Structure and Activation Mechanism of the Visual Pigment Rhodopsin Rhodopsin is a specialized G protein-coupled receptor (GPCR) found in vertebrate rod cells. Absorption of light by its 11-cis retinal chromophore leads to rapid photochemical isomerization and receptor activation. Structural changes on the extracellular side of rhodopsin induced by the retinal isomerization are coupled to motion of the membrane-spanning helices to create a G-protein binding pocket on the intracellular side of the receptor. The existing crystal structures of rhodopsin provide a high-resolution framework to study in detail the role of specific residues and motifs in receptor activation. Because of the high conservation of many of the key residues involved activation of rhodopsin, the emerging model indicates that rather than being unique, the visual receptors provide a basis for understanding the common structural and dynamic elements in the class A GPCRs. The general experimental strategy is to use solid-state NMR spectroscopy in combination with mutational, optical and biochemical methods to target specific regions in the inactive and active states of the receptor. The goal is to understand in atomic detail the interplay between specific signature, group-conserved and subfamily-conserved motifs in the activation mechanism of rhodopsin and derive the basis of a working model for the activation of other GPCRs. Three specific aims address structure-function questions involving regions on the extracellular side of the receptor (Aim 1), within the transmembrane (TM) core (Aim 2) and on the intracellular side of the receptor (Aim 3). In Aim 1, we describe two hydrogen-bonding networks that tether extracellular loop 2 (EL2) to the ends of the TM helices H5-H7. We propose NMR measurements to quantify the displacement of EL2 upon activation and to establish how this displacement is coupled to helix motion. In Aim 2, we target the conserved stable core of rhodopsin composed of interlocking signature and group-conserved residues. We hypothesize that H6 rotates in the conversion to Meta I and then tilts outward upon deprotonation of the retinal Schiff base and associated motion of EL2. In Aim 3, we focus on the G-protein and its interactions with residues on the intracellular surface of Meta I and Meta II. The experiments target the structural transitions between inactive and active complexes of rhodopsin with G1 peptide or G-protein. In addition, our studies address how specific mutations lead to retinal diseases through constitutive activation, receptor misfolding or stabilization of non-functional receptor conformations. PUBLIC HEALTH RELEVANCE: Structure and Activation Mechanism of the Visual Pigment Rhodopsin Rhodopsin is the visual receptor in the retina responsible for converting light into a chemical signal. Two diseases - autosomal dominant retinitis pigmentosa and congenital stationary night blindness - are associated with mutations in the receptor. The proposed research will uncover the mechanism for light- activation of rhodopsin and how specific mutations lead to rhodopsin-linked diseases. Rhodopsin is also a prototypical member of the family of class A G protein-coupled receptors, which are the largest target for current pharmaceuticals. The proposed studies will provide a framework for drug development within this large family of receptors.