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      How Do Branched Detergents Stabilize GPCRs in Micelles?

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          Abstract

          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 A 2A receptor (WT-A 2AR), thermostabilized A 2AR (tA 2AR), and wild type β 2-adrenoceptor (β 2AR) in a variety of detergents (LMNG, DMNG, OGNG, and DDM). Analysis of molecular dynamics simulations of tA 2AR 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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          Most cited references31

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          CHARMM-GUI: a web-based graphical user interface for CHARMM.

          CHARMM is an academic research program used widely for macromolecular mechanics and dynamics with versatile analysis and manipulation tools of atomic coordinates and dynamics trajectories. CHARMM-GUI, http://www.charmm-gui.org, has been developed to provide a web-based graphical user interface to generate various input files and molecular systems to facilitate and standardize the usage of common and advanced simulation techniques in CHARMM. The web environment provides an ideal platform to build and validate a molecular model system in an interactive fashion such that, if a problem is found through visual inspection, one can go back to the previous setup and regenerate the whole system again. In this article, we describe the currently available functional modules of CHARMM-GUI Input Generator that form a basis for the advanced simulation techniques. Future directions of the CHARMM-GUI development project are also discussed briefly together with other features in the CHARMM-GUI website, such as Archive and Movie Gallery. 2008 Wiley Periodicals, Inc.
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            High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor.

            Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the beta2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.
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              Crystal structure of rhodopsin: A G protein-coupled receptor.

              Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) respond to a variety of different external stimuli and activate G proteins. GPCRs share many structural features, including a bundle of seven transmembrane alpha helices connected by six loops of varying lengths. We determined the structure of rhodopsin from diffraction data extending to 2.8 angstroms resolution. The highly organized structure in the extracellular region, including a conserved disulfide bridge, forms a basis for the arrangement of the seven-helix transmembrane motif. The ground-state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Interactions of the chromophore with a cluster of key residues determine the wavelength of the maximum absorption. Changes in these interactions among rhodopsins facilitate color discrimination. Identification of a set of residues that mediate interactions between the transmembrane helices and the cytoplasmic surface, where G-protein activation occurs, also suggests a possible structural change upon photoactivation.
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                Author and article information

                Journal
                Biochemistry
                Biochemistry
                bi
                bichaw
                Biochemistry
                American Chemical Society
                0006-2960
                1520-4995
                21 May 2020
                16 June 2020
                : 59
                : 23
                : 2125-2134
                Affiliations
                []Department of Computational and Quantitative Medicine, Beckman Research Institute of the City of Hope , 1500 East Duarte Road, Duarte, California 91010, United States
                []Heptares Therapeutics Ltd. , BioPark, Broadwater Road, Welwyn Garden City, AL7 3AX, U.K.
                [§ ]MRC Laboratory of Molecular Biology , Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, U.K.
                Author notes
                Article
                10.1021/acs.biochem.0c00183
                7302508
                32437610
                c3eb2685-476c-42c3-befa-fe6d7e1996a5
                Copyright © 2020 American Chemical Society

                This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.

                History
                : 04 March 2020
                : 21 May 2020
                Categories
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                Custom metadata
                bi0c00183
                bi0c00183

                Biochemistry
                Biochemistry

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