膜包裹的纳米颗粒上g蛋白信号通路的膜相关组分的重构

Michael J. Irwin; Xin Wang; Rick H. Cote

doi:10.21769/BioProtoc.4303

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Reconstitution of Membrane-associated Components of a G-protein Signaling Pathway on Membrane-coated Nanoparticles (Lipobeads)

膜包裹的纳米颗粒上g蛋白信号通路的膜相关组分的重构

MI Michael J. Irwin

XW Xin Wang

RC Rick H. Cote email

发布: 2022年01月20日第12卷第2期 DOI: 10.21769/BioProtoc.4303 浏览次数: 1823

评审: Khyati Hitesh ShahSaravanan KolandaiveluKathleen Boesze-Battaglia

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Abstract

G-protein coupled signaling pathways are organized into multi-protein complexes called signalosomes that are located within and on cellular membranes. We describe the use of silica nanoparticles coated with a unilamellar phospholipid bilayer (lipobeads) to reconstitute the activated photoreceptor G-protein α-subunit (Gtα*) with its cognate effector (phosphodiesterase-6; PDE6) for biochemical and structural studies of the activation mechanism regulating this GPCR signaling pathway. Lipobeads are prepared by resuspending dried-down phospholipid mixtures with monodisperse 70 nm silica particles, followed by extrusion through a 100 nm membrane filter. This uniform and supported liposomal preparation is easily sedimented, permitting the separation of soluble from membrane-associated proteins. Upon loading lipobeads with Gtα* and PDE6, we find that activation of PDE6 catalysis by Gtα* occurs much more efficiently than in the absence of membranes. Chemical cross-linking of membrane-confined proteins allows detection of changes in protein-protein interactions, resulting from G-protein activation of PDE6. The advantages of using lipobeads over partially purified membrane preparations or traditional liposomal preparations are generally applicable to the study of other membrane-confined signal transduction pathways.

Keywords: Signalosome (信号体)

Signal transduction (信号转导)

G-protein (G蛋白)

Peripheral membrane protein (外周膜蛋白)

Liposome (脂质体)

Protein-protein interactions (蛋白质-蛋白质相互作用)

Nanoparticle (纳米粒子)

Background

Supported phospholipid bilayers have been used for a variety of fundamental and applied applications, including membrane biophysics, biosensors, cell-cell interactions, screening assays of membrane proteins, and as drug-delivery vehicles (Ng et al., 2004; Troutier and Ladaviere, 2007; Chemburu et al., 2010; Crites et al., 2015; Schadauer et al., 2015). Microspheres consisting of silica or polymeric materials (e.g., hydrogels) have been used as a solid support for forming unilamellar phospholipid bilayers whose size, density, membrane curvature, and other biophysical properties can be optimized for their intended applications.

We have utilized 70 nm diameter silica particles coated with a unilamellar lipid bilayer (termed “lipobeads”; Figure 1A) to bind the prenylated photoreceptor phosphodiesterase (PDE6) holoenzyme (Figure 1B), as well as the acylated photoreceptor G-protein α-subunit (Gtα), for enzymological and structural studies of the Gtα*-PDE6 activation complex in a membrane-confined environment (Figure 1C) (Irwin et al., 2019). Activation of PDE6 by its cognate G-protein (transducin) occurs on the membrane surface in the light-harvesting outer segment of rod and cone photoreceptors upon photoactivation of rhodopsin (Cote et al., 2021). Studies have shown that the efficiency with which Gtα* can activate PDE6 is greatly enhanced when both proteins are tethered to either the photoreceptor membrane or to liposomal preparations (Melia et al., 2000). Reduction in the dimensionality of diffusional encounters between Gtα* and PDE6, in conjunction with high local protein concentrations, likely account for this phenomenon. The ability to reconstitute the proteins comprising GPCR signalosomes in vitro on well-defined lipobeads enables the integration of biochemical, biophysical, and structural studies to advance our mechanistic understanding of GPCR signaling pathways, in the context of their membrane environment.

