Thermal destabilization of rhodopsin and opsin by proteolytic cleavage in bovine rod outer segment disk membranes

The G-protein coupled receptor, rhodopsin, consists of seven transmembrane helices which are buried in the lipid bilayer and are connected by loop dom ains extending out of the hydrophobic core. The thermal stability of rhodop sin and its bleached form, opsin, was investigated using differential scann ing calorimetry (DSC). The thermal transitions were asymmetric, and the tem peratures of the thermal transitions were scan rate dependent. This depende nce exhibited characteristics of a two-state irreversible denaturation in w hich intermediate states rapidly proceed to the final irreversible state. T hese studies suggest that the denaturation of both rhodopsin and opsin is k inetically 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 carbo xyl terminus and a portion of the third cytoplasmic loop, and chymotrypsin cleaved cytoplasmic loops 2 and 3. In each of these cases the fragments rem ained 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 de pendence of the T-m indicated that the transition was kinetically controlle d. Trypsin-proteolyzed protein differed little from the intact protein. How ever, the activation energy for denaturation was decreased when cytoplasmic loop 3 was cleaved by papain or chymotrypsin. This was observed for both b leached and unbleached samples. In the presence of the chromophore, 11-cis- retinal, the noncovalent interactions among the proteolytic fragments produ ced by papain and chymotrypsin cleavage were sufficiently strong such that each of the complexes denatured as a unit. Upon bleaching, the papain fragm ents exhibited a single thermal transition. However, after bleaching, the c hymotrypsin fragments exhibited two calorimetric transitions. These data su ggest that the loops of rhodopsin exert a stabilizing effect on the protein .