Opsin Stability and Folding: Modulation by Phospholipid Bicelles

McKibbin, Craig; Farmer, Nicola A; Jeans, Chris; Reeves, Philip J.; Khorana, H. G.; Wallace, B.A.; Edwards, Patricia C.; Villa, Claudio; Booth, Paula J.

doi:10.1016/j.jmb.2007.10.018

Cited by 74 publications

(92 citation statements)

References 46 publications

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“…Here, we purified the newly reported unstable RP mutants, G90V and N55K, in DMPC/DHPC bicelles. This bicelles system has been proposed to support folding and thermal stability of Rho and opsin 26 . Such a system has been used in solid-state NMR studies of membrane-associated molecules and its use extended to non-NMR-based biophysical studies of transmembrane proteins 27,[47][48][49] .…”

Section: Discussionmentioning

confidence: 99%

“…Final 2% (w/v) DMPC/DHPC (1:1) mixtures were mixed briefly, heated to 42°C for 10 min, and then left to stir at room temperature for 1 h until the mixtures were clear (bicelles buffer). All bicelles solutions were used within 36 h from preparation 26 .…”

Section: Bicelles Preparationmentioning

confidence: 99%

“…DMPC/DHPC bicelles were previously used to increase the thermal stability of purified Rho obtained from ROS membranes 20,26,37 . Here, ROS Rho, solubilized in DM (Rho DM ), was purified and used merely as a standard control.…”

Section: Dmpc/dhpc Stabilization Of Ros Rhomentioning

confidence: 99%

“…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). G90V (causing RP phenotype) and N55K (associated with sector RP) show structural instability in the dark and thermal sensitivity 21,29,30 .…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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Mutations in the visual photoreceptor rhodopsin are the cause of the retinal degenerative disease retinitis pigmentosa. Some naturally occurring mutations can lead to protein conformational instability. Two such mutations, N55K and G90V, in the first and second transmembrane helices of the receptor, have been associated with sector and classical retinitis pigmentosa, respectively, and showed enhanced thermal sensitivity. We have carefully analyzed the effect of phospholipid bicelles on the stability and ligand binding properties of these two mutants and compared it with those of the detergent-solubilized samples. We have used a phospholipid bilayer consisting of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2dihexanoyl-sn-glycero-3-phosphocholine (DHPC). We find that DMPC/DHPC bicelles dramatically increase the thermal stability of the rhodopsin mutants G90V and N55K. The chromophore stability and regeneration of the mutants were also increased in bicelles when compared to their behavior in a dodecyl maltoside detergent solution. The retinal release process was slowed in bicelles, and chromophore entry, after illumination, was improved for the G90V mutant but not for N55K. Furthermore, fluorescence spectroscopy measurements showed that bicelles allowed more exogenous retinal binding to the photoactivated G90V mutant than in a detergent solution. In contrast, N55K could not reposition any chromophore either in the detergent or in bicelles. The results demonstrate that DMPC/DHPC bicelles can counteract the destabilizing effect of the disease-causing mutations and can modulate the structural changes in the ensuing receptor photoactivation in a distinct specific manner for different retinitis pigmentosa mutant phenotypes.

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Section: Discussionmentioning

confidence: 99%

Section: Bicelles Preparationmentioning

confidence: 99%

Section: Dmpc/dhpc Stabilization Of Ros Rhomentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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“…Bicelles are bilayer-like discs that form in a mixture of lipid and certain amphiphiles. The average size of the disk can be varied by changing the ratio of lipid to amphiphile-the so-called q ratio (19). We used steric trapping to measure bR stability at q ratios of 1, 2, 2.5, and 3.…”

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confidence: 99%

Measuring membrane protein stability under native conditions

Chang

Bowie

2013

Proc. Natl. Acad. Sci. U.S.A.

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The thermodynamic stability of proteins is typically measured at high denaturant concentrations and then extrapolated back to zero denaturant conditions to obtain unfolding free energies under native conditions. For membrane proteins, the extrapolations are fraught with considerable uncertainty as the denaturants may have complex effects on the membrane or micellar structure. We therefore sought to measure stability under native conditions, using a method that does not perturb the properties of the membrane or membrane mimetics. We use a technique called steric trapping to measure the thermodynamic stability of bacteriorhodopsin in bicelles and micelles. We find that bacteriorhodopsin has a high thermodynamic stability, with an unfolding free energy of ∼11 kcal/mol in dimyristoyl phosphatidylcholine bicelles. Nevertheless, the stability is much lower than predicted by extrapolation of measurements made at high denaturant concentrations. We investigated the discrepancy and found that unfolding free energy is not linear with denaturant concentration. Apparently, long extrapolations of helical membrane protein unfolding free energies must be treated with caution. Steric trapping, however, provides a method for making these measurements.membrane protein folding | steric trap M ethods to measure the thermodynamic stability of membrane proteins have largely followed methods developed for soluble protein folding (1). The fraction unfolded is first measured as a function of denaturant concentration (urea, guanidine-HCl, etc.), which in turn provides the unfolding free energy (ΔG U ) as a function of denaturant. The fraction unfolded, however, can be accurately measured only at high denaturant concentration, where the amount of unfolded protein is large enough-the so-called transition zone. Thus, obtaining a measure of the unfolding free energy in the absence of denaturant requires extrapolation from the transition zone. For chemical denaturation, the unfolding free energy is typically linearly dependent on the denaturant concentration in the transition zone, allowing a linear extrapolation back to zero denaturant. However, although there is now considerable experimental and theoretical validation of this approach for soluble proteins (2-4), the validity of these extrapolations is not clear for measuring stability of membrane proteins.Since the observation of Braiman et al. that bacteriorhodopsin (bR) can be refolded from an SDS-denatured state (5), SDS has been commonly used to study the folding of helical membrane proteins. The Booth laboratory has pioneered and extensively studied the refolding kinetics of bR from an SDS-denatured state (6-8). We introduced SDS unfolding to measure the thermodynamic stability of the membrane enzyme diacylglycerol kinase (9) and a similar approach can be used to measure bR thermodynamic stability (10, 11). bR contains a covalently bound retinal chromophore that complicates unfolding analysis because it can slowly hydrolyze off in the SDS unfolded protein (12). We and others originally ...

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An experimental comparison of human and bovine rhodopsin provides insight into the molecular basis of retinal disease

et al. 2017

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Rhodopsin is the visual pigment that mediates dim-light vision in vertebrates and is a model system for the study of retinal disease. The majority of rhodopsin experiments are performed using bovine rhodopsin; however, recent evidence suggests that significant functional differences exist among mammalian rhodopsins. In this study, we identify differences in both thermal decay and light-activated retinal release rates between bovine and human rhodopsin and perform mutagenesis studies to highlight two clusters of substitutions that contribute to these differences. We also demonstrate that the retinitis pigmentosa-associated mutation G51A behaves differently in human rhodopsin compared to bovine rhodopsin and determine that the thermal decay rate of an ancestrally reconstructed mammalian rhodopsin displays an intermediate phenotype compared to the two extant pigments.

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Opsin Stability and Folding: Modulation by Phospholipid Bicelles

Cited by 74 publications

References 46 publications

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

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

Measuring membrane protein stability under native conditions

An experimental comparison of human and bovine rhodopsin provides insight into the molecular basis of retinal disease

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