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NMR investigation of structures of G-protein coupled receptor folding intermediates


Poms, Martin; Ansorge, Philipp; Martinez-Gil, Luis; Jurt, Simon; Gottstein, David; Fracchiolla, Katarina; Cohen, Leah S; Güntert, Peter; Mingarro, Ismael; Naider, Fred; Zerbe, Oliver (2016). NMR investigation of structures of G-protein coupled receptor folding intermediates. Journal of Biological Chemistry, 291(53):27170-27186.

Abstract

Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot et al., Biochemistry 29(1990), 4031) is postulated to proceed in 2 steps: Partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two and the first three transmembrane (TM) segments, as well as a construct comprising TM1-TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin et al., Biophys. J. 96(2009), 3187), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all
GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123 and TM127 adopt predominantly native- like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior.

Abstract

Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot et al., Biochemistry 29(1990), 4031) is postulated to proceed in 2 steps: Partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two and the first three transmembrane (TM) segments, as well as a construct comprising TM1-TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin et al., Biophys. J. 96(2009), 3187), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all
GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123 and TM127 adopt predominantly native- like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Uncontrolled Keywords:NMR, , G-protein coupled receptor, , GPCR, , Folding
Language:English
Date:30 December 2016
Deposited On:13 Dec 2016 07:56
Last Modified:18 Apr 2018 11:48
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:0021-9258
Funders:Swiss National Science Foundation
Additional Information:This research was originally published in Journal of Biological Chemistry. Poms M et al: NMR investigation of structures of G-protein coupled receptor folding intermediates. Journal of Biological Chemistry. 2016 © the American Society for Biochemistry and Molecular Biology.
OA Status:Green
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1074/jbc.M116.740985
PubMed ID:27864365
Project Information:
  • : FunderSNSF
  • : Grant ID
  • : Project TitleSwiss National Science Foundation

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