Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor

Chung, Ka Young; Kim, Tae Hun; Manglik, Aashish; Alvares, Rohan D. A.; Kobilka, Brian K.; Prosser, R. Scott

doi:10.1074/jbc.m112.406371

Cited by 96 publications

(88 citation statements)

References 36 publications

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“…Our data are in contrast with interpretations of 19 F-NMR data on β 2 AR labeled at C265 in ternary complexes with ligands and nanobodies (Chung et al, 2012; Kim et al, 2013; Staus et al, 2016), which were analyzed as showing evidence for hidden signals of additional states. These apparent discrepancies may be due to the use of chemically different 19 F-NMR probes and different detergents.…”

Section: Discussioncontrasting

confidence: 99%

“…Such data provide deepened insight into the mechanisms of GPCR signal transduction, as described in detail, for example, in studies of the β 2 AR (Bokoch et al, 2010; Chung et al, 2012; Didenko et al, 2013; Horst et al, 2012, 2013; Kim et al, 2013; Kofuku et al, 2014; Liu et al, 2012a; Nygaard et al, 2013; Sounier et al, 2015), the μ-opioid receptor (Okude et al, 2015; Sounier et al, 2015), the turkey β 1 -adrenergic receptor (Isogai et al, 2016), and the A 2A AR (Ye et al, 2016). Extension of such studies to β 2 AR-T4L seemed to be of special interest, considering the extensive database of crystal structures determined with the ICL3 fusion approach (Table 1).…”

Section: Introductionmentioning

confidence: 98%

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β 2 -Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop

et al. 2016

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SUMMARY Fluorine-19 nuclear magnetic resonance (NMR) was used to study conformational equilibria at the intracellular tips of helices VI and VII in a variant β2-adrenergic receptor (β2AR) containing T4-lysozyme fused into the third intracellular loop (β2AR-T4L), a G protein-coupled receptor (GPCR) modification widely used in crystal structure determination. G-protein signaling at helix VI showed nearly complete population of an active-like state for all ligand efficacies in the absence of an intracellular protein. For arrestin signaling at helix VII, a native-like equilibrium was observed, except for complexes with ligands devoid of a hydrophobic moiety at the ethanolamine end. These data confirm that response of G-protein and arrestin signaling to ligand efficacy is not coupled, and presents evidence for long-range effects between fusion protein and orthosteric binding cavity, which are suppressed by voluminous bound ligands. Solution NMR thus provides complementary information, which should be considered in functional interpretations of GPCR crystal structures obtained with ICL3 fusions.

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Section: Discussioncontrasting

confidence: 99%

Section: Introductionmentioning

confidence: 98%

β 2 -Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop

et al. 2016

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“…35 It has been also reported that the muscarinic acetylcholine receptor requires cholesterol or its derivatives for protein stability and that MNG-3 stabilizes the receptor without cholesterol derivatives. 36 In addition, the off-rate of MNG-3 from the G protein-coupled beta(2)-adrenoreceptor has been shown to be four orders of magnitude lower than that of DDM, 37 clearly demonstrating that MNG-3 binds the membrane protein tightly.…”

Section: Discussionmentioning

confidence: 99%

Effect of Detergents on Galactoside Binding by Melibiose Permeases

et al. 2015

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The effect of various detergents on the stability and function of melibiose permeases of Escherichia coli (MelBEc) or Salmonella typhimurium (MelBSt) were studied. In n-dodecyl-β-d-maltoside (DDM) or n-undecyl-β-d-maltoside (UDM), WT MelBSt binds melibiose with an affinity similar to that in the membrane. However, with WT MelBEc or MelBSt mutants (Arg141→Cys, Arg295→Cys or Arg363→Cys), galactoside binding is not detected in these detergents, but binding to the phosphotransferase protein IIAGlc is maintained. In the amphiphiles lauryl maltose neopentyl glycol (MNG-3) or glyco-diosgenin (GDN), galactoside binding with all the MelB proteins is observed, with slightly reduced affinities. MelBSt is more thermostable than MelBEc, and the thermostability of either MelB is largely increased in MNG-3 or GDN. Therefore, the functional defect with DDM or UDM likely results from relative instability of the sensitive MelB proteins, and stability, as well as galactoside binding, is retained in MNG-3 or GDN. Furthermore, isothermal titration calorimetry of melibiose binding with MelBSt shows that the favorable entropic contribution to the binding free energy is decreased in MNG-3, indicating that the conformational dynamics of MelB is restricted in this detergent.

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“…Absence of free detergent micelles usually leads to the rapid desorption of detergent from the protein-detergent complex, resulting in rapid aggregation of IMPs (Rasmussen et al, 2011). However, LMNG exhibits an extremely slow off-rate, four orders of magnitude lower than that of commonly used dodecyl maltoside (DDM) (Chung et al, 2012). The high stability of LMNG detergent micelles (Figure S1A) precludes detergent removal by conventional methods such as dialysis or BioBeads adsorption, and the extremely low CMC of LMNG renders simple dilution unfeasible.…”

Section: Introductionmentioning

confidence: 98%

GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM

et al. 2015

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We developed a method, named GraDeR, which substantially improves the preparation of membrane protein complexes for structure determination by single-particle cryo-electron microscopy (cryo-EM). In GraDeR, glycerol gradient centrifugation is used for the mild removal of free detergent monomers and micelles from lauryl maltose-neopentyl glycol detergent stabilized membrane complexes, resulting in monodisperse and stable complexes to which standard processes for water-soluble complexes can be applied. We demonstrate the applicability of the method on three different membrane complexes, including the mammalian FoF1 ATP synthase. For this highly dynamic and fragile rotary motor, we show that GraDeR allows visualizing the asymmetry of the F1 domain, which matches the ground state structure of the isolated domain. Therefore, the present cryo-EM structure of FoF1 ATP synthase provides direct structural evidence for Boyer's binding change mechanism in the context of the intact enzyme.

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Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor

Cited by 96 publications

References 36 publications

β 2 -Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop

β 2 -Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop

Effect of Detergents on Galactoside Binding by Melibiose Permeases

GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM

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