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Biosynthesis and Spectroscopic Characterization of 2-TM Fragments Encompassing the Sequence of a Human GPCR, the Y4 Receptor


Kocherla, Harsha; Marino, Jacopo; Shao, Xuan; Graf, Jon; Zou, Chao; Zerbe, Oliver (2012). Biosynthesis and Spectroscopic Characterization of 2-TM Fragments Encompassing the Sequence of a Human GPCR, the Y4 Receptor. ChemBioChem, 13(6):818-828.

Abstract

This paper presents a divide-and-conquer approach towards obtaining solution structures of G protein-coupled receptors. The human Y4 receptor was dissected into two to three trans-membrane helix fragments, which were individually studied by solution NMR. We systematically compared various biosynthetic routes for the expression of the fragments in Escherichia coli and discuss purification strategies. In particular, we have compared the production of transmembrane (TM) fragments as inclusion bodies by using the DTrp leader sequence, with membrane-directed expression by using Mistic as the fusion partner, and developed methods for enzymatic cleavage. In addition, direct expression of two-TM fragments into inclusion bodies is a successful route in some cases. With the exception of TM13, we could produce all fragments in isotope-labeled form in quantities sufficient for NMR studies. Almost complete back-bone resonance assignment was obtained for the first two helices, as well as for helices 5 and 7, and a high degree was obtained for TM6, while conformational exchange processes resulted in the disappearance of many signals from TM4. In addition, complete assignments were obtained for all residues of the N-terminal domain, as well as the extracellular and cytosolic loops (with the exception of an undecapeptide segment in the second extracellular loop, EC2) and for the complete cytosolic C-terminal tail. In total, backbone resonances of 78% of all residues were assigned for the Y4 receptor. Predictions of secondary structure based on backbone chemical shifts indicate that most residues from the TM regions adopt helical conformations, with exception of those around polar residues or prolines. However, the domain boundaries differ slightly from those predicted for homology models. We suggest that the obtained chemical shifts might be useful in assigning the full- length receptor.

Abstract

This paper presents a divide-and-conquer approach towards obtaining solution structures of G protein-coupled receptors. The human Y4 receptor was dissected into two to three trans-membrane helix fragments, which were individually studied by solution NMR. We systematically compared various biosynthetic routes for the expression of the fragments in Escherichia coli and discuss purification strategies. In particular, we have compared the production of transmembrane (TM) fragments as inclusion bodies by using the DTrp leader sequence, with membrane-directed expression by using Mistic as the fusion partner, and developed methods for enzymatic cleavage. In addition, direct expression of two-TM fragments into inclusion bodies is a successful route in some cases. With the exception of TM13, we could produce all fragments in isotope-labeled form in quantities sufficient for NMR studies. Almost complete back-bone resonance assignment was obtained for the first two helices, as well as for helices 5 and 7, and a high degree was obtained for TM6, while conformational exchange processes resulted in the disappearance of many signals from TM4. In addition, complete assignments were obtained for all residues of the N-terminal domain, as well as the extracellular and cytosolic loops (with the exception of an undecapeptide segment in the second extracellular loop, EC2) and for the complete cytosolic C-terminal tail. In total, backbone resonances of 78% of all residues were assigned for the Y4 receptor. Predictions of secondary structure based on backbone chemical shifts indicate that most residues from the TM regions adopt helical conformations, with exception of those around polar residues or prolines. However, the domain boundaries differ slightly from those predicted for homology models. We suggest that the obtained chemical shifts might be useful in assigning the full- length receptor.

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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:GPCR, NMR, membrane protein
Language:English
Date:16 April 2012
Deposited On:16 Apr 2012 10:27
Last Modified:07 Dec 2017 13:48
Publisher:Wiley-Blackwell
ISSN:1439-4227
Funders:SNF, OPO Foundation
Publisher DOI:https://doi.org/10.1002/cbic.201100776
PubMed ID:22438305

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