title: Topogenesis of heterologously expressed fragments of the human Y4 GPCR.
abstract: Fragments of large membrane proteins have the potential to facilitate structural analysis by NMR, but their folding state remains a concern. Here we determined the quality of folding upon heterologous expression for a series of N- or C-terminally truncated fragments of the human Y4 G-protein coupled receptor, amounting to six different complementation pairs. As the individual fragments lack a specific function that could be used to ascertain proper folding, we instead assessed folding on a basic level by studying their membrane topology and by comparing it to well-established structural models of GPCRs. The topology of the fragments was determined using a reporter assay based on C-terminal green fluorescent protein- or alkaline phosphatase-fusions. N-terminal fusions to Lep or Mistic were used if a periplasmic orientation of the N-terminus of the fragments was expected based on predictions. Fragments fused to Mistic expressed at comparably high levels, whereas Lep fusions were produced to a much lower extent. Though none of the fragments exclusively adopted one orientation, often the correct topology predominated. In addition, systematic analysis of the fragment series suggested that the C-terminal half of the Y4 receptor is more important for adopting the correct topology than the N-terminal part. Using the detergent dodecylphosphocholine, selected fragments were solubilized from the membrane and proved sufficiently stable to allow purification. Finally, as a first step toward reconstituting a functional receptor from two fragments, we observed a physical interaction between complementing fragments pairs upon co-expression.
authors.affiliation: Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, CH 8057, Zurich, Switzerland.
authors.forename: Jacopo
authors.initials: J
authors.lastname: Marino
authors.forename: Eric R
authors.initials: ER
authors.lastname: Geertsma
authors.forename: Oliver
authors.initials: O
authors.lastname: Zerbe
mesh_terms: D002462:Cell Membrane; D049452:Green Fluorescent Proteins; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry*; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D017510:Protein Folding; D017476:Receptors, Neuropeptide Y / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D008565:Membrane Proteins; D010446:Peptide Fragments; D017476:Receptors, Neuropeptide Y; D011993:Recombinant Fusion Proteins; D010767:Phosphorylcholine; D049452:Green Fluorescent Proteins; C028810:dodecylphosphocholine; C109491:neuropeptide Y4 receptor