title: Biosynthesis and spectroscopic characterization of 2-TM fragments encompassing the sequence of a human GPCR, the Y4 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 transmembrane 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 ΔTrp 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 backbone 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.
authors.affiliation: Institute of Organic Chemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
authors.forename: Harsha
authors.initials: H
authors.lastname: Kocherla
authors.forename: Jacopo
authors.initials: J
authors.lastname: Marino
authors.forename: Xuan
authors.initials: X
authors.lastname: Shao
authors.forename: Jon
authors.initials: J
authors.lastname: Graf
authors.forename: Chao
authors.initials: C
authors.lastname: Zou
authors.forename: Oliver
authors.initials: O
authors.lastname: Zerbe
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017476:Receptors, Neuropeptide Y / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D017476:Receptors, Neuropeptide Y; C109491:neuropeptide Y4 receptor