A class-A GPCR solubilized under high hydrostatic pressure retains its ligand binding ability
Yukie Katayama1, Tatsuya Suzuki1, Tatsuki Ebisawa1
1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Biochimica Et Biophysica Acta
|June 26, 2016
Summary
High hydrostatic pressure (HHP) enhances the solubilization of silkmoth pheromone biosynthesis-activating neuropeptide receptor (PBANR). This method preserves PBANR-EGFP ligand binding, unlike traditional solubilization methods.
Area of Science:
- Biochemistry
- Structural Biology
- Membrane Protein Research
Background:
- G protein-coupled receptors (GPCRs) are crucial drug targets.
- Solubilizing membrane proteins like PBANR for study is challenging.
- Preserving receptor function during solubilization is essential for research.
Purpose of the Study:
- To investigate the efficacy of high hydrostatic pressure (HHP) in solubilizing the silkmoth PBANR.
- To assess the impact of HHP on PBANR's ligand-binding ability.
- To compare HHP-assisted solubilization with conventional methods.
Main Methods:
- Expression of silkmoth pheromone biosynthesis-activating neuropeptide receptor (PBANR) as a PBANR-EGFP fusion protein in insect cells.
- Treatment of membrane fractions with high hydrostatic pressure (200 MPa) at room temperature.
- Solubilization using varying concentrations of n-dodecyl-β-D-maltopyranoside (DDM) with and without HHP.
- Analysis of solubilization yield and ligand-binding ability using fluorescence-detection size-exclusion chromatography.
Main Results:
- HHP treatment (200 MPa, 1h) with 0.6% DDM increased PBANR-EGFP solubilization yield by approximately 1.5-fold.
- PBANR-EGFP solubilized using HHP (1.0% DDM, 6h) retained its ligand-binding capacity.
- Conventional solubilization without HHP led to denaturation of PBANR-EGFP.
Conclusions:
- HHP is an effective method for solubilizing class-A GPCRs like PBANR.
- HHP-mediated solubilization preserves the functional integrity of PBANR-EGFP.
- This technique offers a promising alternative for studying membrane proteins that are prone to denaturation.
Keywords:
G protein-coupled receptorHigh hydrostatic pressureLigand binding abilityPheromone biosynthesis-activating neuropeptide receptorSolubilizationMore Related Videos
Related Concept Videos
01:12GPCR Desensitization
8.6K
G protein-coupled receptor (GPCR) signaling plays a crucial role in cell functioning. GPCR desensitization is an equally essential process. It allows cells to respond to changing environments and regain sensitivity to new stimuli while preventing unnecessary stimulation when no longer needed. Prolonged exposure to stimuli leads to GPCR desensitization. It involves blocking the receptors from binding and activating additional G proteins. This inhibits activation of downstream effectors, thereby...
8.6K
01:09GPCRs Regulate Adenylyl Cylase Activity
8.1K
Some GPCRs transmit signals through adenylyl cyclase (AC), a transmembrane enzyme. AC helps synthesize second messenger cyclic adenosine monophosphate (cAMP). AC catalyzes cyclization reaction and converts ATP to cAMP by releasing a pyrophosphate. The pyrophosphate is further hydrolyzed to phosphate by the enzyme pyrophosphatase, which drives cAMP synthesis to completion. However, cAMP is rapidly degraded to 5′ AMP by the enzymes phosphodiesterase (PDE), preventing overstimulation of...
8.1K
01:15G Protein-coupled Receptors
19.3K
G Protein-Coupled Receptors or GPCRs are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to sensory stimuli such as light, odors, hormones, cytokines, or neurotransmitters.
GPCRs are also called heptahelical, 7TM, or serpentine receptors, and consist of seven (H1-H7) transmembrane alpha-helices that span the bilayer to form a cylindrical core. The transmembrane helices are connected by three extracellular loops and three...
GPCRs are also called heptahelical, 7TM, or serpentine receptors, and consist of seven (H1-H7) transmembrane alpha-helices that span the bilayer to form a cylindrical core. The transmembrane helices are connected by three extracellular loops and three...
19.3K
01:30Transducer Mechanism: G Protein–Coupled Receptors
7.1K
G Protein–Coupled Receptors (GPCRs) are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to various stimuli. GPCRs regulate critical physiological pathways and are excellent drug targets for treating diseases such as diabetes, cancer, obesity, depression, or Alzheimer's. Nearly 35% of approved drugs implement their therapeutic effects by selectively interacting with specific GPCRs.
GPCRs are also called heptahelical,...
GPCRs are also called heptahelical,...
7.1K
01:21G-protein Coupled Receptors
133.8K
G-protein coupled receptors are ligand binding receptors that indirectly affect changes in the cell. The actual receptor is a single polypeptide that transverses the cell membrane seven times creating intracellular and extracellular loops. The extracellular loops create a ligand specific pocket which binds to neurotransmitters or hormones. The intracellular loops holds onto the G-protein.
133.8K
01:22Activation and Inactivation of G Proteins
12.2K
Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, beta, and gamma. They remain GDP-bound or GTP-bound inside the cells and switch between inactive/active states. The Gα subunit possesses the nucleotide-binding pocket that binds guanine nucleotides and switches between GDP or GTP-bound states. In contrast, the Gꞵ and Gγ subunits are always bound together with high...
12.2K