Conservation of Molecular Interactions Stabilizing Bovine and Mouse Rhodopsin

Abstract

Rhodopsin is the light receptor that initiates phototransduction in rod photoreceptor cells. The structure and function of rhodopsin is tightly linked to molecular interactions that stabilize and determine the receptor's functional state. Single-molecule force spectroscopy (SMFS) was used to localize and quantify molecular interactions that structurally stabilize bovine and mouse rhodopsin from native disc membranes of rod photoreceptor cells. The mechanical unfolding of bovine and mouse rhodopsin revealed nine major unfolding intermediates, each intermediate defining a structurally stable segment in the receptor. These stable structural segments had similar localization and occurrence in both bovine and mouse samples. For each structural segment, parameters describing their unfolding energy barrier were determined by dynamic SMFS. No major differences were observed between bovine and mouse rhodopsin thereby implying that the structures of both rhodopsins are largely stabilized by similar molecular interactions.Rhodopsin is a G protein-coupled receptor (GPCR)1 residing in rod outer segments (ROS) of photoreceptor cells, where it initiates phototransduction upon light-activation. Several crystal structures are now available for a handful of GPCRs (1-5). These structures highlight the conservation of the general architecture of GPCRs, which display seven transmembrane α-helices. Some of the mechanisms underlying receptor activation and function are likely conserved across members of this family of membrane proteins (4,6,7). Despite low amino acid sequence similarities, comparison of GPCR crystal structures reveals only relatively small deviations in the position of transmembrane α-helices (2,5,8). Yet those small differences are significant enough to facilitate the specific roles and functions of those †

Cholesterol increases kinetic, energetic, and mechanical stability of the human β2-adrenergic receptor

“…As theoretically predicted (46,47) and experimentally verified using membrane proteins (35,36,(48)(49)(50)(51), increasing the loading rate increases the unfolding forces. We observed a linear relationship between the most probable unfolding force and the logarithm of the loading rate for every interaction.…”

Structural, Energetic, and Mechanical Perturbations in Rhodopsin Mutant That Causes Congenital Stationary Night Blindness

“…Mouse Rod Outer Segment (ROS) Disc Membrane Preparation-ROS disc membranes were prepared from the eyes of mice as described (8). Wild-type rhodopsin was obtained from C57BL/6J mice from Jackson Laboratories (Bar Harbor, ME).…”

“…SMFS and DFS-SMFS was performed on ROS disc membranes prepared from wild-type and G90D mice similar to procedures reported previously (8), except for the following modifications. 10 l of washed ROS disc membranes was diluted in 200 l of Ringer's buffer.…”

“…Additionally, SMFS has revealed stabilizing effects on receptor structure promoted by zinc ions and post-translational modifications (6,7). More recently, SMFS and DFS have been used to determine the impact of amino acid sequence differences among rhodopsins from different vertebrate species (8). These studies revealed that despite a difference of 23 amino acid residues in the sequences of bovine and mouse rhodopsin, the network of molecular interactions in the homologous receptors are largely conserved and that both receptors maintain similar functional properties (8).…”

“…More recently, SMFS and DFS have been used to determine the impact of amino acid sequence differences among rhodopsins from different vertebrate species (8). These studies revealed that despite a difference of 23 amino acid residues in the sequences of bovine and mouse rhodopsin, the network of molecular interactions in the homologous receptors are largely conserved and that both receptors maintain similar functional properties (8). Thus, rhodopsin can accommodate certain amino acid substitutions with minimal structural and functional perturbations.…”