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Expression and biophysical analysis of two double transmembrane domain-containing fragments from a yeast G protein-coupled receptor


Cohen, L S; Arshava, B; Estephan, R; Englander, J; Kim, H; Hauser, M; Zerbe, O; Ceruso, M; Becker, J M; Naider, F (2008). Expression and biophysical analysis of two double transmembrane domain-containing fragments from a yeast G protein-coupled receptor. Biopolymers, 90(2):117-130.

Abstract

Fragments of G protein-coupled receptors (GPCRs) are widely used as models to investigate these polytopic integral–membrane, signal-transducing molecules but have proven difficult to prepare in quantities necessary for NMR analyses. We report on the biosynthesis of two double transmembrane (TM) containing fragments of Ste2p, the -
factor GPCR from the yeast Saccharomyces cerevisiae. Ste2p(G31-G110) [TM1-TM2] and Ste2p(A231-S339) [TM6-TM7-CT40] were expressed as TrpLE fusion proteins in
Escherichia coli and released by CNBr cleavage. Expression yields were optimized using different strains and induction parameters, and by performing CNBr cleavage directly on
inclusion bodies. Non-labeled and uniformly labeled [15N]-TM1-TM2 and TM6-TM7-CT40, as well as uniformly labeled [15N,13C]-TM1-TM2 and TM1-TM2 selectively
labeled with [15N-Ala], [15N-Phe], [15N-Leu], [15N-Ile], and [15N-Val] were prepared. Yields of target peptides with >95% homogeneity varied from 3 mg/L of fermentation
([15N]-TM6-TM7-CT40) to 20 mg/L (selectively labeled TM1-TM2). The high level biosynthesis, CNBr processing and the efficient purification yields allowed the initiation
of a comprehensive biophysical analysis of TM1-TM2 and TM6-TM7-CT40. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that TM1-TM2 was
monomeric in this micellar environment whereas TM6-TM7-CT40 migrated as a dimer. CD analysis indicated that TM1-TM2 was highly helical in SDS and1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] but has a tendency to aggregate in dodecylphosphocholine micelles. Similar results were found with TM6-TM7-CT40. Conditions for NMR measurements were optimized, and both TM1-TM2 and TM6-TM7-
CT40 exhibited more that 90% of the expected crosspeaks in the [15N,1H]-HSQC spectrum. These findings set the stage for the determination of the 3D structure of these
large domains of a GPCR in micelles using high-resolution NMR.

Fragments of G protein-coupled receptors (GPCRs) are widely used as models to investigate these polytopic integral–membrane, signal-transducing molecules but have proven difficult to prepare in quantities necessary for NMR analyses. We report on the biosynthesis of two double transmembrane (TM) containing fragments of Ste2p, the -
factor GPCR from the yeast Saccharomyces cerevisiae. Ste2p(G31-G110) [TM1-TM2] and Ste2p(A231-S339) [TM6-TM7-CT40] were expressed as TrpLE fusion proteins in
Escherichia coli and released by CNBr cleavage. Expression yields were optimized using different strains and induction parameters, and by performing CNBr cleavage directly on
inclusion bodies. Non-labeled and uniformly labeled [15N]-TM1-TM2 and TM6-TM7-CT40, as well as uniformly labeled [15N,13C]-TM1-TM2 and TM1-TM2 selectively
labeled with [15N-Ala], [15N-Phe], [15N-Leu], [15N-Ile], and [15N-Val] were prepared. Yields of target peptides with >95% homogeneity varied from 3 mg/L of fermentation
([15N]-TM6-TM7-CT40) to 20 mg/L (selectively labeled TM1-TM2). The high level biosynthesis, CNBr processing and the efficient purification yields allowed the initiation
of a comprehensive biophysical analysis of TM1-TM2 and TM6-TM7-CT40. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that TM1-TM2 was
monomeric in this micellar environment whereas TM6-TM7-CT40 migrated as a dimer. CD analysis indicated that TM1-TM2 was highly helical in SDS and1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] but has a tendency to aggregate in dodecylphosphocholine micelles. Similar results were found with TM6-TM7-CT40. Conditions for NMR measurements were optimized, and both TM1-TM2 and TM6-TM7-
CT40 exhibited more that 90% of the expected crosspeaks in the [15N,1H]-HSQC spectrum. These findings set the stage for the determination of the 3D structure of these
large domains of a GPCR in micelles using high-resolution NMR.

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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:membrane peptides • double transmembrane domains • GPCRs • expression of peptides • NMR in detergents
Language:English
Date:7 February 2008
Deposited On:13 Nov 2008 16:24
Last Modified:05 Apr 2016 12:33
Publisher:Wiley-Blackwell
ISSN:0006-3525
Additional Information:This is a preprint of an article accepted for publication in Biopolymers © copyright 2008 John Wiley & Sons
Publisher DOI:10.1002/bip.20950
Official URL:http://www3.interscience.wiley.com/journal/117909227/abstract
Permanent URL: http://doi.org/10.5167/uzh-5173

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