Determination of Quaternary Structure of Rhodopsin at Room and Body Temperature using Spectral FRET

Abstract

Rhodopsin is a prototypical G-protein coupled receptor that initiates photo-transduction in the retina of the eye. Like many other G protein-coupled receptors, rhodopsin appears to form oligomers in the membrane. The notion that rhodopsin forms oligomers in its native membrane, however, is still quite controversial. The clearest demonstration that rhodopsin forms oligomers comes from atomic force microscopy images displaying large arrays of rhodopsin monomers. It has been speculated, however, that oligomers observed in these images are due to phase separation occurring in membranes at temperatures below 37 C. In the work presented here, we explored in detail the oligomeric status of rhodopsin and its dependency on temperatures using spectrally resolved Resonance Energy Transfer (RET) in Chinese hamster ovary cells. As a control, we also investigated a G188R mutant rhodopsin. This mutation causes misfolding of rhodopsin and leads to the retinal degenerative disorder retinitis pigmentosa. Our results suggest that the quaternary structure of wild-type rhodopsin is vastly different compared to that of the misfolded mutant rhodopsin.