Thermal Destabilization of Rhodopsin and Opsin by Proteolytic Cleavage in Bovine Rod Outer Segment Disk Membranes

Abstract

The G-protein coupled receptor, rhodopsin, consists of seven transmembrane helices which are buried in the lipid bilayer and are connected by loop domains extending out of the hydrophobic core. The thermal stability of rhodopsin and its bleached form, opsin, was investigated using differential scanning calorimetry (DSC). The thermal transitions were asymmetric, and the temperatures of the thermal transitions were scan rate dependent. This dependence exhibited characteristics of a two-state irreversible denaturation in which intermediate states rapidly proceed to the final irreversible state. These studies suggest that the denaturation of both rhodopsin and opsin is kinetically controlled. The denaturation of the intact protein was compared to three proteolytically cleaved forms of the protein. Trypsin removed nine residues of the carboxyl terminus, papain removed 28 residues of the carboxyl terminus and a portion of the third cytoplasmic loop, and chymotrypsin cleaved cytoplasmic loops 2 and 3. In each of these cases the fragments remained associated as a complex in the membrane. DSC studies were carried out on each of the fragmented proteins. In all of the samples the scan rate dependence of the Tm indicated that the transition was kinetically controlled. Trypsin-proteolyzed protein differed little from the intact protein. However, the activation energy for denaturation was decreased when cytoplasmic loop 3 was cleaved by papain or chymotrypsin. This was observed for both bleached and unbleached samples. In the presence of the chromophore, 11-cis-retinal, the noncovalent interactions among the proteolytic fragments produced by papain and chymotrypsin cleavage were sufficiently strong such that each of the complexes denatured as a unit. Upon bleaching, the papain fragments exhibited a single thermal transition. However, after bleaching, the chymotrypsin fragments exhibited two calorimetric transitions. These data suggest that the loops of rhodopsin exert a stabilizing effect on the protein.

Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

“…Recent studies (43,71,72) have begun to address carefully the thermodynamics of rhodopsin protein stability. In the present work, our conclusions about rhodopsin thermal stability are primarily based on monitoring the stability of the retinal Schiff base linkage, which we measure as the loss of absorbance at 500 nm.…”

The role of cholesterol in rod outer segment membranes

“…The temperature of rhodopsin thermal denaturation as determined by DSC is scan rate dependent. That is, it is sensitive to the rate of heating and is therefore a kinetic process [83]. (For this reason, the transition temperatures for rhodopsin and opsin provided above are given as approximate values.)…”

Retinoids Assist the Cellular Folding of the Autosomal Dominant Retinitis Pigmentosa Opsin Mutant P23H

“…Pharmacological chaperones appear to act mainly in the endoplasmic reticulum (ER), releasing the proteins they target from the ER quality control machinery and increasing their flux to other cellular compartments (10). Because opsin stability in vitro is increased by the binding of 11-cis-retinal and other retinoids (11)(12)(13), these compounds may act as pharmacological chaperones and increase the yield of Class II and III rhodopsin mutants. In support of this idea, the level of T17M rhodopsin (a Class III mutant (8)) regenerated from cell membranes increased 10-fold when the protein was expressed in the presence of 11-cis-retinal (14).…”