Characterization of De Novo Synthesized GPCRs Supported in Nanolipoprotein Discs
Abstract
The protein family known as G-protein coupled receptors (GPCRs) comprises an important class of membrane-associated proteins, which remains a difficult family of proteins to characterize because their function requires a native-like lipid membrane environment. This paper focuses on applying a single step method leading to the formation of nanolipoprotein particles (NLPs) capable of solubilizing functional GPCRs for biophysical characterization. NLPs were used to demonstrate increased solubility for multiple GPCRs such as the Neurokinin 1 Receptor (NK1R), the Adrenergic Receptor aˆ2 (ADRB2) and the Dopamine Receptor D1 (DRD1). All three GPCRs showed affinity for their specific ligands using a simple dot blot assay. The NK1R was characterized in greater detail to demonstrate correct folding of the ligand pocket with nanomolar specificity. Electron paramagnetic resonance (EPR) spectroscopy validated the correct folding of the NK1R binding pocket for Substance P (SP). Fluorescence correlation spectroscopy (FCS) was used to identify SP-bound NK1R-containing NLPs and measure their dissociation rate in an aqueous environment. The dissociation constant was found to be 83 nM and was consistent with dot blot assays. This study represents a unique combinational approach involving the single step de novo production of a functional GPCR combined with biophysical techniques to demonstrate receptor associationmore »
- Authors:
-
- Univ. of California, Davis, CA (United States). Medical Center. NSF Center for Biophotonics Science and Technology; Univ. of California, Davis, CA (United States). Medical Center. Dept. of Biochemistry and Molecular Medicine
- Univ. of California, Davis, CA (United States). Medical Center. Dept. of Biochemistry and Molecular Medicine
- Univ. of California, Sacramento, CA (United States). Medical Center. Dept. of Radiation Oncology
- Univ. of California, Davis, CA (United States). Medical Center. NSF Center for Biophotonics Science and Technology
- Life Technologies, Carlsbad, CA (United States)
- Univ. of California, Davis, CA (United States). Medical Center. NSF Center for Biophotonics Science and Technology; Univ. of California, Sacramento, CA (United States). Medical Center. Dept. of Radiation Oncology; Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
- Publication Date:
- Research Org.:
- Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1627550
- Grant/Contract Number:
- AC52-07NA27344
- Resource Type:
- Accepted Manuscript
- Journal Name:
- PLoS ONE
- Additional Journal Information:
- Journal Volume: 7; Journal Issue: 9; Journal ID: ISSN 1932-6203
- Publisher:
- Public Library of Science
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; Science & Technology - Other Topics
Citation Formats
Gao, Tingjuan, Petrlova, Jitka, He, Wei, Huser, Thomas, Kudlick, Wieslaw, Voss, John, and Coleman, Matthew A. Characterization of De Novo Synthesized GPCRs Supported in Nanolipoprotein Discs. United States: N. p., 2012.
Web. doi:10.1371/journal.pone.0044911.
Gao, Tingjuan, Petrlova, Jitka, He, Wei, Huser, Thomas, Kudlick, Wieslaw, Voss, John, & Coleman, Matthew A. Characterization of De Novo Synthesized GPCRs Supported in Nanolipoprotein Discs. United States. https://doi.org/10.1371/journal.pone.0044911
Gao, Tingjuan, Petrlova, Jitka, He, Wei, Huser, Thomas, Kudlick, Wieslaw, Voss, John, and Coleman, Matthew A. Fri .
"Characterization of De Novo Synthesized GPCRs Supported in Nanolipoprotein Discs". United States. https://doi.org/10.1371/journal.pone.0044911. https://www.osti.gov/servlets/purl/1627550.
@article{osti_1627550,
title = {Characterization of De Novo Synthesized GPCRs Supported in Nanolipoprotein Discs},
author = {Gao, Tingjuan and Petrlova, Jitka and He, Wei and Huser, Thomas and Kudlick, Wieslaw and Voss, John and Coleman, Matthew A.},
abstractNote = {The protein family known as G-protein coupled receptors (GPCRs) comprises an important class of membrane-associated proteins, which remains a difficult family of proteins to characterize because their function requires a native-like lipid membrane environment. This paper focuses on applying a single step method leading to the formation of nanolipoprotein particles (NLPs) capable of solubilizing functional GPCRs for biophysical characterization. NLPs were used to demonstrate increased solubility for multiple GPCRs such as the Neurokinin 1 Receptor (NK1R), the Adrenergic Receptor aˆ2 (ADRB2) and the Dopamine Receptor D1 (DRD1). All three GPCRs showed affinity for their specific ligands using a simple dot blot assay. The NK1R was characterized in greater detail to demonstrate correct folding of the ligand pocket with nanomolar specificity. Electron paramagnetic resonance (EPR) spectroscopy validated the correct folding of the NK1R binding pocket for Substance P (SP). Fluorescence correlation spectroscopy (FCS) was used to identify SP-bound NK1R-containing NLPs and measure their dissociation rate in an aqueous environment. The dissociation constant was found to be 83 nM and was consistent with dot blot assays. This study represents a unique combinational approach involving the single step de novo production of a functional GPCR combined with biophysical techniques to demonstrate receptor association with the NLPs and binding affinity to specific ligands. Such a combined approach provides a novel path forward to screen and characterize GPCRs for drug discovery as well as structural studies outside of the complex cellular environment.},
doi = {10.1371/journal.pone.0044911},
journal = {PLoS ONE},
number = 9,
volume = 7,
place = {United States},
year = {Fri Sep 28 00:00:00 EDT 2012},
month = {Fri Sep 28 00:00:00 EDT 2012}
}
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