Structure and function in rhodopsin: Kinetic studies of retinal binding to purified opsin mutants in defined phospholipid–detergent mixtures serve as probes of the retinal binding pocket

Abstract

In the current standard procedure for preparation of mammalian rhodopsin mutants, transfected COS-1 cells expressing the mutant opsin genes are treated with 5 M 11-cis-retinal before detergent solubilization for purification. We found that binding of 11-cis-retinal to opsin mutants with single amino acid changes at Trp-265 (W265F,Y,A) and a retinitis pigmentosa mutant (A164V) was far from complete and required much higher concentrations of 11-cis-retinal. By isolation of the expressed opsins in a stable form, kinetic studies of retinal binding to the opsins in vitro have been carried out by using defined phospholipid-detergent mixtures. The results show wide variation in the rates of 11-cisretinal binding. Thus, the in vitro reconstitution procedure serves as a probe of the retinal-binding pocket in the opsins. Further, a method is described for purification and characterization of the rhodopsin mutants after retinal binding to the opsins in vitro.

Opsin Stability and Folding: The Role of Cys185 and Abnormal Disulfide Bond Formation in the Intradiscal Domain

“…The apoprotein opsin is highly unstable in detergent solution, especially in the absence of any lipids, and previous work has identified denatured states of opsin. 6 Successful attempts to stabilise opsin in vitro have focused both on developing stabilising lipid/detergent systems 7 and on mutations that pin the intradiscal domain of the protein together. 8 Research into the intramolecular interactions of the rhodopsin structure that affect folding, function and stability has also greatly improved our understanding of GPCRs, predominately through rational mutation and comparison to disease-associated mutants of opsin.…”

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

“…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 . Phospholipid bicelles have been shown to improve the stability of Rho [26][27][28] , and for this reason we have studied two Rho mutants, G90V and N55K, and compared their properties in DM detergent and in DMPC/DHPC bicelles ( Figure 1).…”

G Protein-Coupled Receptors as Direct Targets of Inhaled Anesthetics

“…Trp265 is known to interact with retinal (Lin and Sakmar, 1996;Kochendoerfer et al, 1997;Palczewski et al, 2000) and to play an important role in rhodopsin regeneration (Reeves et al, 1999). To illustrate the relationship between the retinal cavity formed in the interhelical space of rhodopsin and the photolabeled residues, the Gravitational Radiation Analysis and Simulation Package (GRASP; http://www.lsc-group.phys.…”