Unusual mode of dimerization of retinitis pigmentosa-associated F220C rhodopsin

Abstract

Mutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wildtype and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.

CD19 CAR antigen engagement mechanisms and affinity tuning

“…MM-GBSA free-energy calculations were carried out on 20- to 150-ns time intervals of the all-atom trajectories, with the same settings as described previously ( 68 ). Briefly, the dielectric constant inside the protein was set to 2, and the accurate Generalized Born Molecular Volume II (GBMVII) method ( 69 ) was used to calculate the polar solvation free energies.…”

A single-molecule method for measuring fluorophore labeling yields for the study of membrane protein oligomerization in membranes

“…Indeed, for some proteins, oligomerization has been linked to regulatory impact on protein function (4), and introduces the idea of membrane proteins participating in dynamic association reactions that may be tuned by physiological factors (5,6).…”

“…Many of the proteins that adopt parallel pathway assemblies form as dimers, trimers, tetramers and beyond, raising the question as to why such complexes are evolutionarily favored. Indeed, for some proteins, oligomerization has been linked to regulatory impact on protein function (4), and introduces the idea of membrane proteins participating in dynamic association reactions that may be tuned by physiological factors (5,6).…”