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Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

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Abstract

The human Y4 receptor, a class A G-protein coupled receptor (GPCR) primarily targeted by the pancreatic polypeptide (PP), is involved in a large number of physiologically important functions. This paper investigates a Y4 receptor fragment (N-TM1-TM2) comprising the N-terminal domain, the first two transmembrane (TM) helices and the first extracellular loop followed by a (His)6 tag, and addresses synthetic problems encountered when recombinantly producing such fragments from GPCRs in Escherichia coli. Rigorous purification and usage of the optimized detergent mixture 28 mM dodecylphosphocholine (DPC)/118 mM% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) resulted in high quality TROSY spectra indicating protein conformational homogeneity. Almost complete assignment of the backbone, including all TM residue resonances was obtained. Data on internal backbone dynamics revealed a high secondary structure content for N-TM1-TM2. Secondary chemical shifts and sequential amide proton nuclear Overhauser effects defined the TM helices. Interestingly, the properties of the N-terminal domain of this large fragment are highly similar to those determined on the isolated N-terminal domain in the presence of DPC micelles.

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Acknowledgements

We would like to thank for financial support from the Swiss National Science Foundation (grant No. 3100A0-11173 to CZ), from the Alfred Werner Legat (to OZ) and from the National Institutes of Health (GM22086 to FN).

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  1. Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland

    Chao Zou & Oliver Zerbe

  2. Department of Chemistry, College of Staten Island, CUNY, Staten Island, NY, 10314, USA

    Fred Naider

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Zou, C., Naider, F. & Zerbe, O. Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR. J Biomol NMR 42, 257–269 (2008). https://doi.org/10.1007/s10858-008-9281-z

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