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Detergents stabilize the conformation of phosphodiesterase 6.

ABSTRACT: Membrane-bound phosphodiesterase 6 (PDE6) plays an important role in visual signal transduction by regulating cGMP levels in rod photoreceptor cells. Our understanding of PDE6 catalysis and structure suffers from inadequate characterization of the ? and ? subunit catalytic core, interactions of the core with two intrinsically disordered, proteolysis-prone inhibitory PDE? (P?) subunits, and binding of two types of isoprenyl-binding protein ?, called PrBP/?, to the isoprenylated C-termini of the catalytic core. Structural studies of native PDE6 have been also been hampered by the lack of a heterologous expression system for the holoenzyme. In this work, we purified PDE6 in the presence of PrBP/? and screened for additives and detergents that selectively suppress PDE6 basal activity while sparing that of the trypsin-activated enzyme. Some detergents removed PrBP/? from the PDE complex, separating it from the holoenzyme after PDE6 purification. Additionally, selected detergents also significantly reduced the level of dissociation of PDE6 subunits, increasing their homogeneity and stabilizing the holoenzyme by substituting for its native membrane environment.

SUBMITTER: Baker BY

PROVIDER: S-EPMC3215081 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Detergents stabilize the conformation of phosphodiesterase 6.

Baker Bo Y BY Palczewski Krzysztof K

Biochemistry 20111014 44

Membrane-bound phosphodiesterase 6 (PDE6) plays an important role in visual signal transduction by regulating cGMP levels in rod photoreceptor cells. Our understanding of PDE6 catalysis and structure suffers from inadequate characterization of the α and β subunit catalytic core, interactions of the core with two intrinsically disordered, proteolysis-prone inhibitory PDEγ (Pγ) subunits, and binding of two types of isoprenyl-binding protein δ, called PrBP/δ, to the isoprenylated C-termini of the c ...[more]

PMID: 21978030

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Project description:The structural and functional properties of G protein-coupled receptors (GPCRs) are often studied in a detergent micellar environment, but many GPCRs tend to denature or aggregate in short alkyl chain detergents. In our previous work [Lee, S., et al. (2016) J. Am. Chem. Soc. 138, 15425-15433], we showed that GPCRs in alkyl glucosides were highly dynamic, resulting in the penetration of detergent molecules between transmembrane α-helices, which is the initial step in receptor denaturation. Although this was not observed for GPCRs in dodecyl maltoside (DDM, also known as lauryl maltoside), even this detergent is not mild enough to preserve the integrity of many GPCRs during purification. Lauryl maltose neopentylglycol (LMNG) detergents have been found to have significant advantages for purifying GPCRs in a native state as they impart more stability to the receptor than DDM. To gain insights into how they stabilize GPCRs, we used atomistic molecular dynamics simulations of wild type adenosine A2A receptor (WT-A2AR), thermostabilized A2AR (tA2AR), and wild type β2-adrenoceptor (β2AR) in a variety of detergents (LMNG, DMNG, OGNG, and DDM). Analysis of molecular dynamics simulations of tA2AR in LMNG, DMNG, and OGNG showed that this series of detergents exhibited behavior very similar to that of an analogous series of detergents DDM, DM, and OG in our previous study. However, there was a striking difference upon comparison of the behavior of LMNG to that of DDM. LMNG showed considerably less motion than DDM, which resulted in the enhanced density of the aliphatic chains around the hydrophobic regions of the receptor and considerably more hydrogen bond formation between the head groups. This contributed to enhanced interaction energies between both detergent molecules and between the receptor and detergent, explaining the enhanced stability of GPCRs purified in this detergent. Branched detergents occlude between transmembrane helices and reduce their flexibility. Our results provide a rational foundation to develop detergent variants for stabilizing membrane proteins.

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Detergents stabilize the conformation of phosphodiesterase 6.

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Detergents stabilize the conformation of phosphodiesterase 6.

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