Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs.

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...

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Autores principales:	Tingjuan Gao, Jitka Petrlova, Wei He, Thomas Huser, Wieslaw Kudlick, John Voss, Matthew A Coleman
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a6fe307cd79e4db1823a132c3cd155d5
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spelling	oai:doaj.org-article:a6fe307cd79e4db1823a132c3cd155d52021-11-18T07:04:06ZCharacterization of de novo synthesized GPCRs supported in nanolipoprotein discs.1932-620310.1371/journal.pone.0044911https://doaj.org/article/a6fe307cd79e4db1823a132c3cd155d52012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23028674/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203The 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 â2 (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.Tingjuan GaoJitka PetrlovaWei HeThomas HuserWieslaw KudlickJohn VossMatthew A ColemanPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 9, p e44911 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Tingjuan Gao Jitka Petrlova Wei He Thomas Huser Wieslaw Kudlick John Voss Matthew A Coleman Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs.
description	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 â2 (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.
format	article
author	Tingjuan Gao Jitka Petrlova Wei He Thomas Huser Wieslaw Kudlick John Voss Matthew A Coleman
author_facet	Tingjuan Gao Jitka Petrlova Wei He Thomas Huser Wieslaw Kudlick John Voss Matthew A Coleman
author_sort	Tingjuan Gao
title	Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs.
title_short	Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs.
title_full	Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs.
title_fullStr	Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs.
title_full_unstemmed	Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs.
title_sort	characterization of de novo synthesized gpcrs supported in nanolipoprotein discs.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/a6fe307cd79e4db1823a132c3cd155d5
work_keys_str_mv	AT tingjuangao characterizationofdenovosynthesizedgpcrssupportedinnanolipoproteindiscs AT jitkapetrlova characterizationofdenovosynthesizedgpcrssupportedinnanolipoproteindiscs AT weihe characterizationofdenovosynthesizedgpcrssupportedinnanolipoproteindiscs AT thomashuser characterizationofdenovosynthesizedgpcrssupportedinnanolipoproteindiscs AT wieslawkudlick characterizationofdenovosynthesizedgpcrssupportedinnanolipoproteindiscs AT johnvoss characterizationofdenovosynthesizedgpcrssupportedinnanolipoproteindiscs AT matthewacoleman characterizationofdenovosynthesizedgpcrssupportedinnanolipoproteindiscs
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Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs.

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