Salt Effects on the Conformational Stability of the Visual G-Protein-Coupled Receptor Rhodopsin

Abstract

Membrane protein stability is a key parameter with important physiological and practical implications. Inorganic salts affect protein stability, but the mechanisms of their interactions with membrane proteins are not completely understood. We have undertaken the study of a prototypical G-protein-coupled receptor, the α-helical membrane protein rhodopsin from vertebrate retina, and explored the effects of inorganic salts on the thermal decay properties of both its inactive and photoactivated states. Under high salt concentrations, rhodopsin significantly increased its activation enthalpy change for thermal bleaching, whereas acid denaturation affected the formation of a denatured loose-bundle state for both the active and inactive conformations. This behavior seems to correlate with changes in protonated Schiff-base hydrolysis. However, chromophore regeneration with the 11-cis-retinal chromophore and MetarhodopsinII decay kinetics were slower only in the presence of sodium chloride, suggesting that in this case, the underlying phenomenon may be linked to the activation of rhodopsin and the retinal release processes. Furthermore, the melting temperature, determined by means of circular dichroism and differential scanning calorimetry measurements, was increased in the presence of high salt concentrations. The observed effects on rhodopsin could indicate that salts favor electrostatic interactions in the retinal binding pocket and indirectly favor hydrophobic interactions at the membrane protein receptor core. These effects can be exploited in applications where the stability of membrane proteins in solution is highly desirable.

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

“…Rhodopsin purification: Rod outer segment (ROS) membranes were prepared under dim red light from frozen bovine retinas by using a sucrose-gradient method as described previously. [21] ROS membranes were suspended in potassium phosphate (70 mm, pH 6.9), MgCl 2 (1 mm) and EDTA (0.1 mm), and pelleted by centrifugation. The membrane pellets were resuspended in Tris-HCl (5 mm, pH 7.5) and MgCl 2 (0.5 mm).…”

“…Functional and structural studies require purified rhodopsin, which is usually studied in the presence of the mild neutral detergent n-dodecyl-b-d-maltoside (DM). [9] Several factors have been studied over the past two decades in order to increase the stability of rhodopsin: mutations, [10] salts [11] and nanodiscs (soluble nanoscale particles of lipid bilayer surrounded by two circular lipoproteins), [12] among others. The lipid environment is known to affect the stability and molecular arrangement of the transmembrane domain of rhodopsin because of very specific interactions between the rhodopsin hydrophobic transmembrane helices and the lipid bilayer.…”

Differential Light-induced Responses in Sectorial Inherited Retinal Degeneration

“…Thermal Bleaching Assay-Rh thermal stability was monitored as described previously (39). Briefly, Rh bleaching rates, in the dark, were obtained by monitoring the decrease of absorbance at max of the visible spectral band as a function of time at 48°C.…”

Probing the remarkable thermal kinetics of visual rhodopsin with E181Q and S186A mutants

“…1 Since thermal stability is key to rhodopsin's function of dim-light vision, thermal properties of rhodopsin have long been a subject of interest. [2][3][4][5][6][7] In order to better understand the factors responsible for rhodopsin's thermal stability, we previously carried out measurements of the rate of decay as a function of temperature for wild type (WT) bovine rhodopsin over the temperature range 37.0 -64.6°C. As we reported, 8 the resulting Arrhenius plot exhibits a distinct elbow at about 47°C.…”