Figure 1. Reconstitution of the Gtα*-PDE6 activation complex on lipobeads. A. Lipobead composed of a silica nanoparticle coated with a unilamellar phospholipid bilayer. B. PDE6 (purple) attached to lipobeads by their prenyl moieties. C. Addition of acylated Gtα* (red) to lipobeads with pre-bound PDE6 results in Gtα* binding and the formation of the Gtα*-PDE6 activation complex.

Materials and Reagents

Amicon Ultra-4 30 kDa or 50 kDa ultrafiltration units (Millipore-Sigma, catalog numbers: UFC803024 and UFC805024)
Phospholipids (Avanti Polar Lipids, Inc.)
1. DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt; Avanti Polar Lipids, catalog number: 890890C-200 mg) is supplied as 100 mg portions dissolved in chloroform at 25 mg/mL (FW 698.5; 35.8 mM).
2. DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, catalog number: 850375C-500 mg) is supplied as 100 mg portions dissolved in chloroform at 25 mg/mL (FW 786.1; 31.8 mM).
Monodisperse silica particles [70 nm diameter; 25 mg/mL aqueous suspension (0.11 μM; 7 × 10¹³ particles/mL); density = 2.0 g/cm³] are obtained from DiagNano (CD Bioparticles, catalog number: DNG-B004).
Purified PDE6 is prepared as described previously (Pentia et al., 2005) and stored at -20°C in PDE6 storage buffer.
Purified Gtα-GDP or purified Gtα*-GTPγS is prepared as described previously (Ting et al., 1993; Irwin et al., 2019) and stored at -20°C in Gtα storage buffer.
NuPAGE 4-12% Bis-Tris gel (Invitrogen, catalog number: NP0321BOX)
Bis(sulfosuccinimidyl) suberate (BS3) (Thermo Scientific, catalog number: 21580)
NaCl (Electron Microscopy Sciences, catalog number: EM-7760)
MgCl₂·6H₂O (Electron Microscopy Sciences, catalog number: EM-5980)
Dithiothreitol (Electron Microscopy Sciences, catalog number: EM-3860)
Glycerol (IBI Scientific, catalog number: IB15760)
Tris base (J.T. Baker, catalog number: JT4109)
AlCl₃·6H₂O (Millipore Sigma, catalog number: A0718)
NaF (J.T. Baker, catalog number: JT3689)
HEPES, hemisodium salt (MP Biomedicals, catalog number: IC15245180)
Guanosine-5’-diphosphate, sodium salt (Millipore Sigma, catalog number: G7127)
HNM buffer (see Recipes)
PDE6 storage buffer (see Recipes)
Gtα storage buffer (see Recipes)
745 µM AlCl₃in HNM buffer (see Recipes)
250 mM NaF in HNM buffer (see Recipes)
1 M MgCl₂ (see Recipes)
50 mM GDP (see Recipes)
10 mM BS3 (see Recipes)
0.5 mM BS3 (see Recipes)

Equipment

Eppendorf 5415D centrifuge
Vortex mixer (VWR, catalog number: 10153-838)
Avanti Mini Extruder Set (Avanti, catalog number: 610023)
1. 0.1 µm, 19 mm diameter polycarbonate membrane (Avanti, catalog number: 610005 or Whatman catalog number: 800309)
2. 10 mm filter supports (Avanti, catalog number: 610014)
3. 1,000 μL gas-tight syringe (Avanti, catalog number: 610017)

Procedure

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Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Irwin, M. J., Wang, X. and Cote, R. H. (2022). Reconstitution of Membrane-associated Components of a G-protein Signaling Pathway on Membrane-coated Nanoparticles (Lipobeads). Bio-protocol 12(2): e4303. DOI: 10.21769/BioProtoc.4303.
Irwin, M. J., Gupta, R., Gao, X. Z., Cahill, K. B., Chu, F. and Cote, R. H. (2019). The molecular architecture of photoreceptor phosphodiesterase 6 (PDE6) with activated G protein elucidates the mechanism of visual excitation. J Biol Chem 294(51): 19486-19497.