non-communicable, age-related diseases are becoming increasingly prevalent in Western societies. They represent a societal burden that urgently must be addressed (National Institute on Aging, 2007 "Why Population Aging Matters"). The combination of our novel NMR methods with molecular dynamics computer simulations has a huge potential for biological insights of far-reaching significance (N. Leioatts et al. (2014) Biochemistry 53, 376−385). We will apply these technologies to the discovery of the dynamic structural changes in the native photosequence of rhodopsin. It will allow us for the first time to peer into the shape changes of rhodopsin that underlie the process of visual signaling. A product of this work will be a molecular dynamics movie showing rhodopsin activation in vivid detail, which will be disseminated to the public at large for educational purposes. Indeed, the GPCR field is entering its most exciting phase: molecular information together with modern spectroscopic approaches brings us closer to revealing and understanding the processes of biological signaling.