Improved Conformational Stability of the Visual G Protein‐Coupled Receptor Rhodopsin by Specific Interaction with Docosahexaenoic Acid Phospholipid

Abstract

Rhodopsin is the photoreceptor located in the rod cells of the retina. It has seven transmembrane helices and is a prototypic member of the G protein-coupled receptor superfamily. The structures and functions of these receptors are clearly affected by the lipid composition of the cell membrane, and their study in a purified recombinant form is usually performed in detergent solution. There is a need to study these receptors in a physiologically relevant environment because the lipid environment is known to have an important effect on their function. In this work, rhodopsin reconstituted in docosahexaenoic acid (DHA) liposomes is shown to have more thermal stability than when it is solubilised with the neutral detergent dodecyl maltoside. Moreover, the specific interaction between rhodopsin and DHA was followed by means of Langmuir experiments with insertion of rhodopsin into lipid monolayers; this showed high affinity for the lipid-receptor interaction. Furthermore, fluorescence spectroscopy measurements indicate that the conformation of opsin obtained after photobleaching is preserved in DHA-containing liposomes, thereby allowing retinal to re-enter the binding pocket even long after bleaching. Overall, our results demonstrate that liposomes of this specific lipid provide a more stable environment for ground-state inactive rhodopsin in the dark, than dodecyl maltoside detergent, and that this lipid can also preserve the native correctly folded ligand-free opsin conformation obtained after illumination. This strategy will be used in further studies on mutations of rhodopsin associated with congenital retinopathies.

Phospholipid Bicelles Improve the Conformational Stability of Rhodopsin Mutants Associated with Retinitis Pigmentosa

“…Some of these mutations can cause structural instability and misfolding 42 . The stability of visual photoreceptors, and other GPCRs, has been extensively studied, and several experimental factors, like temperature, pH, salts, detergents, and lipids have been shown to affect the stability and function of these receptors 8,22,28,[43][44][45][46] . In spite of this, only limited information is available concerning the molecular causes of the structural instability of mutations in Rho associated with RP.…”

“…However, membrane proteins often show poor 5 conformational stability, and can lose activity or even denature in detergent micelles 22,23 . Membrane-like environments help maintaining the proper structure and biochemical function of membrane proteins and their mutants 24,25 .…”

Bioorthogonal Fluorescent Labeling of Functional G‐Protein‐Coupled Receptors

“…[17b, 54] However, this approach relies intrinsic Trp residues that are sensitive to ligand-binding or ligand-induced conformational changes and thus has significant limitations. First, due to the low quantum yield of Trp, studying intrinsic fluorescence requires up to 10 times more protein (>10 µg or 0.5 µ m ) [54] than needed when the receptor is labeled with extrinsic fluorophores with higher quantum yields (<1 µg/50 n m in our study). In addition, native Trp residues are not necessarily well positioned to serve as active-site probes.…”

Differential Light-induced Responses in Sectorial Inherited Retinal Degeneration

“…6B). To improve protein stability, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine lipid was used, previously shown to increase Rh stability (46), but it did not help to stabilize N55K, when compared with Rh from rod outer segments (data not shown).…”