title: NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates.
abstract: Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031-4037) 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, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187-3196), 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.
authors.affiliation: From the Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Poms
authors.affiliation: From the Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Philipp
authors.initials: P
authors.lastname: Ansorge
authors.affiliation: the Department of Biochemistry and Molecular Biology, ERI BioTecMed, University of Valencia, E-46100 Burjassot, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Martinez-Gil
authors.affiliation: From the Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Simon
authors.initials: S
authors.lastname: Jurt
authors.affiliation: the Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Gottstein
authors.affiliation: the Department of Chemistry, The College of Staten Island, City University of New York (CUNY), Staten Island, New York 10314, the Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, New York 10016, and.
authors.forename: Katrina E
authors.initials: KE
authors.lastname: Fracchiolla
authors.affiliation: the Department of Chemistry, The College of Staten Island, City University of New York (CUNY), Staten Island, New York 10314, the Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, New York 10016, and.
authors.forename: Leah S
authors.initials: LS
authors.lastname: Cohen
authors.affiliation: the Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany.|the Laboratory of Physical Chemistry, ETH Zürich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland.
authors.forename: Peter
authors.initials: P
authors.lastname: Güntert
authors.affiliation: the Department of Biochemistry and Molecular Biology, ERI BioTecMed, University of Valencia, E-46100 Burjassot, Spain.
authors.forename: Ismael
authors.initials: I
authors.lastname: Mingarro
authors.affiliation: the Department of Chemistry, The College of Staten Island, City University of New York (CUNY), Staten Island, New York 10314, the Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, New York 10016, and.
authors.forename: Fred
authors.initials: F
authors.lastname: Naider
authors.affiliation: From the Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland, oliver.zerbe@chem.uzh.ch.
authors.forename: Oliver
authors.initials: O
authors.lastname: Zerbe
mesh_terms: D000595:Amino Acid Sequence; D008823:Micelles; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry*; D011487:Protein Conformation; D000072417:Protein Domains; D051964:Receptors, Mating Factor / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
chemical_list: D008823:Micelles; D010446:Peptide Fragments; D051964:Receptors, Mating Factor; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins
keywords: G protein-coupled receptor (GPCR); Ste2p; Topology; membrane protein; nuclear magnetic resonance (NMR); protein folding; structural biology