A reconstitution protocol for the in vitro folded human G protein-coupled Y2 receptor into lipid environment.
Although highly resolved crystal structures of G protein-coupled receptors have become available within the last decade, the need for studying these molecules in their natural membrane environment, where the molecules are rather dynamic, has been widely appreciated. Solid-state NMR spectroscopy is an excellent method to study structure and dynamics of membrane proteins in their native lipid environment. We developed a reconstitution protocol for the uniformly (15)N labeled Y(2) receptor into a bicelle-like lipid structure with high yields suitable for NMR studies. Milligram quantities of target protein were expressed in Escherichia coli using an optimized fermentation process in defined medium yielding in over 10mg/L medium of purified Y(2) receptor solubilized in SDS micelles. The structural integrity of the receptor molecules was strongly increased through refolding and subsequent reconstitution into phospholipid membranes. Specific ligand binding to the integrated receptor was determined using radioligand affinity assay. Further, by NMR measurement a dispersion of the (15)N signals comparable to native rhodopsin was shown. The efficiency of the reconstitution could also be inferred from the fact that reasonable (13)C NMR spectra at natural abundance could be acquired.
Schmidt
Peter
P
Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, D-04107 Leipzig, Germany.
Berger
Christian
C
Scheidt
Holger A
HA
Berndt
Sandra
S
Bunge
Andreas
A
Beck-Sickinger
Annette G
AG
Huster
Daniel
D
D002462:Cell Membrane / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008823:Micelles; D019906:Nuclear Magnetic Resonance, Biomolecular; D010743:Phospholipids / Q000737:chemistry*; D017510:Protein Folding; D017476:Receptors, Neuropeptide Y / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D008823:Micelles; D010743:Phospholipids; D017476:Receptors, Neuropeptide Y; C095889:neuropeptide Y2 receptor; D012967:Sodium Dodecyl Sulfate